Javier Ruiz-Sanz

Crystallographic structure of the SH3 domain of the human c‐Yes tyrosine kinase: Loop flexibility and amyloid aggregation

SH3 domains from the Src family of tyrosine kinases represent an interesting example of the delicate balance between promiscuity and specificity characteristic of proline‐rich ligand recognition by SH3 domains. The development of inhibitors of therapeutic potential requires a good understanding of the molecular determinants of binding affinity and specificity and relies on the availability of high quality structural information. Here, we present the first high‐resolution crystal structure of the SH3 domain of the c‐Yes oncogen. Comparison with other SH3 domains from the Src family revealed significant deviations in the loop regions. In particular, the n‐Src loop, highly flexible and partially disordered, is stabilized in an unusual conformation by the establishment of several intramolecular hydrogen bonds. Additionally, we present here the first report of amyloid aggregation by an SH3 domain from the Src family.

Two-state dynamics of the SH3–SH2 tandem of Abl kinase and the allosteric role of the N-cap

Significance There is increasing interest in developing pharmacological strategies to inhibit the allosteric regulatory mechanisms of signaling enzymes. The Abl tyrosine kinase is a prominent target, due to its ubiquitous cellular role and its involvement in cancer. Here, computational and experimental methods are used in synergy to probe the mechanism of the regulatory unit of Abl, whose dual function is to inhibit the enzyme and to mediate its interaction with other signaling proteins. Our results provide insights into the thermodynamic basis for the mechanism of Abl autoinhibition and activation and expand our understanding of the principles that govern modular domain organization. The regulation and localization of signaling enzymes is often mediated by accessory modular domains, which frequently function in tandems. The ability of these tandems to adopt multiple conformations is as important for proper regulation as the individual domain specificity. A paradigmatic example is Abl, a ubiquitous tyrosine kinase of significant pharmacological interest. SH3 and SH2 domains inhibit Abl by assembling onto the catalytic domain, allosterically clamping it in an inactive state. We investigate the dynamics of this SH3–SH2 tandem, using microsecond all-atom simulations and differential scanning calorimetry. Our results indicate that the Abl tandem is a two-state switch, alternating between the conformation observed in the structure of the autoinhibited enzyme and another configuration that is consistent with existing scattering data for an activated form. Intriguingly, we find that the latter is the most probable when the tandem is disengaged from the catalytic domain. Nevertheless, an amino acid stretch preceding the SH3 domain, the so-called N-cap, reshapes the free-energy landscape of the tandem and favors the interaction of this domain with the SH2-kinase linker, an intermediate step necessary for assembly of the autoinhibited complex. This allosteric effect arises from interactions between N-cap and the SH2 domain and SH3–SH2 connector, which involve a phosphorylation site. We also show that the SH3–SH2 connector plays a determinant role in the assembly equilibrium of Abl, because mutations thereof hinder the engagement of the SH2-kinase linker. These results provide a thermodynamic rationale for the involvement of N-cap and SH3–SH2 connector in Abl regulation and expand our understanding of the principles of modular domain organization.

Interfacial water molecules in SH3 interactions: a revised paradigm for polyproline recognition.

In spite of its biomedical relevance, polyproline recognition is still not fully understood. The disagreement between the current description of SH3 (Src homology 3) complexes and their thermodynamic behaviour calls for a revision of the SH3-binding paradigm. Recently, Abl-SH3 was demonstrated to recognize its ligands by a dual binding mechanism involving a robust network of water-mediated hydrogen bonds that complements the canonical hydrophobic interactions. The systematic analysis of the SH3 structural database in the present study reveals that this dual binding mode is universal to SH3 domains. Tightly bound buried-interfacial water molecules were found in all SH3 complexes studied mediating the interaction between the peptide ligand and the domain. Moreover, structural waters were also identified in a high percentage of the free SH3 domains. A detailed analysis of the pattern of water-mediated interactions enabled the identification of conserved hydration sites in the polyproline-recognition region and the establishment of relationships between hydration profiles and the sequence of both ligands and SH3 domains. Water-mediated interactions were also systematically observed in WW (protein-protein interaction domain containing two conserved tryptophan residues), UEV (ubiquitin-conjugating enzyme E2 variant) and EVH-1 [Ena/VASP (vasodilator-stimulated phosphoprotein) homology 1] structures. The results of the present study clearly indicate that the current description of proline-rich sequence recognition by protein-protein interaction modules is incomplete and insufficient for a correct understanding of these systems. A new binding paradigm is required that includes interfacial water molecules as relevant elements in polyproline recognition.

The promiscuous binding of the Fyn SH3 domain to a peptide from the NS5A protein.

The hepatitis C virus nonstructural 5A (NS5A) protein is a large zinc-binding phosphoprotein that plays an important role in viral RNA replication and is involved in altering signal transduction pathways in the host cell. This protein interacts with Fyn tyrosine kinase in vivo and regulates its kinase activity. The 1.5 Å resolution crystal structure of a complex between the SH3 domain of the Fyn tyrosine kinase and the C-terminal proline-rich motif of the NS5A-derived peptide APPIPPPRRKR has been solved. Crystals were obtained in the presence of ZnCl(2) and belonged to the tetragonal space group P4(1)2(1)2. The asymmetric unit is composed of four SH3 domains and two NS5A peptide molecules; only three of the domain molecules contain a bound peptide, while the fourth molecule seems to correspond to a free form of the domain. Additionally, two of the SH3 domains are bound to the same peptide chain and form a ternary complex. The proline-rich motif present in the NS5A protein seems to be important for RNA replication and virus assembly, and the promiscuous interaction of the Fyn SH3 domain with the NS5A C-terminal proline-rich peptide found in this crystallographic structure may be important in the virus infection cycle.

Intertwined dimeric structure for the SH3 domain of the c-Src tyrosine kinase induced by polyethylene glycol binding

Here we report the first crystal structure of the SH3 domain of the cellular Src tyrosine kinase (c‐Src‐SH3) domain on its own. In the crystal two molecules of c‐Src‐SH3 exchange their –RT loops generating an intertwined dimer, in which the two SH3 units, preserving the binding site configuration, are oriented to allow simultaneous binding of two ligand molecules. The dimerization of c‐Src‐SH3 is induced, both in the crystal and in solution, by the binding of a PEG molecule at the dimer interface, indicating that this type of conformations are energetically close to the native structure. These results have important implications respect to in vivo oligomerization and amyloid aggregation.

Thermodynamic characterization of the folding equilibrium of the human Nedd4-WW4 domain: at the frontiers of cooperative folding.

WW domains are the smallest naturally independent beta-sheet protein structures available to date and constitute attractive model systems for investigating the determinants of beta-sheet folding and stability. Nonetheless, their small size and low cooperativity pose a difficult challenge for a quantitative analysis of the folding equilibrium. We describe here a comprehensive thermodynamic characterization of the conformational equilibrium of the fourth WW domain from the human ubiquitin ligase Nedd4 (hNedd4-WW4) using a combination of calorimetric and spectroscopic techniques with several denaturing agents (temperature, pH, and chemical denaturants). Our results reveal that even though the experimental data can be described in terms of a two-state equilibrium, spectral data together with anomalous values for some thermodynamic parameters (a strikingly low temperature of maximum stability, a higher than expected native-state heat capacity, and a small specific enthalpy of unfolding) could be indicative of more complex types of equilibria, such as one-state downhill folding or alternative native conformations. Moreover, double-perturbation experiments reveal some features that, in spite of the apparent linear correlation between the thermodynamic parameters, seem to be indicative of a complex conformational equilibrium in the presence of urea. In summary, the data presented here point toward the existence of a low-energy barrier between the different macrostates of hNedd4-WW4, placing it at the frontier of cooperative folding.

Isothermal Titration Calorimetry: Thermodynamic Analysis of the Binding Thermograms of Molecular Recognition Events by Using Equilibrium Models

© 2013 Martinez et al., licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Isothermal Titration Calorimetry: Thermodynamic Analysis of the Binding Thermograms of Molecular Recognition Events by Using Equilibrium Models

Role of the surface charges D72 and K8 in the function and structural stability of the cytochrome c6 from Nostoc sp. PCC 7119

We investigated the role of electrostatic charges at positions D72 and K8 in the function and structural stability of cytochrome c6 from Nostoc sp. PCC 7119 (cyt c6). A series of mutant forms was generated to span the possible combinations of charge neutralization (by mutation to alanine) and charge inversion (by mutation to lysine and aspartate, respectively) in these positions. All forms of cyt c6 were functionally characterized by laser flash absorption spectroscopy, and their stability was probed by urea‐induced folding equilibrium relaxation experiments and differential scanning calorimetry. Neutralization or inversion of the positive charge at position K8 reduced the efficiency of electron transfer to photosystem I. This effect could not be reversed by compensating for the change in global charge that had been introduced by the mutation, indicating a specific role for K8 in the formation of the electron transfer complex between cyt c6 and photosystem I. Replacement of D72 by asparagine or lysine increased the efficiency of electron transfer to photosystem I, but destabilized the protein. D72 apparently participates in electrostatic interactions that stabilize the structure of cyt c6. The destabilizing effect was reduced when aspartate was replaced by the small amino acid alanine. Complementing the mutation D72A with a charge neutralization or inversion at position K8 led to mutant forms of cyt c6 that were more stable than the wild‐type under all tested conditions.

Approaching the thermodynamic view of protein folding through the reproduction of Anfinsen's experiment by undergraduate physical biochemistry students

In 1972 Christian B. Anfinsen received the Nobel Prize in Chemistry for “…his work on ribonuclease, especially concerning the connection between the amino acid sequence and the biologically active conformation.” The understanding of this principle is crucial for physical biochemistry students, since protein folding studies, bio‐computing sciences and protein design approaches are founded on such a well‐demonstrated connection. Herein, we describe a detailed and easy‐to‐follow experiment to reproduce the most relevant assays carried out at Anfinsens laboratory in the 60s. This experiment provides students with a platform to interpret by themselves the structural and kinetic experiments conceived to understand the protein folding problem. In addition, this three‐day experiment brings students a nice opportunity for protein manipulation as well as for the setting up of spectroscopic and chromatographic techniques.

Chapter 12:Biocalorimetry: Differential Scanning Calorimetry of Protein Solutions

The term biocalorimetry refers to the application of calorimetry to the study of the energetics of biological processes. Current high-sensitivity commercially available calorimeters offer the possibility of using small sample volumes of very dilute solutions and are able to directly measure very small amounts of heat, which allow the analysis of the thermodynamics of non-covalent interactions, such as those involved in biological macromolecules. This chapter presents an overview of the most relevant applications of DSC to protein solution studies. A survey of the main theoretical models to interpret and analyze protein calorimetric thermograms is described, together with an evaluation of their applicability. Here the main goal of DSC is to characterize protein stability and analyze protein thermal unfolding. From the DSC measurements all the meaningful thermodynamic parameters of such processes, together with the temperature-dependent population of the initial, final and possible intermediate states can be determined. The wider possibilities of DSC to investigate the effect of protein self-association as well as that of the presence of protein ligands during the thermal processes are also described. Finally, when the unfolding process does not occur under equilibrium, DSC is also a very useful technique to study protein denaturation through a kinetic approach.