Portrait of Dr Jose Luis Ortega Roldan

Dr Jose Luis Ortega Roldan

Lecturer in Biological NMR
NMR Facility Manager

About

Dr. Ortega-Roldan graduated in Chemistry at the University of Granada in 2005, where he conducted his PhD research in the laboratory of Prof. Nico van Nuland and Dr. Ana I. Azuaga-Fortes. During his PhD, Dr. Ortega-Roldan studied the structure, folding properties and interactions of the SH3 domains of the CD2 adaptor protein using NMR as the main technique and was awarded with an EMBO short-term fellowship to develop new NMR methodologies for the study of weak protein complexes in the laboratory of Dr. Martin Blackledge at the Institut the Biologie Structurale (Grenoble, France).
In 2011 he obtained a FEBS long-term fellowship to conduct his postdoctoral research in Dr. Jason Schnell laboratory at the University of Oxford studying membrane proteins by solution NMR. Subsequently in 2014 Dr. Ortega-Roldan was awarded a Marie Curie IEF fellowship to continue his research on the structure and function of the human Sigma-1 receptor.
Dr. Ortega-Roldan joined the School of Biosciences in September 2016 as a Lecturer in Biological NMR and head of the NMR facility, where his research group specialises in the structural and functional studies of proteins able to insert in lipid membranes, forming pores and channels.

ORCID ID: 0000-0002-6316-4390

Research interests

Integral membrane proteins and membrane-associated proteins are essential players in biological processes. Their functions range from transport across the membrane, cell adhesion and regulation of the cell shape. They represent around 40% of all proteins, and given their importance in the cell is not surprising that around 50% of the current drugs target membrane proteins. However, only a small number of structures of membrane proteins have been elucidated so far due to their low expression levels and the requirement of membrane mimetic environments for their study.
Our lab is interested in proteins with the ability to exists in a soluble form and in a membrane-bound form, where they usually form channels of pores. We study their mechanism of membrane insertion, the factors that trigger their activation and their inhibition using integrative structural biology tools, with a particular emphasis in solution NMR, as well as biophysics and fluorescence microscopy.
We are currently studying two protein families, the Chloride Intracellular Channel (CLIC) family, and MLKL:

  • Chloride ion channels control several cellular processes required for the normal function of the cell, and growing evidence supports the role of this channels in the development of different types of cancer. The Chloride Intracellular Channel (CLIC) has been linked with glioblastoma proliferative capacity and cardiac disease. We seek to identify which CLIC conformer is involved in disease and inhibitor binding, to understand the factors governing the equilibrium between the different CLIC forms and to determine the mechanism regulating such equilibrium with atomic detail. These information can be of great use for the development of conformation-specific pharmacological inhibitors and regulators that could lead to new avenues for cancer treatment. 
  •  Necroptosis is a process of controlled cell death that is part of the immune response to pathogens such as viruses. MLKL is a critical mediator of necroptosis that ruptures cellular membranes leading to cell death. Our work aims to elucidate how MLKL ruptures the membrane at molecular detail. This will enable the understanding and therapeutic manipulation/ exploitation of MLKL-mediated necroptosis for the treatment of viral infections.   

Teaching

Undergraduate:    

  • Biological Chemistry BI3212/BI3210/BI322/BI3220
  • BI514 Pharmacology
  • BI604 Biomembranes  

Postgraduate 

  • BI852 Advanced Analytical and Emerging Technologies for Biotechnology and Bioengineering 

Supervision

MSc-R projects available for 2019/20

Investigating the rupture the plasma membrane in necrosis? 
The ultimate aim of this work is to establish the mechanism of cell death triggered by necroptosis with atomic detail. We will utilise a range of structural biology tools, including NMR, X-Ray crystallography and Electron Microscopy, to study the membrane-bound form of MLKL.
Additional research costs: £1200 

In-cell structural biology: CLIC1 structure, function and drug binding inside tumour cells CLIC1 is a chloride channel that gets upregulated in different tumour cells and whose inhibition has been shown to halt tumour progression. The aim of this project is to study the activation and inhibition mechanisms with atomic detail using a range of structural biology techniques, including NMR, X-Ray crystallography, EM and fluorescence microscopy
Additional research costs: £1200   

Investigating the pharmacology of Sigma-1 receptors 
The s1 receptor represents one of the most pharmacologically diverse and poorly understood drug targets in humans. It is associated with a number of pathologies, ranging from stroke and cancer through to depression and addiction. This project will use a range of structural biology tools to investigate the changes is structure and dynamics that result upon agonist and antagonist binding
Additional research costs: £1200   

Investigating the lipid modulation of membrane proteins activity Membrane protein structure and function has been shown to be modulated by the lipid composition, but the complexity of the lipid bilayer has prevented the investigation of specific lipid-protein interactions. This projects aims to develop new methodologies to investigate the lipid dependence of membrane proteins in their native environment using NMR
Additional research costs: £1200  

Publications

Article

  • Ossa, F., Schnell, J. and Ortega-Roldan, J. (2017). A Review of the Human Sigma-1 Receptor Structure. Advances in experimental medicine and biology [Online] 964:15-29. Available at: http://dx.doi.org/10.1007/978-3-319-50174-1_3.
    The Sigma-1 Receptor (S1R) is a small, ligand-regulated integral membrane protein involved in cell homeostasis and the cellular stress response. The receptor has a multitude of protein and small molecule interaction partners with therapeutic potential. Newly reported structures of the human S1R in ligand-bound states provides essential insights into small molecule binding in the context of the overall protein structure. The structure also raises many interesting questions and provides an excellent starting point for understanding the molecular tricks employed by this small membrane receptor to modulate a large number of signaling events. Here, we review insights from the structures of ligand-bound S1R in the context of previous biochemical studies and propose, from a structural viewpoint, a set of important future directions.
  • Ortega-Roldan, J., Ossa, F., Amin, N. and Schnell, J. (2015). Solution NMR studies reveal the location of the second transmembrane domain of the human sigma-1 receptor. FEBS Letters [Online] 589:659-665. Available at: http://doi.org/10.1016/j.febslet.2015.01.033.
    The sigma-1 receptor (S1R) is a ligand-regulated membrane chaperone protein associated with endoplasmic reticulum stress response, and modulation of ion channel activities at the plasma membrane. We report here a solution NMR study of a S1R construct (S1R(?35)) in which only the first transmembrane domain and the eight-residue N-terminus have been removed. The second transmembrane helix is found to be composed of residues 91–107, which corresponds to the first steroid binding domain-like region. The cytosolic domain is found to contain three helices, and the secondary structure and backbone dynamics of the chaperone domain are consistent with that determined previously for the chaperone domain alone. The position of TM2 provides a framework for ongoing studies of S1R ligand binding and oligomerisation.
  • Ortega-Roldan, J., Casares, S., Ringkjøbing Jensen, M., Cárdenes, N., Bravo, J., Blackledge, M., Azuaga, A. and van Nuland, N. (2013). Distinct Ubiquitin Binding Modes Exhibited by SH3 Domains: Molecular Determinants and Functional Implications. PLoS ONE [Online] 8:e73018. Available at: https://doi.org/10.1371/journal.pone.0073018.
    SH3 domains constitute a new type of ubiquitin-binding domains. We previously showed that the third SH3 domain (SH3-C) of CD2AP binds ubiquitin in an alternative orientation. We have determined the structure of the complex between first CD2AP SH3 domain and ubiquitin and performed a structural and mutational analysis to decipher the determinants of the SH3-C binding mode to ubiquitin. We found that the Phe-to-Tyr mutation in CD2AP and in the homologous CIN85 SH3-C domain does not abrogate ubiquitin binding, in contrast to previous hypothesis and our findings for the first two CD2AP SH3 domains. The similar alternative binding mode of the SH3-C domains of these related adaptor proteins is characterised by a higher affinity to C-terminal extended ubiquitin molecules. We conclude that CD2AP/CIN85 SH3-C domain interaction with ubiquitin constitutes a new ubiquitin-binding mode involved in a different cellular function and thus changes the previously established mechanism of EGF-dependent CD2AP/CIN85 mono-ubiquitination.
  • Ortega-Roldan, J., Ossa, F. and Schnell, J. (2013). Characterization of the Human Sigma-1 Receptor Chaperone Domain Structure and Binding Immunoglobulin Protein (BiP) Interactions. Journal of Biological Chemistry [Online] 288:21448-21457. Available at: http://doi.org/10.1074/jbc.M113.450379.
    The sigma-1 receptor (S1R) is a ligand-regulated membrane protein chaperone involved in the ER stress response. S1R activity is implicated in diseases of the central nervous system including amnesia, schizophrenia, depression, Alzheimer disease, and addiction. S1R has been shown previously to regulate the Hsp70 binding immunoglobulin protein (BiP) and the inositol triphosphate receptor calcium channel through a C-terminal domain. We have developed methods for bacterial expression and reconstitution of the chaperone domain of human S1R into detergent micelles that enable its study by solution NMR spectroscopy. The chaperone domain is found to contain a helix at the N terminus followed by a largely dynamic region and a structured, helical C-terminal region that encompasses a membrane associated domain containing four helices. The helical region at residues ?198–206 is strongly amphipathic and proposed to anchor the chaperone domain to micelles and membranes. Three of the helices in the C-terminal region closely correspond to previously identified cholesterol and drug recognition sites. In addition, it is shown that the chaperone domain interacts with full-length BiP or the isolated nucleotide binding domain of BiP, but not the substrate binding domain, suggesting that the nucleotide binding domain is sufficient for S1R interactions.
  • Ceregido, M., Garcia-Pino, A., Ortega-Roldan, J., Casares, S., López Mayorga, O., Bravo, J., van Nuland, N. and Azuaga, A. (2013). Multimeric and differential binding of CIN85/CD2AP with two atypical proline-rich sequences from CD2 and Cbl-b*. FEBS Journal [Online] 280:3399-3415. Available at: https://doi.org/10.1111/febs.12333.
    The CD2AP (CD2-associated protein) and CIN85 (Cbl-interacting protein of 85 kDa) adaptor proteins each employ three Src homology 3 (SH3) domains to cluster protein partners and ensure efficient signal transduction and down-regulation of tyrosine kinase receptors. Using NMR, isothermal titration calorimetry and small-angle X-ray scattering methods, we have characterized several binding modes of the N-terminal SH3 domain (SH3A) of CD2AP and CIN85 with two natural atypical proline-rich regions in CD2 (cluster of differentiation 2) and Cbl-b (Casitas B-lineage lymphoma), and compared these data with previous studies and published crystal structures. Our experiments show that the CD2AP-SH3A domain forms a type II dimer with CD2 and both type I and type II dimeric complexes with Cbl-b. Like CD2AP, the CIN85-SH3A domain forms a type II complex with CD2, but a trimeric complex with Cbl-b, whereby the type I and II interactions take place at the same time. Together, these results explain how multiple interactions among similar SH3 domains and ligands produce a high degree of diversity in tyrosine kinase, cell adhesion or T-cell signaling pathways.
  • Guerry, P., Salmon, L., Mollica, L., Ortega-Roldan, J., Markwick, P., van Nuland, N., McCammon, J. and Blackledge, M. (2013). Mapping the population of protein conformational energy sub-states from NMR dipolar couplings. Angewandte Chemie International Edition [Online] 52:3181-3185. Available at: http://dx.doi.org/10.1002/anie.201209669.
  • Salmon, L., Pierce, L., Grimm, A., Ortega-Roldan, J., Mollica, L., Jensen, M., van Nuland, N., Markwick, P., McCammon, J. and Blackledge, M. (2012). Multi-Timescale Conformational Dynamics of the SH3 Domain of CD2-Associated Protein using NMR Spectroscopy and Accelerated Molecular Dynamics. Angewandte Chemie International Edition [Online] 51:6103-6106. Available at: https://doi.org/10.1002/anie.201202026.
  • Jensen, M., Ortega-Roldan, J., Salmon, L., van Nuland, N. and Blackledge, M. (2011). Characterizing weak protein–protein complexes by NMR residual dipolar couplings. European Biophysics Journal [Online] 40:1371-1381. Available at: https://doi.org/10.1007/s00249-011-0720-5.
    Protein–protein interactions occur with a wide range of affinities from tight complexes characterized by femtomolar dissociation constants to weak, and more transient, complexes of millimolar affinity. Many of the weak and transiently formed protein–protein complexes have escaped characterization due to the difficulties in obtaining experimental parameters that report on the complexes alone without contributions from the unbound, free proteins. Here, we review recent developments for characterizing the structures of weak protein–protein complexes using nuclear magnetic resonance spectroscopy with special emphasis on the utility of residual dipolar couplings.
  • Ortega-Roldan, J., Blackledge, M., van Nuland, N. and Azuaga, A. (2011). Solution structure, dynamics and thermodynamics of the three SH3 domains of CD2AP. Journal of Biomolecular NMR [Online] 50:103-117. Available at: https://doi.org/10.1007/s10858-011-9505-5.
    CD2 associated protein (CD2AP) is an adaptor protein that plays an important role in cell to cell union needed for the kidney function. It contains three N-terminal SH3 domains that are able to interact among others with CD2, ALIX, c-Cbl and Ubiquitin. To understand the role of the individual SH3 domains of this adaptor protein we have performed a complete structural, thermodynamic and dynamic characterization of the separate domains using NMR and DSC. The energetic contributions to the stability and the backbone dynamics have been related to the structural features of each domain using the structure-based FoldX algorithm. We have found that the N-terminal SH3 domain of both adaptor proteins CD2AP and CIN85 are the most stable SH3 domains that have been studied until now. This high stability is driven by a more extensive network of intra-molecular interactions. We believe that this increased stabilization of N-terminal SH3 domains in adaptor proteins is crucial to maintain the necessary conformation to establish the proper interactions critical for the recruitment of their natural targets.
  • Salmon, L., Ortega-Roldan, J., Lescop, E., Licinio, A., van Nuland, N., Jensen, M. and Blackledge, M. (2011). Structure, Dynamics, and Kinetics of Weak Protein-Protein Complexes from NMR Spin Relaxation Measurements of Titrated Solutions. Angewandte Chemie International Edition [Online] 50:3755-3759. Available at: https://doi.org/10.1002/anie.201100310.
  • Ortega-Roldan, J., Jensen, M., Brutscher, B., Azuaga, A., Blackledge, M. and van Nuland, N. (2009). Accurate characterization of weak macromolecular interactions by titration of NMR residual dipolar couplings: application to the CD2AP SH3-C:ubiquitin complex. Nucleic Acids Research [Online] 37:e70. Available at: https://doi.org/10.1093/nar/gkp211.
    The description of the interactome represents one of key challenges remaining for structural biology. Physiologically important weak interactions, with dissociation constants above 100??M, are remarkably common, but remain beyond the reach of most of structural biology. NMR spectroscopy, and in particular, residual dipolar couplings (RDCs) provide crucial conformational constraints on intermolecular orientation in molecular complexes, but the combination of free and bound contributions to the measured RDC seriously complicates their exploitation for weakly interacting partners. We develop a robust approach for the determination of weak complexes based on: (i) differential isotopic labeling of the partner proteins facilitating RDC measurement in both partners; (ii) measurement of RDC changes upon titration into different equilibrium mixtures of partially aligned free and complex forms of the proteins; (iii) novel analytical approaches to determine the effective alignment in all equilibrium mixtures; and (iv) extraction of precise RDCs for bound forms of both partner proteins. The approach is demonstrated for the determination of the three-dimensional structure of the weakly interacting CD2AP SH3-C:Ubiquitin complex (Kd?=?132?±?13??M) and is shown, using cross-validation, to be highly precise. We expect this methodology to extend the remarkable and unique ability of NMR to study weak protein–protein complexes.
  • Ortega-Roldan, J., Romero Romero, M., Ora, A., AB, E., Lopez Mayorga, O., Azuaga, A. and van Nuland, N. (2007). The high resolution NMR structure of the third SH3 domain of CD2AP. Journal of Biomolecular NMR [Online] 39:331-336. Available at: https://doi.org/10.1007/s10858-007-9201-7.
    CD2 associated protein (CD2AP) is an adaptor protein that plays an important role in cell to cell union needed for the kidney function. CD2AP interacts, as an adaptor protein, with different natural targets, such as CD2, nefrin, c-Cbl and podocin. These proteins are believed to interact to one of the three SH3 domains that are positioned in the N-terminal region of CD2AP. To understand the network of interactions between the natural targets and the three SH3 domains (SH3-A, B and C), we have started to determine the structures of the individual SH3 domains. Here we present the high-resolution structure of the SH3-C domain derived from NMR data. Full backbone and side-chain assignments were obtained from triple-resonance spectra. The structure was determined from distance restraints derived from high-resolution 600 and 800 MHz NOESY spectra, together with phi and psi torsion angle restraints based on the analysis of 1HN, 15N, 1H?, 13C?, 13CO and 13C? chemical shifts. Structures were calculated using CYANA and refined in water using RECOORD. The three-dimensional structure of CD2AP SH3-C contains all the features that are typically found in other SH3 domains, including the general binding site for the recognition of polyproline sequences.

Book section

  • Ortega-Roldan, J., Blackledge, M. and Ringkjøbing Jensen, M. (2018). Characterizing Protein-Protein Interactions Using Solution NMR Spectroscopy. In: Marsh, J. A. ed. Protein Complex Assembly: Methods and Protocols. Humana Press. Available at: https://doi.org/10.1007/978-1-4939-7759-8_5.
    In this chapter, we describe how NMR chemical shift titrations can be used to study the interaction between two proteins with emphasis on mapping the interface of the complex and determining the bind- ing affinity from a quantitative analysis of the experimental data. In particular, we discuss the appearance of NMR spectra in different chemical exchange regimes (fast, intermediate, and slow) and how these regimes affect NMR data analysis.
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