Structure, dynamics, and binding thermodynamics of the v-Src SH2 domain: implications for drug design

SH2 domains provide fundamental recognition sites in tyrosine kinase-mediated signaling pathways which, when aberrant, give rise to disease states such as cancer, diabetes, and immune deficiency. Designing specific inhibitors that target the SH2 domain-binding site, however, have presented a major challenge. Despite well over a decade of intensive research, clinically useful SH2 domain inhibitors have yet to become available. A better understanding of the structural, dynamic, and thermodynamic contributions to ligand binding of individual SH2 domains will provide some insight as to whether inhibitor development is possible. We report the first high resolution solution structure of the apo-v-Src SH2 domain. This is accompanied by the analysis of backbone dynamics and pK(a) values within the apo- and peptide-bound states. Our results indicate that the phosphotyrosine (pY) pocket is tightly structured and hence not adaptable to exogenous ligands. On the other hand, the pocket which accommodates residues proximal and C-terminal of the pY (pY + 3) or so-called specificity determining region, is a large dynamic-binding surface. This appears to allow a high level of promiscuity in binding. Binding of a series of synthetic, phosphotyrosyl, peptidomimetic compounds designed to explore interactions in the pY + 3 pocket further demonstrates the ability of the SH2 domain to accommodate diverse ligands. The thermodynamic parameters of these interactions show dramatic enthalpy/entropy compensation. These data suggest that the v-Src SH2 domain does not have a highly specific secondary-binding site, which clearly presents a major hurdle to design selective inhibitors.     

The response of internal dynamics to hydrophobic core mutations in the SH3 domain from the Fyn tyrosine kinase

We have used (15)N- and (2)H-NMR spin relaxation experiments to study the response of backbone and side-chain dynamics when a leucine or valine is substituted for a completely buried phenylalanine residue in the SH3 domain from the Fyn tyrosine kinase. Several residues show differences in the time scales and temperature dependences of internal motions when data for the three proteins are compared. Changes were also observed in the magnitude of dynamics, with the valine, and to a lesser extent leucine mutant, showing enhanced flexibility compared to the wild-type (WT) protein. The motions of many of the same amide and methyl groups are affected by both mutations, identifying a set of loci where dynamics are sensitive to interactions involving the targeted side chain. These results show that contacts within the hydrophobic core affect many aspects of internal mobility throughout the Fyn SH3 domain.     

The EF-hand domain: A globally cooperative structural unit

EF-hand Ca(2+)-binding proteins participate in both modulation of Ca(2+) signals and direct transduction of the ionic signal into downstream biochemical events. The range of biochemical functions of these proteins is correlated with differences in the way in which they respond to the binding of Ca(2+). The EF-hand domains of calbindin D(9k) and calmodulin are homologous, yet they respond to the binding of calcium ions in a drastically different manner. A series of comparative analyses of their structures enabled the development of hypotheses about which residues in these proteins control the calcium-induced changes in conformation. To test our understanding of the relationship between protein sequence and structure, we specifically designed the F36G mutation of the EF-hand protein calbindin D(9k) to alter the packing of helices I and II in the apoprotein. The three-dimensional structure of apo F36G was determined in solution by nuclear magnetic resonance spectroscopy and showed that the design was successful. Surprisingly, significant structural perturbations also were found to extend far from the site of mutation. The observation of such long-range effects provides clear evidence that four-helix EF-hand domains should be treated as a single globally cooperative unit. A hypothetical mechanism for how the long-range effects are transmitted is described. Our results support the concept of energetic and structural coupling of the key residues that are crucial for a protein's fold and function.     

Solution structure and proposed domain domain recognition interface of an acyl carrier protein domain from a modular polyketide synthase

Polyketides are a medicinally important class of natural products. The architecture of modular polyketide synthases (PKSs), composed of multiple covalently linked domains grouped into modules, provides an attractive framework for engineering novel polyketide-producing assemblies. However, impaired domain-domain interactions can compromise the efficiency of engineered polyketide biosynthesis. To facilitate the study of these domain-domain interactions, we have used nuclear magnetic resonance (NMR) spectroscopy to determine the first solution structure of an acyl carrier protein (ACP) domain from a modular PKS, 6-deoxyerythronolide B synthase (DEBS). The tertiary fold of this 10-kD domain is a three-helical bundle; an additional short helix in the second loop also contributes to the core helical packing. Superposition of residues 14-94 of the ensemble on the mean structure yields an average atomic RMSD of 0.64 +/- 0.09 Angstrom for the backbone atoms (1.21 +/- 0.13 Angstrom for all non-hydrogen atoms). The three major helices superimpose with a backbone RMSD of 0.48 +/- 0.10 Angstrom (0.99 +/- 0.11 Angstrom for non-hydrogen atoms). Based on this solution structure, homology models were constructed for five other DEBS ACP domains. Comparison of their steric and electrostatic surfaces at the putative interaction interface (centered on helix II) suggests a model for protein-protein recognition of ACP domains, consistent with the previously observed specificity. Site-directed mutagenesis experiments indicate that two of the identified residues influence the specificity of ACP recognition.     

Structure,dynamics, and stability of the globular domain of human linker histone H1.0 and the role of positive charges

Linker histone H1 (H1) is an abundant chromatin‐binding protein that acts as an epigenetic regulator binding to nucleosomes and altering chromatin structures and dynamics. Nonetheless, the mechanistic details of its function remain poorly understood. Recent work suggest that the number and position of charged side chains on the globular domain (GD) of H1 influence chromatin structure and hence gene repression. Here, we solved the solution structure of the unbound GD of human H1.0, revealing that the structure is almost completely unperturbed by complex formation, except for a loop connecting two antiparallel β‐strands. We further quantified the role of the many positive charges of the GD for its structure and conformational stability through the analysis of 11 charge variants. We find that modulating the number of charges has little effect on the structure, but the stability is affected, resulting in a difference in melting temperature of 26 K between GD of net charge +5 versus +13. This result suggests that the large number of positive charges on H1‐GDs have evolved for function rather than structure and high stability. The stabilization of the GD upon binding to DNA can thus be expected to have a pronounced electrostatic component, a contribution that is amenable to modulation by posttranslational modifications, especially acetylation and phosphorylation.     

Probing the ligand binding domain of the GluR2 receptor by proteolysis and deletion mutagenesis defines domain boundaries and yields a crystallizable construct.

Ionotropic glutamate receptors constitute an important family of ligand-gated ion channels for which there is little biochemical or structural data. Here we probe the domain structure and boundaries of the ligand binding domain of the AMPA-sensitive GluR2 receptor by limited proteolysis and deletion mutagenesis. To identify the proteolytic fragments, Maldi mass spectrometry and N-terminal amino acid sequencing were employed. Trypsin digestion of HS1S2 (Chen GQ, Gouaux E. 1997. Proc Natl Acad Sci USA 94:13431-13436) in the presence and absence of glutamate showed that the ligand stabilized the S1 and S2 fragments against complete digestion. Using limited proteolysis and multiple sequence alignments of glutamate receptors as guides, nine constructs were made, folded, and screened for ligand binding activity. From this screen, the S1S21 construct proved to be trypsin- and chymotrypsin-resistant, stable to storage at 4 degrees C, and amenable to three-dimensional crystal formation. The HS1S21 variant was readily prepared on a large scale, the His tag was easily removed by trypsin, and crystals were produced that diffracted to beyond 1.5 A resolution. These experiments, for the first time, pave the way to economical overproduction of the ligand binding domains of glutamate receptors and more accurately map the boundaries of the ligand binding domain.     

Crystal structure of zinc-finger domain of Nanos and its functional implications

Nanos is an RNA-binding protein that is involved in the development and maintenance of germ cells. In combination with Pumilio, Nanos binds to the 3' untranslated region of a messenger RNA and represses its translation. Nanos has two conserved Cys-Cys-His-Cys zinc-finger motifs that are indispensable for its function. In this study, we have determined the crystal structure of the zinc-finger domain of zebrafish Nanos, for the first time revealing that Nanos adopts a novel zinc-finger structure. In addition, Nanos has a conserved basic surface that is directly involved in RNA binding. Our results provide the structural basis for further studies to clarify Nanos function.     

Merozoite surface protein 2 of Plasmodium falciparum: expression, structure, dynamics, and fibril formation of the conserved N-terminal domain

Merozoite surface protein 2 (MSP2) is a GPI-anchored protein on the surface of the merozoite stage of the malaria parasite Plasmodium falciparum. It is largely disordered in solution, but has a propensity to form amyloid-like fibrils under physiological conditions. The N-terminal conserved region (MSP2(1-25)) is part of the protease-resistant core of these fibrils. To investigate the structure and dynamics of this region, its ability to form fibrils, and the role of individual residues in these properties, we have developed a bacterial expression system that yields > or =10 mg of unlabeled or (15)N-labeled peptide per litre of culture. Two recombinant versions of MSP2(1-25), wild-type and a Y7A/Y16A mutant, have been produced. Detailed conformational analysis of the wild-type peptide and backbone (15)N relaxation data indicated that it contains beta-turn and nascent helical structures in the central and C-terminal regions. Residues 6-21 represent the most ordered region of the structure, although there is some flexibility around residues 8 and 9. The 10-residue sequence (MSP2(7-16)) (with two Tyr residues) was predicted to have a higher propensity for beta-aggregation than the 8-mer sequence (MSP2(8-15)), but there was no significant difference in conformation between MSP2(1-25) and [Y7A,Y16A]MSP2(1-25) and the rate of fibril formation was only slightly slower in the mutant. The peptide expression system described here will facilitate further mutational analyses to define the roles of individual residues in transient structural elements and fibril formation, and thus contribute to the further development of MSP2 as a malaria vaccine candidate.     

The role of the turn in beta-hairpin formation during WW domain folding

The folding of WW domains is rate limited by formation of a beta-hairpin comprising residues from strands 1 and 2. Residues in the turn of this hairpin have reported Phi-values for folding close to 1 and have been proposed to nucleate folding. High Phi-values do not necessarily imply that the energetics of formation are a driving force for initiating folding. We demonstrate by NMR studies and molecular dynamics simulations that the first turn of the hYAP, FBP28, and PIN1 WW domains is structurally dynamic and solvent exposed in the native and folding transition states. It is, therefore, unlikely that the formation of the beta-turn per se provides the energetic driving force for hairpin folding. It is more likely that the turn acts as an easily formed hinge that facilitates the formation of the hairpin; it is a nucleus as defined by the nucleation-condensation mechanism whereby a diffuse nucleus is stabilized by associated interactions.     

Effects of Phe-to-Trp mutation and fluorotryptophan incorporation on the solution structure of cardiac troponin C, and analysis of its suitability as a potential probe for in situ NMR studies

19F NMR spectroscopy is potentially a powerful tool for probing protein properties in situ. However, results obtained using this technique are relevant only if the 19F probe offers minimal perturbation to the surrounding environment. In this paper, we examine the effect of 5-fluorotryptophan (5fW) incorporation on the three-dimensional structure of cardiac troponin-C (cTnC), with the intention of developing a 19F-labeled TnC for use in in situ 19FNMR. We find that, in general, 5fW does not perturb the structure of the protein significantly. Replacement of residue Phe 153 with 5fW produces no noticeable change in protein conformation. However, replacement of residue Phe 104 with 5fW produces a folding behavior that is dependent on the Escherichia coli strain used to express the mutant. The orientations of the indole rings in these mutants are such that the Trp residue adopts a chi2 of approximately 90 degrees in the F104W mutant and approximately -100 degrees in the F153W mutant. Using results from 19F-1H heteronuclear NOE experiment, we show the replacement of L-Trp with 5fW at these positions does not change the orientation of the indole ring and the spread of the 5fW side-chain dihedral angles increases moderately for the F104(5fW) mutant and not at all for the F153(5fW) mutant. Based on these structures, we conclude that the substitution of Phe by 5fW at these two positions has minimal effects on the structure of cTnC and that the 5fW indole rings in both mutants have well defined orientation, making the two mutants viable candidates for use in in situ 19F NMR spectroscopy.     

Structure and dynamics of the human pleckstrin DEP domain: distinct molecular features of a novel DEP domain subfamily

Pleckstrin1 is a major substrate for protein kinase C in platelets and leukocytes, and comprises a central DEP (disheveled, Egl-10, pleckstrin) domain, which is flanked by two PH (pleckstrin homology) domains. DEP domains display a unique alpha/beta fold and have been implicated in membrane binding utilizing different mechanisms. Using multiple sequence alignments and phylogenetic tree reconstructions, we find that 6 subfamilies of the DEP domain exist, of which pleckstrin represents a novel and distinct subfamily. To clarify structural determinants of the DEP fold and to gain further insight into the role of the DEP domain, we determined the three-dimensional structure of the pleckstrin DEP domain using heteronuclear NMR spectroscopy. Pleckstrin DEP shares main structural features with the DEP domains of disheveled and Epac, which belong to different DEP subfamilies. However, the pleckstrin DEP fold is distinct from these structures and contains an additional, short helix alpha4 inserted in the beta4-beta5 loop that exhibits increased backbone mobility as judged by NMR relaxation measurements. Based on sequence conservation, the helix alpha4 may also be present in the DEP domains of regulator of G-protein signaling (RGS) proteins, which are members of the same DEP subfamily. In pleckstrin, the DEP domain is surrounded by two PH domains. Structural analysis and charge complementarity suggest that the DEP domain may interact with the N-terminal PH domain in pleckstrin. Phosphorylation of the PH-DEP linker, which is required for pleckstrin function, could regulate such an intramolecular interaction. This suggests a role of the pleckstrin DEP domain in intramolecular domain interactions, which is distinct from the functions of other DEP domain subfamilies found so far.     

Insertion of the cytochrome b5 heme-binding loop into an SH3 domain. Effects on structure and stability, and clues about the cytochrome's architecture

Under native conditions, apocytochrome b(5) exhibits a stable core and a disordered heme-binding region that refolds upon association with the cofactor. The termini of this flexible region are in close proximity, suggesting that loop closure may contribute to the thermodynamic properties of the apocytochrome. A chimeric protein containing 43 residues encompassing the cytochrome loop was constructed using the cyanobacterial photosystem I accessory protein E (PsaE) from Synechococcus sp. PCC 7002 as a structured scaffold. PsaE has the topology of an SH3 domain, and the insertion was engineered to replace its 14-residue CD loop. NMR and optical spectroscopies showed that the hybrid protein (named EbE1) was folded under native conditions and that it retained the characteristics of an SH3 domain. NMR spectroscopy revealed that structural and dynamic differences were confined near the site of loop insertion. Variable-temperature 1D NMR spectra of EbE1 confirmed the presence of a kinetic unfolding barrier. Thermal and chemical denaturations of PsaE and EbE1 demonstrated cooperative, two-state transitions; the stability of the PsaE scaffold was found only moderately compromised by the insertion, with a DeltaT(m) of 8.3 degrees C, a DeltaC(m) of 1.5 M urea, and a DeltaDeltaG degrees of 4.2 kJ/mole. The data implied that the penalty for constraining the ends of the inserted region was lower than the approximately 6.4 kJ/mole calculated for a self-avoiding chain. Extrapolation of these results to cytochrome b(5) suggested that the intrinsic stability of the folded portion of the apoprotein reflected only a small detrimental contribution from the large heme-binding domain.     

The NLRP12 pyrin domain: structure, dynamics, and functional insights

The initial line of defense against infection is sustained by the innate immune system. Together, membrane-bound Toll-like receptors and cytosolic nucleotide-binding domain and leucine-rich repeat-containing receptors (NLR) play key roles in the innate immune response by detecting bacterial and viral invaders as well as endogenous stress signals. NLRs are multi-domain proteins with varying N-terminal effector domains that are responsible for regulating downstream signaling events. Here, we report the structure and dynamics of the N-terminal pyrin domain of NLRP12 (NLRP12 PYD) determined using NMR spectroscopy. NLRP12 is a non-inflammasome NLR that has been implicated in the regulation of Toll-like receptor-dependent nuclear factor-κB activation. NLRP12 PYD adopts a typical six-helical bundle death domain fold. By direct comparison with other PYD structures, we identified hydrophobic residues that are essential for the stable fold of the NLRP PYD family. In addition, we report the first in vitro confirmed non-homotypic PYD interaction between NLRP12 PYD and the pro-apoptotic protein Fas-associated factor 1 (FAF-1), which links the innate immune system to apoptotic signaling. Interestingly, all residues that participate in this protein:protein interaction are confined to the α2-α3 surface, a region of NLRP12 PYD that differs most between currently reported NLRP PYD structures. Finally, we experimentally highlight a significant role for tryptophan 45 in the interaction between NLRP12 PYD and the FAF-1 UBA domain.     

Ligand-dependent dynamics and intramolecular signaling in a PDZ domain

Allosteric communication is a fundamental process that proteins use to propagate signals from one site to functionally important distal sites. Although allostery is usually associated with multimeric proteins and enzymes, “long-range” communication may be a fundamental property of proteins. In some cases, communication occurs with minimal structural change. PDZ (post-synaptic density-95/discs large/zonula occludens-1) domains are small, protein-protein binding modules that can use multiple surfaces for docking diverse molecules. Furthermore, these domains have long-range energetic couplings that link the ligand-binding site to distal regions of the structure. Here, we show that allosteric behavior in a representative member of the PDZ domain family may be directly detected using side-chain methyl dynamics measurements. The changes in side-chain dynamics parameters in the second PDZ domain from the human tyrosine phosphatase 1E (hPTP1E) were determined upon binding a peptide target. Long-range dynamic effects were detected that correspond to previously observed pair-wise energetic couplings. These results provide one of the first experimental examples for the potential role of ps-ns timescale dynamics in propagating long-range signals within a protein, and reinforce the idea that dynamic fluctuations in proteins contribute to allosteric signal transduction.     

Solution studies of the SH2 domain from the fyn tyrosine kinase: secondary structure,backbone dynamics and protein association

The SH2 domain from Fyn tyrosine kinase, corresponding to residues 155–270 of the human enzyme, was expressed as a GST-fusion protein in a pGEX-E. coli system. After thrombin cleavage and removal of GST, the protein was studied by heteronuclear NMR. Two different phosphotyrosyl-peptides were synthesized and added to the SH2 domain. One peptide corresponded to the regulatory C-terminal tail region of Fyn. Sequence-specific assignment of NMR spectra was achieved using a combination of¹H-¹⁵N-correlated 2D HSQC,¹⁵N-edited 3D TOCSY-HMQC, and¹⁵N-edited 3D NOESY-HMQC spectra. By analysis of the -proton chemical shifts and NOE intensities, the positions of secondary structural elements were determined and found to correspond closely to that seen in the crystal structure of the, homologous, Src-SH2 domain.To investigate the internal dynamics of the protein backbone, T₁ and T₂ relaxation parameters were measured on the free protein, as well as on both peptide complexes. Analytical ultracentrifugation and dynamic light scattering were employed to measure the effect of concentration and peptide-binding on self-association. The results suggest that, at NMR-sample concentrations, the free protein is present in at least dimeric form. Phosphopeptide binding and lower concentration significantly, but not completely, shift the equilibrium towards monomers. The possible role of this protein association in the regulation of the Src-family tyrosine kinases is discussed.Abbreviations SH Src homology - GST glutathione-S-transferase - IPTG isopropyl--D-galactopyranoside - DTT dithiothreitol - PMSF phenyl-methyl-sulphonyl-fluoride - TBS 50 mM Tris, 150 mM NaCl, 5 mM DTT, pH 8.0 - MWCO molecular weight cut off - NMR nuclear magnetic resonance - HSQC heteronuclear single-quantum correlation - NOESY nuclear Overhauser effect spectroscopy     

A functional NMR for membrane proteins: dynamics,ligand binding,and allosteric modulation

By nature of conducting ions, transporting substrates and transducing signals, membrane channels, transporters and receptors are expected to exhibit intrinsic conformational dynamics. It is therefore of great interest and importance to understand the various properties of conformational dynamics acquired by these proteins, for example, the relative population of states, exchange rate, conformations of multiple states, and how small molecule ligands modulate the conformational exchange. Because small molecule binding to membrane proteins can be weak and/or dynamic, structural characterization of these effects is very challenging. This review describes several NMR studies of membrane protein dynamics, ligand‐induced conformational rearrangements, and the effect of ligand binding on the equilibrium of conformational exchange. The functional significance of the observed phenomena is discussed.     

Study of the structure and dynamics of a chimeric variant of the SH3 domain (SHA-Bergerac) by NMR spectroscopy

A structural-dynamic study of one of the chimeric proteins (SHA) belonging to the SH3-Bergerac family and containing the KATANGKTYE sequence instead of the N47D48 β-turn in the spectrin SH3-domain was carried out by high resolution NMR spectroscopy. The spatial structure of the protein was determined and its dynamics in solution was investigated on the basis of the NMR data. The elongation of the SHA polypeptide chain in comparison with the WT-SH3 original protein (by ～17%) exerts practically no effect on the general topology of the molecule. The presence of a stable β-hairpin in the region of insertion was confirmed. This hairpin was shown to have a higher mobility in comparison with other regions of the protein.