Portrait of Dr Ben Goult

Dr Ben Goult

Senior Lecturer in Biochemistry
Sandwich/Professional Year Co-ordinator


Ben joined the School of Biosciences in August 2014 where his research group specialises in the structural and biochemical studies of cell-extracellular matrix (ECM) adhesion complexes.

  • 1995-1998 University of Sheffield: BSc(Hons) Biochemistry 2:1 
  • 1998-2002 UMIST: PhD in Biological Science 
  • 2003-2005 University of Manchester: Research Associate 
  • 2005-2006 AstraZeneca Alderley Park: Senior Physical Scientist 
  • 2006-2012 University of Leicester: Research Associate 
  • 2012-2014 University of Leicester: Research Fellow 
  • 2014-2017 University of Kent: Lecturer 
  • 2017-present University of Kent: Senior Lecturer. 

Dr Ben Goult obtained his first degree in Biochemistry at the University of Sheffield in 1998, before embarking on a PhD in the labs of Dr Tim Norwood (University of Leicester) and Professor Lu-Yun Lian (University of Leicester/ Manchester) developing NMR based approaches for detecting small molecule binding to target proteins, a first step in drug discovery. Following a 2year postdoctoral position at the University of Manchester he moved to AstraZeneca Alderley Park as a Senior Physical Scientist.
In 2005, Ben returned to Leicester to work with Professor David Critchley on the proteins that regulate cell adhesion and migration, in particular the FERM domain containing proteins talin and kindlin; key players in integrin mediated adhesion. In 2014, Ben moved to the University of Kent to set up his own research group.

ORCID: 0000-0002-3438-2807 

Research interests

Research Interests: 
Cell-extracellular matrix (ECM) adhesion complexes, FERM domains 

Structural Biology: NMR Spectroscopy, X-Ray Crystallography and Small Angle X-Ray Scattering (SAXS)
My research involves the use of biophysical techniques to understand the structure and function of proteins that are involved in the process of Cell-extracellular matrix (ECM) adhesion. Such adhesion proteins are increasingly recognised as potential targets for therapeutic intervention in a range of pathologies including immune and vascular disorders, blood clotting, skin blistering, wound healing and cancer.

Pubmed Link
Google Scholar
If you are interested in joining the group then please contact: Dr Ben Goult


Year 1 

  • BI532 Skills For Bioscientists 2 

Final Year 

  •  BI602 Cell Signalling (Module Convenor) 


Post-Doctoral Researcher, Ph.D and Research Master applications from UK, EU, US & Overseas are always considered. Various funding sources can be explored. Post-Doc fellowships: EMBO, FEBS, HFSP, Newton, Marie Curie, Wellcome Trust. Please send your CV and summary of your research interests to:  b.t.goult@kent.ac.uk

MSc-R projects available for 2020/21

Deciphering the talin code - a cellular code that enables cells to feel their environment All cells in the human body are held in the correct place via adhesion to neighbouring cells, and to a dense meshwork of proteins that surround cells called the extracellular matrix. It is becoming evident that cells interpret classical signalling pathways in the context of the mechanical forces experienced by the cells attachment to this matrix, and this “mechanosensing" of the environment is a major determinant of cellular function. In cancer cells this “mechanosensing” is misregulated, leading to aberrant cell behaviour and metastasis.
The protein talin forms the core of most adhesive structures that mediate cell adhesion to the matrix, holding the cell in place. Furthermore, when the cell adheres to the matrix, talin then functions as a Mechanosensitive Signalling Hub (MSH), engaging different signalling molecules as a function of mechanical force to elicit different cellular behaviours (Goult et al., 2018). This plasticity of talin enables different signalling complexes to assemble on talin scaffolds in different conditions ultimately leading to alterations in gene expression.
The aim of this project is to determine precisely using a combination of biochemistry, structural biology and mechanobiology approaches how talin is able to adopt different conformations to switch “On” and “Off” different cellular pathways. The project will focus on defining the talin interactions that are misregulated in metastatic cell migration.  

Additional research costs: £1500  

Wiring the brain - deciphering how neurons make the right connections. The human brain is comprised of trillions of neurons, all linked together to form complex networks. Quite how our brains are wired up with such precision is a major question in biology. This project will work on the mechanisms that regulate axon guidance, the process by which neurons send out axons that extend and migrate to their correct targets. The project will focus on defining the talin interactions that are central to its function in axon guidance and neuronal pathfinding. 

Additional research costs £1500

Mechanical signaling mis-regulation in metastasis
Cell migration requires the coordinated assembly and disassembly of adhesions between the cell and the surrounding extracellular matrix, coupled to force exerted by the cell which enables the cell to pull itself forwards. While cell migration is essential for the development of multicellular life, it must be tightly controlled. Cancer metastasis is a product of uncontrolled cell migration.
The aim of this project is to use structural and biochemical techniques to characterise the protein-protein complexes that drive these processes.

Additional research costs £1500  


Showing 50 of 63 total publications in the Kent Academic Repository. View all publications.


  • Cowell, A., Jacquemet, G., Singh, A., Ammon, Y., Brown, D., Akhmanova, A., Ivaska, J. and Goult, B. (2020). Talin Rod Domain Containing Protein 1 (TLNRD1) is a novel actin-bundling protein which promotes filopodia formation. BioRxiv [Online]. Available at: https://doi.org/10.1101/2020.05.19.103606.
    Talin is a mechanosensitive adapter protein which couples integrins to the cytoskeleton and regulates integrin-mediated adhesion. Talin rod domain-containing protein-1 (TLNRD1) shares 22% homology with the R7R8 domains of talin, and is highly conserved throughout vertebrate evolution, however little is known about its function. Here we show that TLNRD1 is an α-helical protein which shares the same atypical topology as talin R7R8, but forms a novel antiparallel dimer arrangement. Actin co-sedimentation assays and electron microscopy reveal TLNRD1 is an actin-bundling protein that forms tight actin bundles. In addition, TLNRD1 binds to the same LD-motif containing proteins, RIAM and KANK, as talin, and thus may act in competition with talin. Filopodia are cell protrusions supported by tightly bundled actin filaments and TLNRD1 localises to filopodia tips, increases filopodia number and promotes cell migration in 2D. Together our results suggest that TLNRD1 has similar functionality to talin R7R8, serving as a nexus between the actin and microtubule cytoskeletons independent of adhesion complexes.
  • Khan, R. and Goult, B. (2019). Adhesions Assemble!—Autoinhibition as a Major Regulatory Mechanism of Integrin-Mediated Adhesion. Frontiers in Molecular Biosciences [Online] 6. Available at: https://doi.org/10.3389/fmolb.2019.00144.
    The advent of cell-cell and cell-extracellular adhesion enabled cells to interact in a coherent manner, forming larger structures and giving rise to the development of tissues, organs and complex multicellular life forms. The development of such organisms required tight regulation of dynamic adhesive structures by signaling pathways that coordinate cell attachment. Integrin-mediated adhesion to the extracellular matrix provides cells with support, survival signals and context-dependent cues that enable cells to run different cellular programs. One mysterious aspect of the process is how hundreds of proteins assemble seemingly spontaneously onto the activated integrin. An emerging concept is that adhesion assembly is regulated by autoinhibition of key proteins, a highly dynamic event that is modulated by a variety of signaling events. By enabling precise control of the activation state of proteins, autoinhibition enables localization of inactive proteins and the formation of pre-complexes. In response to the correct signals, these proteins become active and interact with other proteins, ultimately leading to development of cell-matrix junctions. Autoinhibition of key components of such adhesion complexes—including core components integrin, talin, vinculin, and FAK and important peripheral regulators such as RIAM, Src, and DLC1—leads to a view that the majority of proteins involved in complex assembly might be regulated by intramolecular interactions. Autoinhibition is relieved via multiple different signals including post-translation modification and proteolysis. More recently, mechanical forces have been shown to stabilize and increase the lifetimes of active conformations, identifying autoinhibition as a means of encoding mechanosensitivity. The complexity and scope for nuanced adhesion dynamics facilitated via autoinhibition provides numerous points of regulation. In this review, we discuss what is known about this mode of regulation and how it leads to rapid and tightly controlled assembly and disassembly of cell-matrix adhesion.
  • Bryce, N., Failes, T., Stehn, J., Baker, K., Zahler, S., Arzhaeva, Y., Bischof, L., Lyons, C., Dedova, I., Arndt, G., Gaus, K., Goult, B., Hardeman, E., Gunning, P. and Lock, J. (2019). High content imaging of unbiased chemical perturbations reveals that the phenotypic plasticity of the actin cytoskeleton is constrained. Cell Systems [Online]. Available at: https://doi.org/10.1016/j.cels.2019.09.002.
    Although F-actin has a large number of binding partners and regulators, the number of phenotypic states available to the actin cytoskeleton is unknown. Here, we quantified 74 features defining filamentous actin (F-actin) and cellular morphology in >25 million cells after treatment with a library of 114,400 structurally diverse compounds. After reducing the dimensionality of these data, only ∼25 recurrent F-actin phenotypes emerged, each defined by distinct quantitative features that could be machine learned. We identified 2,003 unknown compounds as inducers of actin-related phenotypes, including two that directly bind the focal adhesion protein, talin. Moreover, we observed that compounds with distinct molecular mechanisms could induce equivalent phenotypes and that initially divergent cellular responses could converge over time. These findings suggest a conceptual parallel between the actin cytoskeleton and gene regulatory networks, where the theoretical plasticity of interactions is nearly infinite, yet phenotypes in vivo are constrained into a limited subset of practicable configurations.
  • Han, S., Dean, K., Whitewood, A., Bachir, A., Guttierrez, E., Groisman, A., Horwitz, R., Goult, B. and Danuser, G. (2019). Formation of talin-vinculin pre-complexes dictates maturation of nascent adhesions by accelerated force transmission and vinculin recruitment. BioRxiv [Online]. Available at: https://www.biorxiv.org/content/10.1101/735183v1.
    Talin, vinculin, and paxillin are mechanosensitive proteins that are recruited early to nascent integrin-based adhesions (NAs). Using machine learning, high-resolution traction force microscopy, single-particle-tracking and fluorescence fluctuation time-series analysis, we find that, only in the NAs that eventually mature to focal adhesions, all three molecules are recruited concurrently and in synchrony with force onset. Thereafter, vinculin assembles at ~5 fold higher rates than in non-maturing NAs. We identify a domain in talin, R8, which exposes a vinculin- binding-site (VBS) without requiring tension. Stabilizing this domain via mutation lowers tension- free vinculin binding in conjunction with talin, impairs maturation of NAs, and reduces the rate of additional vinculin recruitment after force onset. Taken together, our data show that talin forms a complex with vinculin, before association with integrins, which is essential for NA maturation by talin’s effective unfolding and exposure of additional VBSs that induce fast force growth and further vinculin binding.
  • Yu, M., Le, S., Ammon, Y., Goult, B., Akhmanova, A. and Yan, J. (2019). Force-dependent regulation of KANK1-talin complex at focal adhesions. Nano Letters [Online]:5982-5990. Available at: http://dx.doi.org/10.1021/acs.nanolett.9b01732.
    KANK proteins mediate cross-talk between dynamic microtubules and integrin-based adhesions to the extracellular matrix. KANKs interact with the integrin/actin-binding protein talin and with several components of microtubule-stabilizing cortical complexes. Due to actomyosin contractility, the talin-KANK complex is likely under mechanical force, and its mechanical stability is expected to be a critical determinant of KANK recruitment to focal adhesions. Here, we quantified the lifetime of the complex of the talin rod domain R7 and the KN domain of KANK1 under shear-force geometry and found that it can withstand forces for seconds to minutes over a physiological force range up to 10 pN. Complex stability measurements combined with cell biological experiments suggest that shear-force stretching promotes KANK1 localization to the periphery of focal adhesions. These results indicate that the talin-KANK1 complex is mechanically strong, enabling it to support the cross-talk between microtubule and actin cytoskeleton at focal adhesions.
  • Wang, Y., Yan, J. and Goult, B. (2019). Force-dependent binding constants. Biochemistry [Online]. Available at: https://dx.doi.org/10.1021/acs.biochem.9b00453.
    Life is an emergent property of transient interactions between biomolecules and other organic and inorganic molecules that somehow leads to harmony and order. Measurement and quantitation of these biological interactions is of value to scientists, and is a major goal of biochemistry, as affinities provide insight into biological processes. In an organism these interactions occur in the context of forces and the need for a consideration of binding affinities in the context of a changing mechanical landscape necessitates a new way to consider the biochemistry of protein-protein interactions. In the last few decades the field of Mechanobiology has exploded, as both the appreciation, and the technical advances required to facilitate the study, of how forces impact on biological processes has become evident. The aim of this review is to introduce the concept of force-dependence of biomolecular interactions, and the requirement to be able to measure force-dependent binding constants. The focus of this discussion will be on the mechanotransduction that occurs at the integrin-mediated adhesions with the extracellular matrix, and the major mechanosensors talin and vinculin. However, the approaches that the cell uses to sense and respond to forces are applicable to other systems, and therefore provides a general discussion of the force-dependence of biomolecule interactions.
  • Guttula, D., Yao, M., Baker, K., Liang, Y., Goult, B., Doyle, P. and Yan, J. (2019). Calcium-Mediated Protein Folding and Stabilisation of Salmonella Biofilm-Associated Protein a. Journal of Molecular Biology [Online] 431:433-443. Available at: https://doi.org/10.1016/j.jmb.2018.11.014.
    Biofilm-associated proteins (BAPs) are important for early biofilm formation (adhesion) by bacteria and are also found in mature biofilms. BapA from Salmonella is a ~386 kDa surface protein, comprised of 27 tandem repeats predicted to be bacterial Ig-like (BIg) domains. Such tandem repeats are conserved for BAPs across different bacterial species, but the function of these domains is not completely understood. In this work, we report the first study of the mechanical stability of the BapA protein. Using magnetic tweezers, we show that the folding of BapA BIg domains requires calcium- binding and the folded domains have differential mechanical stabilities. Importantly, we identify that >100 nM concentration of calcium is needed for folding of the BIg domains, and the stability of the folded BIg domains is regulated by calcium over a wide concentration range from sub-micromolar (?M) to millimolar (mM). Only at mM calcium concentrations, as found in the extracellular environment, do the BIg domains have the saturated mechanical stability. BapA has been suggested to be involved in Salmonella invasion, and it is likely a crucial mechanical component of biofilms. Therefore, our results provide new insights into the potential roles of BapA as a structural maintenance component of Salmonella biofilm and also Salmonella invasion.
  • Camp, D., Haage, A., Solianova, V., Castle, W., Xu, Q., Lostchuck, E., Goult, B. and Tanentzapf, G. (2018). Direct binding of Talin to Rap1 is required for Cell-ECM adhesion in Drosophila. Journal of cell science [Online] 131. Available at: http://dx.doi.org/10.1242/jcs.225144.
    Attachment of cells to the Extracellular Matrix (ECM) via integrins is essential for animal development and tissue maintenance. The cytoplasmic protein Talin is necessary for linking integrins to the cytoskeleton and its recruitment is a key step in the assembly of the adhesion complex. However, the mechanisms that regulate Talin recruitment to sites of adhesion in vivo are still not well understood. Here we show that Talin recruitment to, and maintenance at, sites of integrin-mediated adhesion requires a direct interaction between Talin and the GTPase Rap1. A mutation that blocks the direct binding of Talin to Rap1 abolished Talin recruitment to sites of adhesion and the resulting phenotype phenocopies null alleles of Talin. Moreover, we show that Rap1 activity modulates Talin recruitment to sites of adhesion via its direct binding to Talin. These results identify the direct Talin-Rap1 interaction as a key in vivo mechanism for controlling integrin-mediated cell-ECM adhesion.
  • Haage, A., Goodwin, K., Whitewood, A., Camp, D., Bogutz, A., Turner, C., Granville, D., Lefebvre, L., Plotnikov, S., Goult, B. and Tanentzapf, G. (2018). Talin Autoinhibition Regulates Cell-ECM Adhesion Dynamics and Wound Healing In Vivo. Cell Reports [Online] 25:2401-2416. Available at: https://doi.org/10.1016/j.celrep.2018.10.098.
    Cells in multicellular organisms are arranged in complex three-dimensional patterns. This requires both transient and stable adhesions with the extracellular matrix (ECM). Integrin adhesion receptors bind ECM ligands outside the cell and then, by binding the protein talin inside the cell, assemble an adhesion complex connecting to the cytoskeleton. The activity of talin is controlled by several mechanisms, but these have not been well studied in vivo. By generating mice containing the activating point mutation E1770A in talin (Tln1), which disrupts autoinhibition, we show that talin autoinhibition controls cell-ECM adhesion, cell migration, and wound healing in vivo. In particular, blocking autoinhibition gives rise to more mature, stable focal adhesions that exhibit increased integrin activation. Mutant cells also show stronger attachment to ECM and decreased traction force. Overall, these results demonstrate that modulating talin function via autoinhibition is an important mechanism for regulating multiple aspects of integrin-mediated cell-ECM adhesion in vivo.
  • Goult, B., Yan, J. and Schwartz, M. (2018). Talin as a mechanosensitive signaling hub. Journal of Cell Biology [Online]. Available at: http://jcb.rupress.org/content/early/2018/09/24/jcb.201808061.
    Cell adhesion to the extracellular matrix (ECM), mediated by transmembrane receptors of the integrin family, is exquisitely sensitive to biochemical, structural, and mechanical features of the ECM. Talin is a cytoplasmic protein consisting of a globular head domain and a series of ?-helical bundles that form its long rod domain. Talin binds to the cytoplasmic domain of integrin ?-subunits, activates integrins, couples them to the actin cytoskeleton, and regulates integrin signaling. Recent evidence suggests switch-like behavior of the helix bundles that make up the talin rod domains, where individual domains open at different tension levels, exerting positive or negative effects on different protein interactions. These results lead us to propose that talin functions as a mechanosensitive signaling hub that integrates multiple extracellular and intracellular inputs to define a major axis of adhesion signaling.
  • Lagarrigue, F., Gingras, A., Paul, D., Valadez, A., Cuevas, M., Sun, H., Lopez-Ramirez, M., Goult, B., Shattil, S., Bergmeier, W. and Ginsberg, M. (2018). Rap1 binding to the talin 1 F0 domain makes a minimal contribution to murine platelet GPIIb-IIIa activation. Blood Advances [Online] 2:2358-2368. Available at: https://doi.org/10.1182/bloodadvances.2018020487.
    Activation of platelet glycoprotein IIb-IIIa (GPIIb-IIIa; integrin aIIbb3) leads to high-affinity fibrinogen binding and platelet aggregation during hemostasis. Whereas GTP-bound Rap1 GTPase promotes talin 1 binding to the b3 cytoplasmic domain to activate platelet GPIIb-IIIa, the Rap1 effector that regulates talin association with b3 in platelets is unknown. Rap1 binding to the talin 1 F0 subdomain was proposed to forge the talin 1–Rap1 link in platelets. Here, we report a talin 1 point mutant (R35E) that significantly reduces Rap1 affinity without a significant effect on its structure or expression. Talin 1 head domain (THD) (R35E) was of similar potency to wild-type THD in activating aIIbb3 in Chinese hamster ovary cells. Coexpression with activated Rap1b increased activation, and coexpression with Rap1GAP1 reduced activation caused by transfection of wild-type THD or THD(R35E). Furthermore, platelets from Tln1R35E/R35E mice showed similar GPIIb-IIIa activation to those from wild- type littermates in response to multiple agonists. Tln1R35E/R35E platelets exhibited slightly reduced platelet aggregation in response to low doses of agonists; however, there was not a significant hemostatic defect, as judged by tail bleeding times. Thus, the Rap1–talin 1 F0 interaction has little effect on platelet GPIIb-IIIa activation and hemostasis and cannot account for the dramatic effects of loss of Rap1 activity on these platelet functions.
  • Michael, M., Begum, R., Chan, G., Whitewood, A., Matthews, D., Goult, B., McGrath, J. and Parsons, M. (2018). Kindlin-1 regulates epidermal growth factor receptor signalling. Journal of Investigative Dermatology [Online]. Available at: https://www.jidonline.org/article/S0022-202X(18)32587-9/pdf.
    Kindler syndrome (KS) is an autosomal recessive genodermatosis that results from mutations in the FERMT1 gene encoding kindlin-1. Kindlin-1 localises to focal adhesion and is known to contribute to the activation of integrin receptors. Most cases of KS show a reduction or complete absence of kindlin-1 in keratinocytes, resulting in defective integrin activation, cell adhesion and migration. However, roles for kindlin-1 beyond integrin activation remain poorly defined. In the current study we show that skin and keratinocytes from KS patients have significantly reduced expression levels of the epidermal growth factor receptor (EGFR), resulting in defective EGF-dependent signalling and cell migration. Mechanistically, we demonstrate that kindlin-1 can associate directly with EGFR in vitro and in keratinocytes in an EGF-dependent, integrin-independent manner and that formation of this complex is required for EGF-dependent migration. We further demonstrate that kindlin-1 acts to protect EGFR from lysosomal-mediated degradation. This reveals a new role for kindlin-1 that has implications for understanding KS disease pathology.
  • De Franceschi, N., Miihkinen, M., Hamidi, H., Alanko, J., Mai, A., Picas, L., Guzman, C., Levy, D., Mattjus, P., Goult, B., Goud, B. and Ivaska, J. (2018). ProLIF: a quantitative assay for investigating integrin cytoplasmic protein interactions and synergistic membrane effects on proteoliposomes. Journal of Cell Science [Online]. Available at: https://doi.org/10.1242/jcs.214270.
    Integrin transmembrane heterodimeric receptors control a wide range of biological interactions by triggering the assembly of large multiprotein complexes at their cytoplasmic interface. A diverse set of methods have been used to investigate cytoplasmic interactions between integrins and intracellular proteins. These predominantly consist of peptide-based pull-downs and biochemical immuno- isolations from detergent-solubilized cell lysates. However, quantitative methods to probe integrin- protein interactions in a more biologically relevant context where the integrin is embedded within a lipid bilayer have been lacking. Here we describe a technique called ProLIF (Protein-Liposome Interactions by Flow cytometry) to reconstitute recombinant integrin transmembrane domain (TMD) and cytoplasmic tail (CT) fragments on liposomes as individual ? or ? subunits or as ?? heterodimers and, using flow cytometry, to rapidly and quantitatively measure protein interactions with these membrane-embedded integrins. Importantly, the assay can analyse binding of fluorescent proteins directly from cell lysates without further purification steps. By combining integrins with membrane lipids to generate proteoliposomes, the effects of membrane composition such as PI(4,5)P2 presence on protein recruitment to the integrin CTs can be analyzed. ProLIF requires no specific instrumentation, apart from a standard flow cytometer and can be applied to measure a broad range of membrane-dependent protein-protein interactions with the potential for high-throughput/multiplex analyses.
  • Gough, R. and Goult, B. (2018). The tale of two talins – two isoforms to fine-tune integrin signalling. FEBS Letters [Online] 592:2108-2125. Available at: http://dx.doi.org/10.1002/1873-3468.13081.
    Talins are cytoplasmic adapter proteins essential for integrin-mediated cell adhesion to the
    extracellular matrix. Talins control the activation state of integrins, link integrins to cytoskeletal
    actin, recruit numerous signalling molecules that mediate integrin signalling, and coordinate
    recruitment of microtubules to adhesion sites via interaction with KANK (kidney ankyrin repeat-
    containing) proteins. Vertebrates have two talin genes, TLN1 and TLN2. Although talin1 and
    talin2 share 76% protein sequence identity (88% similarity), they are not functionally redundant,
    and the differences between the two isoforms are not fully understood. In this Review, we focus
    on the similarities and differences between the two talins in terms of structure, biochemistry
    and function, which hint at subtle differences in fine-tuning adhesion signalling.
  • Whitewood, A., Singh, A., Brown, D. and Goult, B. (2018). Chlamydial virulence factor TarP mimics talin to disrupt the talin-vinculin complex. FEBS Letters [Online] 592:1751-1760. Available at: https://doi.org/10.1002/1873-3468.13074.
    Vinculin is a central component of mechanosensitive adhesive complexes that form between cells and the extracellular matrix. A myriad of infectious agents mimic vinculin binding sites (VBS), enabling them to hijack the adhesion machinery and facilitate cellular entry. Here, we report the structural and biochemical characterisation of a VBS from the chlamydial virulence factor TarP. Whilst the affinities of isolated VBS peptides from TarP and talin for vinculin are similar, their behaviour in larger fragments is markedly different. In talin, VBS are cryptic and require mechanical activation to bind vinculin, whereas the TarP VBS are located in disordered regions, and so are constitutively active. We demonstrate that the TarP VBS can uncouple talin:vinculin complexes, which may lead to adhesion destabilisation.
  • Bokhovchuk, F., Bate, N., Kovalevskaya, N., Goult, B., Spronk, C. and Vuister, G. (2018). The Structural Basis of Calcium Dependent Inactivation of the Transient Receptor Potential Vanilloid 5 Channel. Biochemistry [Online]. Available at: https://pubs.acs.org/doi/10.1021/acs.biochem.7b01287.
    The Transient Receptor Potential Vanilloid Channel subfamily member 5 (TRPV5) is a highly selective calcium ion channel predominately expressed in the kidney epithelium that plays an essential role in calcium reabsorption from renal infiltrate. In order to maintain Ca2+ homeostasis, TRPV5 possesses a tightly regulated negative feedback mechanism, where the ubiquitous Ca2+-binding protein Calmodulin (CaM) directly binds to the intracellular TRPV5 C-terminus, thus regulating TRPV5. Here we report on the characterisation of the TRPV5 C-terminal CaM binding site and its interaction with CaM at an atomistic level. We have solved the de novo solution structure of the TRPV5 C-terminus in complex with a CaM mutant, creating conditions that mimic the cellular basal Ca2+ state. We demonstrate that under these conditions the TRPV5 C-terminus is exclusively bound to the CaM C-lobe only, while conferring conformational freedom to the CaM N-lobe. We also show that at elevated calcium levels, additional interactions between the TRPV5 C-terminus and CaM N-lobe occur, resulting in formation of a tight 1:1 complex, effectively making the N-lobe the calcium sensor. Together, these data are consistent with, and support the novel model for Ca2+/CaM-dependent inactivation of TRPV channels as proposed by Bate et al. (Biochemistry, 2018, in press).
  • Bate, N., Caves, R., Skinner, S., Goult, B., Basran, J., Mitcheson, J. and Vuister, G. (2018). A Novel Mechanism for Calmodulin Dependent Inactivation of Transient Receptor Potential Vanilloid 6. Biochemistry [Online] 57:2611-2622. Available at: http://dx.doi.org/10.1021/acs.biochem.7b01286.
    The paralogues TRPV5 and TRPV6 belong to the vanilloid subfamily of the Transient Receptor Potential (TRP) superfamily of ion channels and both play an important role in overall Cahomeostasis. The functioning of the channels centres on a tightly controlled Ca-dependent feedback mechanism where the direct binding of the universal Ca-binding protein calmodulin (CaM) to the channel's C-terminal tail is required for channel inactivation. We have investigated this interaction at the atomic level and propose that under basal cellular [CaCaM is constitutively bound to the channel's C-tail via CaM C-lobe only contacts. When cytosolic [Ca] increases charging the apo CaM N-lobe with Ca, the CaM:TRPV6 complex rearranges and the TRPV6 C-tail further engages the CaM N-lobe via a crucial interaction involving L707. In a cellular context, mutation of L707 significantly increased the rate of channel inactivation. Finally, we present a model for TRPV6 CaM-dependent inactivation, which involves a novel so-called "two-tail" mechanism whereby CaM bridges between two TRPV6 monomers resulting in closure of the channel pore.
  • Collier, M., Ettelaie, C., Goult, B., Maraveyas, A. and Goodall, A. (2017). Investigation of the filamin-A dependent mechanisms of tissue factor incorporation into microvesicles. Thrombosis and Haemostasis [Online]:2009-2211. Available at: https://doi.org/10.1160/TH17-01-0009.
    We have previously shown that phosphorylation of tissue factor (TF) at Ser253 increases the incorporation of TF into microvesicles (MVs) following protease-activated receptor 2 (PAR2) activation through a process involving filamin-A, whereas Ser258 phosphorylation suppresses this process. Here we examined the contribution of the individual phosphorylation of these serine residues to the interaction between filamin-A and TF, and further examined how filamin-A regulates the incorporation of TF into MVs. In vitro binding assays using recombinant filamin-A C-terminal repeats 22-24 with biotinylated phospho-TF cytoplasmic domain peptides as bait, showed that filamin-A had the highest binding affinities for phospho-Ser253 and double-phosphorylated TF peptides, whilst the phospho-Ser258 TF peptide had the lowest affinity. Analysis of MDA-MB-231 cells using an in situ proximity ligation assay revealed increased proximity between the C-terminus of filamin-A and TF following PAR2 activation, which was concurrent with Ser253 phosphorylation and TF- positive MV release from these cells. Knock-down of filamin-A expression suppressed PAR2-mediated increases in cell surface TF procoagulant activity without reducing cell surface TF antigen expression. Disrupting lipid rafts by pre-incubation with methyl-beta cyclodextrin (M?CD) prior to PAR2 activation reduced TF-positive MV release and cell surface TF procoagulant activity to the same extent as filamin-A knock-down. In conclusion, this study shows that the interaction between TF and filamin-A is dependent on the differential phosphorylation of Ser253 and Ser258. Furthermore the interaction of TF with filamin-A may translocate cell surface TF to cholesterol-rich lipid rafts, increasing cell surface TF activity as well as TF incorporation and release into MVs.
  • Qi, L., Jafari, N., Chen, Z., Li, L., Hyto?nen, V., Goult, B., Zhan, C. and Huang, C. (2016). Talin2-mediated traction force drives matrix degradation and cell invasion. Journal of Cell Science [Online] 129:3661-3674. Available at: http://dx.doi.org/10.1242/jcs.185959.
    Talin binds to ?-integrin tails to activate integrins, regulating cell migration, invasion and metastasis. There are two talin genes, TLN1 and TLN2, encoding talin1 and talin2, respectively. Talin1 regulates focal adhesion dynamics, cell migration and invasion, whereas the biological function of talin2 is not clear and, indeed, talin2 has been presumed to function redundantly with talin1. Here, we show that talin2 has a much stronger binding to ?-integrin tails than talin1. Replacement of talin2 Ser339 with Cys significantly decreased its binding to ?1-integrin tails to a level comparable to that of talin1. Talin2 localizes at invadopodia and is indispensable for the generation of traction force and invadopodium-mediated matrix degradation. Ablation of talin2 suppressed traction force generation and invadopodia formation, which were restored by re-expressing talin2 but not talin1. Furthermore, re-expression of wild-type talin2 (but not talin2S339C) in talin2-depleted cells rescued development of traction force and invadopodia. These results suggest that a strong interaction of talin2 with integrins is required to generate traction, which in turn drives invadopodium-mediated matrix degradation, which is key to cancer cell invasion.
  • Bouchet, B., Gough, R., Ammon, Y., van de Willige, D., Post, H., Jacquemet, G., Altelaar, A., Heck, A., Goult, B. and Akhmanova, A. (2016). Talin-KANK1 interaction controls the recruitment of cortical microtubule stabilizing complexes to focal adhesions. eLife [Online] 10:1-42. Available at: http://dx.doi.org/10.7554/eLife.18124.
    The cross-talk between dynamic microtubules and integrin-based adhesions to the extracellular matrix plays a crucial role in cell polarity and migration. Microtubules regulate the turnover of adhesion sites, and, in turn, focal adhesions promote cortical microtubule capture and stabilization in their vicinity, but the underlying mechanism is unknown. Here, we show that cortical microtubule stabilization sites containing CLASPs, KIF21A, LL5? and liprins are recruited to focal adhesions by the adaptor protein KANK1, which directly interacts with the major adhesion component, talin. Structural studies showed that the conserved KN domain in KANK1 binds to the talin rod domain R7. Perturbation of this interaction, including a single point mutation in talin, which disrupts KANK1 binding but not the talin function in adhesion, abrogates the association of microtubule-stabilizing complexes with focal adhesions. We propose that the talin-KANK1 interaction links the two macromolecular assemblies that control cortical attachment of actin fibers and microtubules.
  • Yao, M., Goult, B., Klapholz, B., Hu, X., Toseland, C., Guo, Y., Cong, P., Sheetz, M. and Yan, J. (2016). The mechanical response of talin. Nature Communications [Online] 7:1-11. Available at: http://dx.doi.org/10.1038/ncomms11966.
    Talin, a force-bearing cytoplasmic adapter essential for integrin-mediated cell adhesion, links the actin cytoskeleton to integrin-based cell–extracellular matrix adhesions at the plasma membrane. Its C-terminal rod domain, which contains 13 helical bundles, plays important roles in mechanosensing during cell adhesion and spreading. However, how the structural stability and transition kinetics of the 13 helical bundles of talin are utilized in the diverse talin-dependent mechanosensing processes remains poorly understood. Here we report the force-dependent unfolding and refolding kinetics of all talin rod domains. Using experimentally determined kinetics parameters, we determined the dynamics of force fluctuation during stretching of talin under physiologically relevant pulling speeds and experimentally measured extension fluctuation trajectories. Our results reveal that force-dependent stochastic unfolding and refolding of talin rod domains make talin a very effective force buffer that sets a physiological force range of only a few pNs in the talin-mediated force transmission pathway.
  • Zacharchenko, T., Qian, X., Goult, B., Jethwa, D., Almeida, T., Ballestrem, C., Critchley, D. and Barsukov, I. (2016). LD Motif Recognition by Talin: Structure of the Talin-DLC1 Complex. Structure [Online]:1-13. Available at: http://www.dx.doi.org/10.1016/j.str.2016.04.016.
    Cell migration requires coordination between integrin-mediated cell adhesion to the extracellular matrix and force applied to adhesion sites. Talin plays a key role in coupling integrin receptors to the actomyosin contractile machinery, while deleted in liver cancer 1 (DLC1) is a Rho GAP that binds talin and regulates Rho, and therefore actomyosin contractility. We show that the LD motif of DLC1 forms a helix that binds to the four-helix bundle of the talin R8 domain in a canonical triple-helix arrangement. We demonstrate that the same R8 surface interacts with the paxillin LD1 and LD2 motifs. We identify key charged residues that stabilize the R8 interactions with LD motifs and demonstrate their importance in vitro and in cells. Our results suggest a network of competitive interactions in adhesion complexes that involve LD motifs, and identify mutations that can be used to analyze the biological roles of specific protein-protein interactions in cell migration.
  • Kumar, A., Ouyang, M., Van den Vries, K., McGhee, E., Tanaka, K., Anderson, M., Groisman, A., Goult, B., Anderson, K. and Schwartz, M. (2016). Talin tension sensor reveals novel features of focal adhesion force transmission and mechanosensitivity. Journal of Cell Biology [Online] 213:371-383. Available at: http://dx.doi.org/10.1083/jcb.201510012.
    Integrin-dependent adhesions are mechanosensitive structures in which talin mediates a linkage to actin laments either directly or indirectly by recruiting vinculin. Here, we report the development and validation of a talin tension sensor. We nd that talin in focal adhesions is under tension, which is higher in peripheral than central adhesions. Tension on talin is increased by vinculin and depends mainly on actin-binding site 2 (ABS2) within the middle of the rod domain, rather than ABS3 at the far C terminus. Unlike vinculin, talin is under lower tension on soft substrates. The difference between central and peripheral adhesions requires ABS3 but not vinculin or ABS2. However, differential stiffness sensing by talin requires ABS2 but not vinculin or ABS3. These results indicate that central versus peripheral adhesions must be orga- nized and regulated differently, and that ABS2 and ABS3 have distinct functions in spatial variations and stiffness sens- ing. Overall, these results shed new light on talin function and constrain models for cellular mechanosensing.
  • Atherton, P., Stuchbury, B., Wang, D., Jethwa, D., Tsang, R., Meiler-Rodriguez, E., Wang, P., Bate, N., Zent, R., Barsukov, I., Goult, B., Critchley, D. and Ballestrem, C. (2015). Vinculin controls talin engagement with the actomyosin machinery. Nature Communications [Online] 6:1-12. Available at: http://dx.doi.org/10.1038/ncomms10038.
    The link between extracellular-matrix-bound integrins and intracellular F-actin is essential for cell spreading and migration. Here, we demonstrate how the actin-binding proteins talin and vinculin cooperate to provide this link. By expressing structure-based talin mutants in talin null cells, we show that while the C-terminal actin-binding site (ABS3) in talin is required for adhesion complex assembly, the central ABS2 is essential for focal adhesion (FA) maturation. Thus, although ABS2 mutants support cell spreading, the cells lack FAs, fail to polarize and exert reduced force on the surrounding matrix. ABS2 is inhibited by the preceding mechanosensitive vinculin-binding R3 domain, and deletion of R2R3 or expression of constitutively active vinculin generates stable force-independent FAs, although cell polarity is compromised. Our data suggest a model whereby force acting on integrin-talin complexes via ABS3 promotes R3 unfolding and vinculin binding, activating ABS2 and locking talin into an actin-binding configuration that stabilizes FAs.
  • Yan, J., Yao, M., Goult, B. and Sheetz, M. (2015). Talin Dependent Mechanosensitivity of Cell Focal Adhesions. Cellular and Molecular Bioengineering [Online] 8:151-159. Available at: http://dx.doi.org/10.1007/s12195-014-0364-5.
    A fundamental question in mechanobiology is how mechanical stimuli are sensed by mechanosensing proteins and converted into signals that direct cells to adapt to the external environment. A key function of cell adhesion to the extracellular matrix (ECM) is to transduce mechanical forces between cells and their extracellular environment. Talin, a cytoplasmic adapter essential for integrin-mediated adhesion to the ECM, links the actin cytoskeleton to integrin at the plasma membrane. Here, we review recent progress in the understanding of talin-dependent mechanosensing revealed by stretching single talin molecules. Rapid progress in single-molecule force manipulation technologies has made it possible to directly study the impact of mechanical force on talin’s conformations and its interactions with other signaling proteins. We also provide our views on how findings from such studies may bring new insights into understanding the principles of mechanobiology on a broader scale, and how such fundamental knowledge may be harnessed for mechanopharmacology.
  • Skinner, S., Goult, B., Fogh, R., Boucher, W., Stevens, T., Laue, E. and Vuister, G. (2015). Structure calculation, refinement and validation using CcpNmr Analysis. Acta Crystallographica Section D-Biological Crystallography [Online] 71:154-161. Available at: http://dx.doi.org/10.1107/S1399004714026662.
    CcpNmr Analysis provides a streamlined pipeline for both NMR chemical shift assignment and structure determination of biological macromolecules. In addition, it encompasses tools to analyse the many additional experiments that make NMR such a pivotal technique for research into complex biological questions. This report describes how CcpNmr Analysis can seamlessly link together all of the tasks in the NMR structure-determination process. It details each of the stages from generating NMR restraints [distance, dihedral,hydrogen bonds and residual dipolar couplings (RDCs)],exporting these to and subsequently re-importing them from structure-calculation software (such as the programs CYANA or ARIA) and analysing and validating the results obtained from the structure calculation to, ultimately, the streamlined deposition of the completed assignments and the refined ensemble of structures into the PDBe repository. Until recently, such solution-structure determination by NMR has been quite a laborious task, requiring multiple stages and programs. However, with the new enhancements to CcpNmr Analysis described here, this process is now much more intuitive and efficient and less error-prone.
  • Villari, G., Jayo, A., Zanet, J., Fitch, B., Serrels, B., Frame, M., Stramer, B., Goult, B. and Parsons, M. (2015). A direct interaction between fascin and microtubules contributes to adhesion dynamics and cell migration. Journal of Cell Science [Online]:1-32. Available at: http://dx.doi.org/10.1242/jcs.175760.
    Fascin is an actin-binding and bundling protein that is highly upregulated in most epithelial cancers. Fascin promotes cell migration and adhesion dynamics in vitro and tumour cell metastasis in vivo. However, potential non-actin bundling roles for fascin remain unknown. Here we show for the first time that fascin can directly interact with the microtubule cytoskeleton and that this does not depend upon fascin-actin bundling. Microtubule binding contributes to fascin-dependent control of focal adhesion dynamics and cell migration speed. We also show that fascin forms a complex with focal adhesion kinase (FAK) and Src, and that this signalling pathway lies downstream of fascin-microtubule association in the control of adhesion stability. These findings shed light on new non actin-dependent roles for fascin and may have implications for the design of therapies to target fascin in metastatic disease.
  • Ellis, S., Lostchuck, E., Goult, B., Bouaouina, M., Fairchild, M., López-Ceballos, P., Calderwood, D. and Tanentzapf, G. (2014). The Talin Head Domain Reinforces Integrin-Mediated Adhesion by Promoting Adhesion Complex Stability and Clustering. PLoS Genetics [Online] 10:e1004756. Available at: http://dx.doi.org/10.1371/journal.pgen.1004756.
    Talin serves an essential function during integrin-mediated adhesion in linking integrins to actin via the intracellular adhesion complex. In addition, the N-terminal head domain of talin regulates the affinity of integrins for their ECM-ligands, a process known as inside-out activation. We previously showed that in Drosophila, mutating the integrin binding site in the talin head domain resulted in weakened adhesion to the ECM. Intriguingly, subsequent studies showed that canonical inside-out activation of integrin might not take place in flies. Consistent with this, a mutation in talin that specifically blocks its ability to activate mammalian integrins does not significantly impinge on talin function during fly development. Here, we describe results suggesting that the talin head domain reinforces and stabilizes the integrin adhesion complex by promoting integrin clustering distinct from its ability to support inside-out activation. Specifically, we show that an allele of talin containing a mutation that disrupts intramolecular interactions within the talin head attenuates the assembly and reinforcement of the integrin adhesion complex. Importantly, we provide evidence that this mutation blocks integrin clustering in vivo. We propose that the talin head domain is essential for regulating integrin avidity in Drosophila and that this is crucial for integrin-mediated adhesion during animal development.
  • Yao, M., Goult, B., Chen, H., Cong, P., Sheetz, M. and Yan, J. (2014). Mechanical activation of vinculin binding to talin locks talin in an unfolded conformation. Scientific reports [Online] 4:4610. Available at: http://dx.doi.org/10.1038/srep04610.
    The force-dependent interaction between talin and vinculin plays a crucial role in the initiation and growth of focal adhesions. Here we use magnetic tweezers to characterise the mechano-sensitive compact N-terminal region of the talin rod, and show that the three helical bundles R1-R3 in this region unfold in three distinct steps consistent with the domains unfolding independently. Mechanical stretching of talin R1-R3 enhances its binding to vinculin and vinculin binding inhibits talin refolding after force is released. Mutations that stabilize R3 identify it as the initial mechano-sensing domain in talin, unfolding at ~5 pN, suggesting that 5 pN is the force threshold for vinculin binding and adhesion progression.
  • Evans, S., Goult, B., Fairall, L., Jamieson, A., Ko Ferrigno, P., Ford, R., Schwabe, J. and Wagner, S. (2014). The ansamycin antibiotic, rifamycin SV, inhibits BCL6 transcriptional repression and forms a complex with the BCL6-BTB/POZ domain. PloS one [Online] 9:e90889. Available at: http://dx.doi.org/10.1371/journal.pone.0090889.
    BCL6 is a transcriptional repressor that is over-expressed due to chromosomal translocations, or other abnormalities, in ~40% of diffuse large B-cell lymphoma. BCL6 interacts with co-repressor, SMRT, and this is essential for its role in lymphomas. Peptide or small molecule inhibitors, which prevent the association of SMRT with BCL6, inhibit transcriptional repression and cause apoptosis of lymphoma cells in vitro and in vivo. In order to discover compounds, which have the potential to be developed into BCL6 inhibitors, we screened a natural product library. The ansamycin antibiotic, rifamycin SV, inhibited BCL6 transcriptional repression and NMR spectroscopy confirmed a direct interaction between rifamycin SV and BCL6. To further determine the characteristics of compounds binding to BCL6-POZ we analyzed four other members of this family and showed that rifabutin, bound most strongly. An X-ray crystal structure of the rifabutin-BCL6 complex revealed that rifabutin occupies a partly non-polar pocket making interactions with tyrosine58, asparagine21 and arginine24 of the BCL6-POZ domain. Importantly these residues are also important for the interaction of BLC6 with SMRT. This work demonstrates a unique approach to developing a structure activity relationship for a compound that will form the basis of a therapeutically useful BCL6 inhibitor.
  • Dhani, D., Goult, B., George, G., Rogerson, D., Bitton, D., Miller, C., Schwabe, J. and Tanaka, K. (2013). Mzt1/Tam4, a fission yeast MOZART1 homologue, is an essential component of the gamma-tubulin complex and directly interacts with GCP3(Alp6). Molecular biology of the cell [Online] 24:3337-3349. Available at: http://dx.doi.org/10.1091/mbc.E13-05-0253.
    In humans, MOZART1 plays an essential role in mitotic spindle formation as a component of the gamma-tubulin ring complex. We report that the fission yeast homologue of MOZART1, Mzt1/Tam4, is located at microtubule-organizing centers (MTOCs) and coimmunoprecipitates with gamma-tubulin Gtb1 from cell extracts. We show that mzt1/tam4 is an essential gene in fission yeast, encoding a 64-amino acid peptide, depletion of which leads to aberrant microtubule structure, including malformed mitotic spindles and impaired interphase microtubule array. Mzt1/Tam4 depletion also causes cytokinesis defects, suggesting a role of the gamma-tubulin complex in the regulation of cytokinesis. Yeast two-hybrid analysis shows that Mzt1/Tam4 forms a complex with Alp6, a fission yeast homologue of gamma-tubulin complex protein 3 (GCP3). Biophysical methods demonstrate that there is a direct interaction between recombinant Mzt1/Tam4 and the N-terminal region of GCP3(Alp6). Together our results suggest that Mzt1/Tam4 contributes to the MTOC function through regulation of GCP3(Alp6).
  • Goult, B., Xu, X., Gingras, A., Swift, M., Patel, B., Bate, N., Kopp, P., Barsukov, I., Critchley, D., Volkmann, N. and Hanein, D. (2013). Structural studies on full-length talin1 reveal a compact auto-inhibited dimer: implications for talin activation. Journal of structural biology [Online] 184:21-32. Available at: http://dx.doi.org/10.1016/j.jsb.2013.05.014.
    Talin is a large adaptor protein that activates integrins and couples them to cytoskeletal actin. Talin contains an N-terminal FERM (band 4.1, ezrin, radixin, moesin) domain (the head) linked to a flexible rod comprised of 13 amphipathic helical bundles (R1-R13) that terminate in a C-terminal helix (DD) that forms an anti-parallel dimer. We derived a three-dimensional structural model of full-length talin at a resolution of approximately 2.5nm using EM reconstruction of full-length talin and the known shapes of the individual domains and inter-domain angles as derived from small angle X-ray scattering. Talin adopts a compact conformation consistent with a dimer in which the two talin rods form a donut-shaped structure, with the two talin heads packed side by side occupying the hole at the center of this donut. In this configuration, the integrin binding site in the head domain and the actin-binding site at the carboxy-terminus of the rod are masked, implying that talin must unravel before it can support integrin activation and engage the actin cytoskeleton.
  • Ellis, S., Goult, B., Fairchild, M., Harris, N., Long, J., Lobo, P., Czerniecki, S., Van Petegem, F., Schöck, F., Peifer, M. and Tanentzapf, G. (2013). Talin autoinhibition is required for morphogenesis. Current biology [Online] 23:1825-1833. Available at: http://dx.doi.org/10.1016/j.cub.2013.07.054.
    The establishment of a multicellular body plan requires coordinating changes in cell adhesion and the cytoskeleton to ensure proper cell shape and position within a tissue. Cell adhesion to the extracellular matrix (ECM) via integrins plays diverse, essential roles during animal embryogenesis and therefore must be precisely regulated. Talin, a FERM-domain containing protein, forms a direct link between integrin adhesion receptors and the actin cytoskeleton and is an important regulator of integrin function. Similar to other FERM proteins, talin makes an intramolecular interaction that could autoinhibit its activity. However, the functional consequence of such an interaction has not been previously explored in vivo. Here, we demonstrate that targeted disruption of talin autoinhibition gives rise to morphogenetic defects during fly development and specifically that dorsal closure (DC), a process that resembles wound healing, is delayed. Impairment of autoinhibition leads to reduced talin turnover at and increased talin and integrin recruitment to sites of integrin-ECM attachment. Finally, we present evidence that talin autoinhibition is regulated by Rap1-dependent signaling. Based on our data, we propose that talin autoinhibition provides a switch for modulating adhesion turnover and adhesion stability that is essential for morphogenesis.
  • Watkins, R., Patil, R., Goult, B., Thomas, M., Gottlob, I. and Shackleton, S. (2013). A novel interaction between FRMD7 and CASK: evidence for a causal role in idiopathic infantile nystagmus. Human molecular genetics [Online] 22:2105-2118. Available at: http://dx.doi.org/10.1093/hmg/ddt060.
    Idiopathic infantile nystagmus (IIN) is a genetically heterogeneous disorder of eye movement that can be caused by mutations in the FRMD7 gene that encodes a FERM domain protein. FRMD7 is expressed in the brain and knock-down studies suggest it plays a role in neurite extension through modulation of the actin cytoskeleton, yet little is known about its precise molecular function and the effects of IIN mutations. Here, we studied four IIN-associated missense mutants and found them to have diverse effects on FRMD7 expression and cytoplasmic localization. The C271Y mutant accumulates in the nucleus, possibly due to disruption of a nuclear export sequence located downstream of the FERM-adjacent domain. While overexpression of wild-type FRMD7 promotes neurite outgrowth, mutants reduce this effect to differing degrees and the nuclear localizing C271Y mutant acts in a dominant-negative manner to inhibit neurite formation. To gain insight into FRMD7 molecular function, we used an IP-MS approach and identified the multi-domain plasma membrane scaffolding protein, CASK, as a FRMD7 interactor. Importantly, CASK promotes FRMD7 co-localization at the plasma membrane, where it enhances CASK-induced neurite length, whereas IIN-associated FRMD7 mutations impair all of these features. Mutations in CASK cause X-linked mental retardation. Patients with C-terminal CASK mutations also present with nystagmus and, strikingly, we show that these mutations specifically disrupt interaction with FRMD7. Together, our data strongly support a model whereby CASK recruits FRMD7 to the plasma membrane to promote neurite outgrowth during development of the oculomotor neural network and that defects in this interaction result in nystagmus.
  • Goult, B., Zacharchenko, T., Bate, N., Tsang, R., Hey, F., Gingras, A., Elliott, P., Roberts, G., Ballestrem, C., Critchley, D. and Barsukov, I. (2013). RIAM and vinculin binding to talin are mutually exclusive and regulate adhesion assembly and turnover. The Journal of biological chemistry [Online] 288:8238-8249. Available at: http://dx.doi.org/10.1074/jbc.M112.438119.
    Talin activates integrins, couples them to F-actin, and recruits vinculin to focal adhesions (FAs). Here, we report the structural characterization of the talin rod: 13 helical bundles (R1-R13) organized into a compact cluster of four-helix bundles (R2-R4) within a linear chain of five-helix bundles. Nine of the bundles contain vinculin-binding sites (VBS); R2R3 are atypical, with each containing two VBS. Talin R2R3 also binds synergistically to RIAM, a Rap1 effector involved in integrin activation. Biochemical and structural data show that vinculin and RIAM binding to R2R3 is mutually exclusive. Moreover, vinculin binding requires domain unfolding, whereas RIAM binds the folded R2R3 double domain. In cells, RIAM is enriched in nascent adhesions at the leading edge whereas vinculin is enriched in FAs. We propose a model in which RIAM binding to R2R3 initially recruits talin to membranes where it activates integrins. As talin engages F-actin, force exerted on R2R3 disrupts RIAM binding and exposes the VBS, which recruit vinculin to stabilize the complex.
  • Banno, A., Goult, B., Lee, H., Bate, N., Critchley, D. and Ginsberg, M. (2012). Subcellular localization of talin is regulated by inter-domain interactions. The Journal of biological chemistry [Online] 287:13799-13812. Available at: http://dx.doi.org/10.1074/jbc.M112.341214.
    Talin, which is composed of head (THD) and rod domains, plays an important role in cell adhesion events in diverse species including most metazoans and Dictyostelium discoideum. Talin is abundant in the cytosol; however, it mediates adhesion by associating with integrins in the plasma membrane where it forms a primary link between integrins and the actin cytoskeleton. Cells modulate the partitioning of talin between the plasma membrane and the cytosol to control cell adhesion. Here, we combine nuclear magnetic resonance spectroscopy (NMR) with subcellular fractionation to characterize two distinct THD-rod domain interactions that control the interaction of talin with the actin cytoskeleton or its localization to the plasma membrane. An interaction between a discrete vinculin-binding region of the rod (VBS1/2a; Tln1(482-787)), and the THD restrains talin from interacting with the plasma membrane. Furthermore, we show that vinculin binding to VBS1/2a results in talin recruitment to the plasma membrane. Thus, we have structurally defined specific inter-domain interactions between THD and the talin rod domain that regulate the subcellular localization of talin.
  • Bate, N., Gingras, A., Bachir, A., Horwitz, R., Ye, F., Patel, B., Goult, B. and Critchley, D. (2012). Talin contains a C-terminal calpain2 cleavage site important in focal adhesion dynamics. PloS one [Online] 7:e34461. Available at: http://dx.doi.org/10.1371/journal.pone.0034461.
    Talin is a large (~2540 residues) dimeric adaptor protein that associates with the integrin family of cell adhesion molecules in cell-extracellular matrix junctions (focal adhesions; FAs), where it both activates integrins and couples them to the actin cytoskeleton. Calpain2-mediated cleavage of talin between the head and rod domains has previously been shown to be important in FA turnover. Here we identify an additional calpain2-cleavage site that removes the dimerisation domain from the C-terminus of the talin rod, and show that an E2492G mutation inhibits calpain cleavage at this site in vitro, and increases the steady state levels of talin1 in vivo. Expression of a GFP-tagged talin1 E2492G mutant in CHO.K1 cells inhibited FA turnover and the persistence of cell protrusion just as effectively as a L432G mutation that inhibits calpain cleavage between the talin head and rod domains. Moreover, incorporation of both mutations into a single talin molecule had an additive effect clearly demonstrating that calpain cleavage at both the N- and C-terminal regions of talin contribute to the regulation of FA dynamics. However, the N-terminal site was more sensitive to calpain cleavage suggesting that lower levels of calpain are required to liberate the talin head and rod fragments than are needed to clip off the C-terminal dimerisation domain. The talin head and rod liberated by calpain2 cleavage have recently been shown to play roles in an integrin activation cycle important in FA turnover and in FAK-dependent cell cycle progression respectively. The half-life of the talin head is tightly regulated by ubiquitination and we suggest that removal of the C-terminal dimerisation domain from the talin rod may provide a mechanism both for terminating the signalling function of the talin rod and indeed for inactivating full-length talin thereby promoting FA turnover at the rear of the cell.
  • Phelan, M., Goult, B., Clayton, J., Hautbergue, G., Wilson, S. and Lian, L. (2012). The structure and selectivity of the SR protein SRSF2 RRM domain with RNA. Nucleic acids research [Online] 40:3232-3244. Available at: http://dx.doi.org/10.1093/nar/gkr1164.
    SRSF2 is a prototypical SR protein which plays important roles in the alternative splicing of pre-mRNA. It has been shown to be involved in regulatory pathways for maintaining genomic stability and play important roles in regulating key receptors in the heart. We report here the solution structure of the RNA recognition motifs (RRM) domain of free human SRSF2 (residues 9-101). Compared with other members of the SR protein family, SRSF2 structure has a longer L3 loop region. The conserved aromatic residue in the RNP2 motif is absent in SRSF2. Calorimetric titration shows that the RNA sequence 5'AGCAGAGUA3' binds SRSF2 with a Kd of 61 ± 1 nM and a 1:1 stoichiometry. NMR and mutagenesis experiments reveal that for SFSF2, the canonical B1 and B3 interactions are themselves not sufficient for effective RNA binding; the additional loop L3 is crucial for RNA complex formation. A comparison is made between the structures of SRSF2-RNA complex with other known RNA complexes of SR proteins. We conclude that interactions involving the L3 loop, N- and C-termini of the RRM domain are collectively important for determining selectivity between the protein and RNA.
  • Bouaouina, M., Goult, B., Huet-Calderwood, C., Bate, N., Brahme, N., Barsukov, I., Critchley, D. and Calderwood, D. (2012). A conserved lipid-binding loop in the kindlin FERM F1 domain is required for kindlin-mediated aIIbB3 integrin coactivation. The Journal of biological chemistry [Online] 287:6979-6990. Available at: http://dx.doi.org/10.1074/jbc.M111.330845.
    The activation of heterodimeric integrin adhesion receptors from low to high affinity states occurs in response to intracellular signals that act on the short cytoplasmic tails of integrin beta subunits. Binding of the talin FERM (four-point-one, ezrin, radixin, moesin) domain to the integrin beta-tail provides one key activation signal, but recent data indicate that the kindlin family of FERM domain proteins also play a central role. Kindlins directly bind integrin beta subunit cytoplasmic domains at a site distinct from the talin-binding site, and target to focal adhesions in adherent cells. However, the mechanisms by which kindlins impact integrin activation remain largely unknown. A notable feature of kindlins is their similarity to the integrin-binding and activating talin FERM domain. Drawing on this similarity, here we report the identification of an unstructured insert in the kindlin F1 FERM domain, and provide evidence that a highly conserved polylysine motif in this loop supports binding to negatively charged phospholipid head groups. We further show that the F1 loop and its membrane-binding motif are required for kindlin-1 targeting to focal adhesions, and for the cooperation between kindlin-1 and -2 and the talin head in aIIbB3 integrin activation, but not for kindlin binding to integrin beta tails. These studies highlight the structural and functional similarities between kindlins and the talin head and indicate that as for talin, FERM domain interactions with acidic membrane phospholipids as well beta-integrin tails contribute to the ability of kindlins to activate integrins.
  • Calkin, A., Goult, B., Zhang, L., Fairall, L., Hong, C., Schwabe, J. and Tontonoz, P. (2011). FERM-dependent E3 ligase recognition is a conserved mechanism for targeted degradation of lipoprotein receptors. Proceedings of the National Academy of Sciences of the United States of America [Online] 108:20107-20112. Available at: http://dx.doi.org/10.1073/pnas.1111589108.
    The E3 ubiquitin ligase IDOL (inducible degrader of the LDL receptor) regulates LDL receptor (LDLR)-dependent cholesterol uptake, but its mechanism of action, including the molecular basis for its stringent specificity, is poorly understood. Here we show that IDOL uses a singular strategy among E3 ligases for target recognition. The IDOL FERM domain binds directly to a recognition sequence in the cytoplasmic tails of lipoprotein receptors. This physical interaction is independent of IDOL's really interesting new gene (RING) domain E3 ligase activity and its capacity for autoubiquitination. Furthermore, IDOL controls its own stability through autoubiquitination of a unique FERM subdomain fold not present in other FERM proteins. Key residues defining the IDOL-LDLR interaction and IDOL autoubiquitination are functionally conserved in their insect homologs. Finally, we demonstrate that target recognition by IDOL involves a tripartite interaction between the FERM domain, membrane phospholipids, and the lipoprotein receptor tail. Our data identify the IDOL-LDLR interaction as an evolutionarily conserved mechanism for the regulation of lipid uptake and suggest that this interaction could potentially be exploited for the pharmacologic modulation of lipid metabolism.
  • Zhang, L., Fairall, L., Goult, B., Calkin, A., Hong, C., Millard, C., Tontonoz, P. and Schwabe, J. (2011). The IDOL-UBE2D complex mediates sterol-dependent degradation of the LDL receptor. Genes & development [Online] 25:1262-1274. Available at: http://dx.doi.org/10.1101/gad.2056211.
    We previously identified the E3 ubiquitin ligase IDOL as a sterol-dependent regulator of the LDL receptor (LDLR). The molecular pathway underlying IDOL action, however, remains to be determined. Here we report the identification and biochemical and structural characterization of an E2-E3 ubiquitin ligase complex for LDLR degradation. We identified the UBE2D family (UBE2D1-4) as E2 partners for IDOL that support both autoubiquitination and IDOL-dependent ubiquitination of the LDLR in a cell-free system. NMR chemical shift mapping and a 2.1 Å crystal structure of the IDOL RING domain-UBE2D1 complex revealed key interactions between the dimeric IDOL protein and the E2 enzyme. Analysis of the IDOL-UBE2D1 interface also defined the stereochemical basis for the selectivity of IDOL for UBE2Ds over other E2 ligases. Structure-based mutations that inhibit IDOL dimerization or IDOL-UBE2D interaction block IDOL-dependent LDLR ubiquitination and degradation. Furthermore, expression of a dominant-negative UBE2D enzyme inhibits the ability of IDOL to degrade the LDLR in cells. These results identify the IDOL-UBE2D complex as an important determinant of LDLR activity, and provide insight into molecular mechanisms underlying the regulation of cholesterol uptake.
  • Clayton, J., Phelan, M., Goult, B., Hautbergue, G., Wilson, S. and Lian, L. (2011). The 1H, 13C and 15N backbone and side-chain assignment of the RRM domain of SC35, a regulator of pre-mRNA splicing. Biomolecular NMR assignments [Online] 5:7-10. Available at: http://dx.doi.org/10.1007/s12104-010-9254-5.
    The serine-arginine rich family of proteins play important roles in the regulation of both constitutive and alternative splicing. SC35 (also known as SFRS2 and PR264) is a member of this family and contains one RNA recognition motif (RRM domain) and a RS domain at the C-terminus which is enriched with arginine and serine residues. SC35 is specifically involved in major regulatory pathways for cell proliferation and cell cycle progression. Determining the structure of SC35 would enable greater understanding of how its structure relates to its many functions. Complete (1)H, (13)C and (15)N assignments of the RRM domain of SC35 are presented. The assignments were obtained using 2D heteronuclear and 3D triple-resonance experiments with the uniformly [(15)N,(13)C]-labelled protein. The chemical shifts are used to predict the 3-dimensional structure of this RRM domain in the absence of RNA.
  • Oberoi, J., Fairall, L., Watson, P., Yang, J., Czimmerer, Z., Kampmann, T., Goult, B., Greenwood, J., Gooch, J., Kallenberger, B., Nagy, L., Neuhaus, D. and Schwabe, J. (2011). Structural basis for the assembly of the SMRT/NCoR core transcriptional repression machinery. Nature structural & molecular biology [Online] 18:177-184. Available at: http://dx.doi.org/10.1038/nsmb.1983.
    Eukaryotic transcriptional repressors function by recruiting large coregulatory complexes that target histone deacetylase enzymes to gene promoters and enhancers. Transcriptional repression complexes, assembled by the corepressor NCoR and its homolog SMRT, are crucial in many processes, including development and metabolic physiology. The core repression complex involves the recruitment of three proteins, HDAC3, GPS2 and TBL1, to a highly conserved repression domain within SMRT and NCoR. We have used structural and functional approaches to gain insight into the architecture and biological role of this complex. We report the crystal structure of the tetrameric oligomerization domain of TBL1, which interacts with both SMRT and GPS2, and the NMR structure of the interface complex between GPS2 and SMRT. These structures, together with computational docking, mutagenesis and functional assays, reveal the assembly mechanism and stoichiometry of the corepressor complex.
  • Elliott, P., Goult, B., Kopp, P., Bate, N., Grossmann, J., Roberts, G., Critchley, D. and Barsukov, I. (2010). The Structure of the talin head reveals a novel extended conformation of the FERM domain. Structure [Online] 18:1289-1299. Available at: http://dx.doi.org/10.1016/j.str.2010.07.011.
    FERM domains are found in a diverse superfamily of signaling and adaptor proteins at membrane interfaces. They typically consist of three separately folded domains (F1, F2, F3) in a compact cloverleaf structure. The crystal structure of the N-terminal head of the integrin-associated cytoskeletal protein talin reported here reveals a novel FERM domain with a linear domain arrangement, plus an additional domain F0 packed against F1. While F3 binds beta-integrin tails, basic residues in F1 and F2 are required for membrane association and for integrin activation. We show that these same residues are also required for cell spreading and focal adhesion assembly in cells. We suggest that the extended conformation of the talin head allows simultaneous binding to integrins via F3 and to PtdIns(4,5)P2-enriched microdomains via basic residues distributed along one surface of the talin head, and that these multiple interactions are required to stabilize integrins in the activated state.
  • Kalli, A., Wegener, K., Goult, B., Anthis, N., Campbell, I. and Sansom, M. (2010). The structure of the talin/integrin complex at a lipid bilayer: an NMR and MD simulation study. Structure [Online] 18:1280-1288. Available at: http://dx.doi.org/10.1016/j.str.2010.07.012.
    Integrins are cell surface receptors crucial for cell migration and adhesion. They are activated by interactions of the talin head domain with the membrane surface and the integrin ? cytoplasmic tail. Here, we use coarse-grained molecular dynamic simulations and nuclear magnetic resonance spectroscopy to elucidate the membrane-binding surfaces of the talin head (F2-F3) domain. In particular, we show that mutations in the four basic residues (K258E, K274E, R276E, and K280E) in the F2 binding surface reduce the affinity of the F2-F3 for the membrane and modify its orientation relative to the bilayer. Our results highlight the key role of anionic lipids in talin/membrane interactions. Simulation of the F2-F3 in complex with the ?/? transmembrane dimer reveals information for its orientation relative to the membrane. Our studies suggest that the perturbed orientation of talin relative to the membrane in the F2 mutant would be expected to in turn perturb talin/integrin interactions.
  • Gingras, A., Bate, N., Goult, B., Patel, B., Kopp, P., Emsley, J., Barsukov, I., Roberts, G. and Critchley, D. (2010). Central region of talin has a unique fold that binds vinculin and actin. The Journal of biological chemistry [Online] 285:29577-29587. Available at: http://dx.doi.org/10.1074/jbc.M109.095455.
    Talin is an adaptor protein that couples integrins to F-actin. Structural studies show that the N-terminal talin head contains an atypical FERM domain, whereas the N- and C-terminal parts of the talin rod include a series of ?-helical bundles. However, determining the structure of the central part of the rod has proved problematic. Residues 1359-1659 are homologous to the MESDc1 gene product, and we therefore expressed this region of talin in Escherichia coli. The crystal structure shows a unique fold comprised of a 5- and 4-helix bundle. The 5-helix bundle is composed of nonsequential helices due to insertion of the 4-helix bundle into the loop at the C terminus of helix ?3. The linker connecting the bundles forms a two-stranded anti-parallel ?-sheet likely limiting the relative movement of the two bundles. Because the 5-helix bundle contains the N and C termini of this module, we propose that it is linked by short loops to adjacent bundles, whereas the 4-helix bundle protrudes from the rod. This suggests the 4-helix bundle has a unique role, and its pI (7.8) is higher than other rod domains. Both helical bundles contain vinculin-binding sites but that in the isolated 5-helix bundle is cryptic, whereas that in the isolated 4-helix bundle is constitutively active. In contrast, both bundles are required for actin binding. Finally, we show that the MESDc1 protein, which is predicted to have a similar fold, is a novel actin-binding protein.


  • Castle, W. (2016). Characterising a Novel Interaction Between Rap1b and Rhea Sheds Light on New Mechanisms for Focal Adhesion Assembly.
    For the first time, we reveal a direct interaction between Rap1b and the fly homolog of talin, Rhea. Using a combination of biochemical and biophysical techniques, the Rap1 binding site on Rhea has been successfully mapped. Additionally, we reveal that an acidic-to-basic K17E substitution, on Rhea, completely abolishes Rap1 binding. Our collaborators have shown that this mutation results in non-viable embryos and our data links the Rap1:Rhea interaction to this lethal phenotype. The implications of our findings support currently proposed mechanisms of RIAM-independent integrin activation, that would challenge our understanding of focal adhesion formation.

    Furthermore, we propose a double-dependent Rap1 integrin-activation pathway, involving Rap1 directly interacting with the FERM domain, alongside the known Rap1-dependent recruitment of talin.
    Optimisations have allowed us to express both the wild-type and mutant Rhea F0 domain in E.coli BL21(DE3) cells. Efficient purification via Ni-NTA-based affinity chromatography results in yields of ~50-60 mg/litre being obtained. Using circular dichroism, it is shown that substitution of the K17 residue does not interfere with the structural integrity of Rhea; both proteins have identical full spectrum measurements and Tm values.

    Optimal expression of the conserved G-domain of mouse Rap1b was achieved in the CK600K cell line. This region is highly conserved to that in fly (90% identical). NMR was used to show direct interaction between drosophila Rhea F0 and Rap1b; whilst additionally confirming that Rap1b was unable to induce chemical shifts in the F0-K17E mutant. Triple resonance NMR experiments revealed the location of the Rap1 binding site on the wild-type Rhea F0, with V15, K17, T18, K37 and E40 being highlighted at the centre of this interaction. Structural models of Rap1:Rhea F0 binding agree with our findings, with the 5 highlighted residues seen to make close contact with the Rap1 switch I domain.

    Together this work confirms a direct interaction between Rhea and Rap1 whilst providing biochemical validation for the lethal phenotypes observed in mutant flies. It also provides further insight into new mechanisms of focal adhesion formation and integrin activation.


  • Goult, B. (2020). The Mechanical Basis of Memory - the MeshCODE Theory. Preprints.org [Online]. Available at: https://www.preprints.org/manuscript/202005.0118/v1.
    The MeshCODE framework outlined here represents a unifying theory of data storage in animals, providing read/write storage of both dynamic and persistent information in a binary format. Mechanosensitive proteins, that contain force-dependent switches, can store information persistently which can be written/updated using small changes in mechanical force. These mechanosensitive proteins, such as talin, scaffold each and every synapse creating a meshwork of switches that forms a code, a MeshCODE. Synaptic transmission and action potential spike trains would operate the cytoskeletal machinery to write and update the synaptic MeshCODEs, propagating this coding throughout the brain and to the entire organism. Based on established biophysical principles, a mechanical basis for memory provides a physical location for data storage in the brain. Furthermore, the conversion and storage of sensory and temporal inputs into a binary format identifies an addressable read/write memory system supporting the view of the mind as an organic supercomputer.
  • Miihkinen, M., Grönloh, M., Vihinen, H., Jokitalo, E., Goult, B., Ivaska, J. and Jacquemet, G. (2020). Myosin-X is required for integrin activation at filopodia tips. BioRxiv [Online]. Available at: https://www.biorxiv.org/content/10.1101/2020.05.05.078733v1.
    Filopodia assemble unique integrin-adhesion complexes as they sense and attach to the surrounding extracellular matrix. Integrin activation is essential for filopodia stability
  • Azizi, L., Cowell, A., Mykuliak, V., Goult, B., Turkki, P. and Hytönen, V. (2020). Cancer associated talin point mutations disorganise cell adhesion and migration. BioRxiv [Online]. Available at: https://doi.org/10.1101/2020.03.25.008193.
    Talin-1 is a key component of the multiprotein adhesion complexes which mediate cell migration, adhesion and integrin signalling and has been linked to cancer in several studies. In this study we analysed mutations in talin-1 reported in the Catalogue of Somatic Mutations in Cancer. A total of 11 talin mutants were selected and expressed in talin-deficient fibroblasts and their functional and structural effects were characterised in detail. An I392N point mutation in the F3 domain caused a three-fold increase in invasion, and enhanced migration compared to wildtype talin. Mutations R1368W and L1539P in the R7 and R8 domains caused increased invasion and proliferation and affected talin-vinculin complexation, but were not linked to changes in their binding affinities with known substrates KANK1 and RIAM measured for isolated talin domains. Lastly, L2509P, a mutation in the dimerisation domain of talin, prevented talin dimer formation, actin recruitment and FAKpTyr397 activation leading to anisotropic cell spreading and loss of random migration. Altogether, this study suggests that cancer derived point mutations in talin-1 can drastically affect cell behaviour and so may contribute to cancer progression.
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