Dr Ben Goult

Senior Lecturer in Biochemistry
Sandwich/Professional Year Co-ordinator

About

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.
Career 

  • 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

Teaching

Year 1 

  • BI532 Skills For Bioscientists 2 

Final Year 

  •  BI602 Cell Signalling (Module Convenor) 

Publications

Article

  • 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 α-helical bundles that form its long rod domain. Talin binds to the cytoplasmic domain of integrin β-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.
  • De Franceschi, N. et al. (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 α or β subunits or as αβ 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.
  • Haage, A. et al. (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.
  • Bokhovchuk, F. et al. (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).
  • Lagarrigue, F. et al. (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.
  • Whitewood, A. et al. (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.
  • Bate, N. et al. (2018). A Novel Mechanism for Calmodulin Dependent Inactivation of Transient Receptor Potential Vanilloid 6. Biochemistry [Online]. Available at: https://pubs.acs.org/doi/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.
  • Michael, M. et al. (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.
  • Collier, M. et al. (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 (MβCD) 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.
  • Kumar, A. et al. (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.
  • Yao, M. et al. (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.
  • Qi, L. et al. (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 β-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 β-integrin tails than talin1. Replacement of talin2 Ser339 with Cys significantly decreased its binding to β1-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. et al. (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, LL5β 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.
  • Zacharchenko, T. et al. (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.
  • Yan, J. et al. (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.
  • Villari, G. et al. (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.
  • Atherton, P. et al. (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.
  • Skinner, S. et al. (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.
  • Evans, S. et al. (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.
  • Yao, M. et al. (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.
  • Ellis, S. et al. (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.
  • Watkins, R. et al. (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. et al. (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.
  • Goult, B. et al. (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.
  • Ellis, S. et al. (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.
  • Dhani, D. et al. (2013). Mzt1/Tam4, a fission yeast MOZART1 homologue, is an essential component of the γ-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 γ-tubulin ring complex. We report that the fission yeast homologue of MOZART1, Mzt1/Tam4, is located at microtubule-organizing centers (MTOCs) and coimmunoprecipitates with γ-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 γ-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 γ-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).
  • Banno, A. et al. (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.
  • Bouaouina, M. et al. (2012). A conserved lipid-binding loop in the kindlin FERM F1 domain is required for kindlin-mediated αIIbβ3 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 β subunits. Binding of the talin FERM (four-point-one, ezrin, radixin, moesin) domain to the integrin β 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 β 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 αIIbβ3 integrin activation, but not for kindlin binding to integrin β 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 β-integrin tails contribute to the ability of kindlins to activate integrins.
  • Bate, N. et al. (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. et al. (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 K(d) of 61 ± 1 nM and a 1:1 stoichiometry. NMR and mutagenesis experiments reveal that for SFSF2, the canonical β1 and β3 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.
  • Zhang, L. et al. (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.
  • Calkin, A. et al. (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.
  • Oberoi, J. et al. (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.
  • Clayton, J. et al. (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.
  • Kopp, P. et al. (2010). Studies on the morphology and spreading of human endothelial cells define key inter- and intramolecular interactions for talin1. European journal of cell biology [Online] 89:661-73. Available at: http://dx.doi.org/10.1016/j.ejcb.2010.05.003.
    Talin binds to and activates integrins and is thought to couple them to cytoskeletal actin. However, functional studies on talin have been restricted by the fact that most cells express two talin isoforms. Here we show that human umbilical vein endothelial cells (HUVEC) express only talin1, and that talin1 knockdown inhibited focal adhesion (FA) assembly preventing the cells from maintaining a spread morphology, a phenotype that was rescued by GFP-mouse talin1. Thus HUVEC offer an ideal model system in which to conduct talin structure/function studies. Talin contains an N-terminal FERM domain (comprised of F1, F2 and F3 domains) and a C-terminal flexible rod. The F3 FERM domain binds beta-integrin tails, and mutations in F3 that inhibited integrin binding (W359A) or activation (L325R) severely compromised the ability of GFP-talin1 to rescue the talin1 knockdown phenotype despite the presence of a second integrin-binding site in the talin rod. The talin rod contains several actin-binding sites (ABS), and mutations in the C-terminal ABS that reduced actin-binding impaired talin1 function, whereas those that increased binding resulted in more stable FAs. The results show that both the N-terminal integrin and C-terminal actin-binding functions of talin are essential to cell spreading and FA assembly. Finally, mutations that relieve talin auto-inhibition resulted in the rapid and excessive production of FA, highlighting the importance of talin regulation within the cell.
  • Kalli, A. et al. (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 β 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 α/β 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.
  • Goult, B. et al. (2010). Structure of a double ubiquitin-like domain in the talin head: a role in integrin activation. The EMBO journal [Online] 29:1069-1080. Available at: http://dx.doi.org/10.1038/emboj.2010.4.
    Talin is a 270-kDa protein that activates integrins and couples them to cytoskeletal actin. Talin contains an N-terminal FERM domain comprised of F1, F2 and F3 domains, but it is atypical in that F1 contains a large insert and is preceded by an extra domain F0. Although F3 contains the binding site for beta-integrin tails, F0 and F1 are also required for activation of beta1-integrins. Here, we report the solution structures of F0, F1 and of the F0F1 double domain. Both F0 and F1 have ubiquitin-like folds joined in a novel fixed orientation by an extensive charged interface. The F1 insert forms a loop with helical propensity, and basic residues predicted to reside on one surface of the helix are required for binding to acidic phospholipids and for talin-mediated activation of beta1-integrins. This and the fact that basic residues on F2 and F3 are also essential for integrin activation suggest that extensive interactions between the talin FERM domain and acidic membrane phospholipids are required to orientate the FERM domain such that it can activate integrins.
  • Gingras, A. et al. (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 α-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 α3. The linker connecting the bundles forms a two-stranded anti-parallel β-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.
  • Elliott, P. et al. (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 β-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.
  • Goult, B. et al. (2010). The domain structure of talin: residues 1815-1973 form a five-helix bundle containing a cryptic vinculin-binding site. FEBS letters [Online] 584:2237-2241. Available at: http://dx.doi.org/10.1016/j.febslet.2010.04.028.
    Talin is a large flexible rod-shaped protein that activates the integrin family of cell adhesion molecules and couples them to cytoskeletal actin. Its rod region consists of a series of helical bundles. Here we show that residues 1815-1973 form a 5-helix bundle, with a topology unique to talin which is optimally suited for formation of a long rod such as talin. This is much more stable than the 4-helix (1843-1973) domain described earlier and as a result its vinculin binding sequence is inaccessible to vinculin at room temperature, with implications for the overall mechanism of the talin-vinculin interaction.
  • Goult, B. et al. (2009). The structure of the N-terminus of kindlin-1: a domain important for alphaiibbeta3 integrin activation. Journal of molecular biology [Online] 394:944-956. Available at: http://dx.doi.org/10.1016/j.jmb.2009.09.061.
    The integrin family of heterodimeric cell adhesion molecules exists in both low- and high-affinity states, and integrin activation requires binding of the talin FERM (four-point-one, ezrin, radixin, moesin) domain to membrane-proximal sequences in the beta-integrin cytoplasmic domain. However, it has recently become apparent that the kindlin family of FERM domain proteins is also essential for talin-induced integrin activation. FERM domains are typically composed of F1, F2, and F3 domains, but the talin FERM domain is atypical in that it contains a large insert in F1 and is preceded by a previously unrecognized domain, F0. Initial sequence alignments showed that the kindlin FERM domain was most similar to the talin FERM domain, but the homology appeared to be restricted to the F2 and F3 domains. Based on a detailed characterization of the talin FERM domain, we have reinvestigated the sequence relationship with kindlins and now show that kindlins do indeed contain the same domain structure as the talin FERM domain. However, the kindlin F1 domain contains an even larger insert than that in talin F1 that disrupts the sequence alignment. The insert, which varies in length between different kindlins, is not conserved and, as in talin, is largely unstructured. We have determined the structure of the kindlin-1 F0 domain by NMR, which shows that it adopts the same ubiquitin-like fold as the talin F0 and F1 domains. Comparison of the kindlin-1 and talin F0 domains identifies the probable interface with the kindlin-1 F1 domain. Potential sites of interaction of kindlin F0 with other proteins are discussed, including sites that differ between kindlin-1, kindlin-2, and kindlin-3. We also demonstrate that F0 is required for the ability of kindlin-1 to support talin-induced alphaIIbbeta3 integrin activation and for the localization of kindlin-1 to focal adhesions.
  • Gingras, A. et al. (2009). Structural determinants of integrin binding to the talin rod. The Journal of biological chemistry [Online] 284:8866-8876. Available at: http://dx.doi.org/10.1074/jbc.M805937200.
    The adaptor protein talin serves both to activate the integrin family of cell adhesion molecules and to couple integrins to the actin cytoskeleton. Integrin activation has been shown to involve binding of the talin FERM domain to membrane proximal sequences in the cytoplasmic domain of the integrin beta-subunit. However, a second integrin-binding site (IBS2) has been identified near the C-terminal end of the talin rod. Here we report the crystal structure of IBS2 (residues 1974-2293), which comprises two five-helix bundles, "IBS2-A" (1974-2139) and "IBS2-B" (2140-2293), connected by a continuous helix with a distinct kink at its center that is stabilized by side-chain H-bonding. Solution studies using small angle x-ray scattering and NMR point to a fairly flexible quaternary organization. Using pull-down and enzyme-linked immunosorbent assays, we demonstrate that integrin binding requires both IBS2 domains, as does binding to acidic phospholipids and robust targeting to focal adhesions. We have defined the membrane proximal region of the integrin cytoplasmic domain as the major binding region, although more membrane distal regions are also required for strong binding. Alanine-scanning mutagenesis points to an important electrostatic component to binding. Thermal unfolding experiments show that integrin binding induces conformational changes in the IBS2 module, which we speculate are linked to vinculin and membrane binding.
  • Anthis, N. et al. (2009). The structure of an integrin/talin complex reveals the basis of inside-out signal transduction. The EMBO journal [Online] 28:3623-3632. Available at: http://dx.doi.org/10.1038/emboj.2009.287.
    Fundamental to cell adhesion and migration, integrins are large heterodimeric membrane proteins that uniquely mediate inside-out signal transduction, whereby adhesion to the extracellular matrix is activated from within the cell by direct binding of talin to the cytoplasmic tail of the beta integrin subunit. Here, we report the first structure of talin bound to an authentic full-length beta integrin tail. Using biophysical and whole cell measurements, we show that a specific ionic interaction between the talin F3 domain and the membrane-proximal helix of the beta tail disrupts an integrin alpha/beta salt bridge that helps maintain the integrin inactive state. Second, we identify a positively charged surface on the talin F2 domain that precisely orients talin to disrupt the heterodimeric integrin transmembrane (TM) complex. These results show key structural features that explain the ability of talin to mediate inside-out TM signalling.
  • Goult, B. et al. (2009). The structure of an interdomain complex that regulates talin activity. The Journal of biological chemistry [Online] 284:15097-15106. Available at: http://dx.doi.org/10.1074/jbc.M900078200.
    Talin is a large flexible rod-shaped protein that activates the integrin family of cell adhesion molecules and couples them to cytoskeletal actin. It exists in both globular and extended conformations, and an intramolecular interaction between the N-terminal F3 FERM subdomain and the C-terminal part of the talin rod contributes to an autoinhibited form of the molecule. Here, we report the solution structure of the primary F3 binding domain within the C-terminal region of the talin rod and use intermolecular nuclear Overhauser effects to determine the structure of the complex. The rod domain (residues 1655-1822) is an amphipathic five-helix bundle; Tyr-377 of F3 docks into a hydrophobic pocket at one end of the bundle, whereas a basic loop in F3 (residues 316-326) interacts with a cluster of acidic residues in the middle of helix 4. Mutation of Glu-1770 abolishes binding. The rod domain competes with beta3-integrin tails for binding to F3, and the structure of the complex suggests that the rod is also likely to sterically inhibit binding of the FERM domain to the membrane.
  • Gingras, A. et al. (2008). The structure of the C-terminal actin-binding domain of talin. The EMBO journal [Online] 27:458-469. Available at: http://dx.doi.org/10.1038/sj.emboj.7601965.
    Talin is a large dimeric protein that couples integrins to cytoskeletal actin. Here, we report the structure of the C-terminal actin-binding domain of talin, the core of which is a five-helix bundle linked to a C-terminal helix responsible for dimerisation. The NMR structure of the bundle reveals a conserved surface-exposed hydrophobic patch surrounded by positively charged groups. We have mapped the actin-binding site to this surface and shown that helix 1 on the opposite side of the bundle negatively regulates actin binding. The crystal structure of the dimerisation helix reveals an antiparallel coiled-coil with conserved residues clustered on the solvent-exposed face. Mutagenesis shows that dimerisation is essential for filamentous actin (F-actin) binding and indicates that the dimerisation helix itself contributes to binding. We have used these structures together with small angle X-ray scattering to derive a model of the entire domain. Electron microscopy provides direct evidence for binding of the dimer to F-actin and indicates that it binds to three monomers along the long-pitch helix of the actin filament.
  • Goult, B. et al. (2008). NMR assignment of the C-terminal actin-binding domain of talin. Biomolecular NMR assignments [Online] 2:17-19. Available at: http://dx.doi.org/10.1007/s12104-007-9073-5.
    Talin is a large dimeric 270 kDa adapter protein which binds the cytoplasmic face of a subset of integrin beta-subunits and couples them to the actin cytoskeleton. Here we report the near complete 15N, 13C and 1H chemical shift assignments for the C-terminal actin-binding domain.
  • Srivastava, J. et al. (2008). Structural model and functional significance of pH-dependent talin-actin binding for focal adhesion remodeling. Proceedings of the National Academy of Sciences of the United States of America [Online] 105:14436-14441. Available at: http://dx.doi.org/10.1073/pnas.0805163105.
    Actin filament binding by the focal adhesion (FA)-associated protein talin stabilizes cell-substrate adhesions and is thought to be rate-limiting in cell migration. Although F-actin binding by talin is known to be pH-sensitive in vitro, with lower affinity at higher pH, the functional significance of this pH dependence is unknown. Because increased intracellular pH (pH(i)) promotes cell migration and is a hallmark of metastatic carcinomas, we asked whether it increases FA remodeling through lower-affinity talin-actin binding. Talin contains several actin binding sites, but we found that only the COOH-terminal USH-I/LWEQ module showed pH-dependent actin binding, with lower affinity and decreased maximal binding at higher pH. Molecular dynamics simulations and NMR of this module revealed a structural mechanism for pH-dependent actin binding. A cluster of titratable amino acids with upshifted pK(a) values, including His-2418, was identified at one end of the five-helix bundle distal from the actin binding site. Protonation of His-2418 induces changes in the conformation and dynamics of the remote actin binding site. Structural analyses of a mutant talin-H2418F at pH 6.0 and 8.0 suggested changes different from the WT protein, and we confirmed that actin binding by talin-H2418F was relatively pH-insensitive. In motile fibroblasts, increasing pH(i) decreased FA lifetime and increased the migratory rate. However, expression of talin-H2418F increased lifetime 2-fold and decreased the migratory rate. These data identify a molecular mechanism for pH-sensitive actin binding by talin and suggest that FA turnover is pH-dependent and in part mediated by pH-dependent affinity of talin for binding actin.

Forthcoming

  • Guttula, D. et al. (2018). Calcium-Mediated Protein Folding and Stabilisation of Salmonella Biofilm-Associated Protein a. Journal of Molecular Biology [Online]. 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. et al. (2018). Direct binding of Talin to Rap1 is required for Cell-ECM adhesion in Drosophila. Journal of Cell Science [Online]. 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.