Ubiquitin C-Terminal Hydrolase L1: Biochemical and Cellular Characterization of a Covalent Cyanopyrrolidine-Based Inhibitor.

The deubiquitinase (DUB) ubiquitin C-terminal hydrolase L1 (UCHL1) is expressed primarily in the central nervous system under normal physiological conditions. However, UCHL1 is overexpressed in various aggressive forms of cancer with strong evidence supporting UCHL1 as an oncogene in lung, glioma, and blood cancers. In particular, the level of UCHL1 expression in these cancers correlates with increased invasiveness and metastatic behavior, as well as poor patient prognosis. Although UCHL1 is considered an oncogene with potential as a therapeutic target, there remains a significant lack of useful small-molecule probes to pharmacologically validate in vivo targeting of the enzyme. Herein, we describe the characterization of a new covalent cyanopyrrolidine-based UCHL1 inhibitory scaffold in biochemical and cellular studies to better understand the utility of this inhibitor in elucidating the role of UCHL1 in cancer biology.

All-atom adaptively biased path optimization of Src kinase conformational inactivation: Switched electrostatic network in the concerted motion of αC helix and the activation loop.

A method to optimize a conformational pathway through a space of well-chosen reduced variables is employed to advance our understanding of protein conformational equilibrium. The adaptively biased path optimization strategy utilizes unrestricted, enhanced sampling in the region of a path in the reduced-variable space to identify a broad path between two stable end-states. Application to the inactivation transition of the Src tyrosine kinase catalytic domain reveals new insight into this well studied conformational equilibrium. The mechanistic description gained from identifying the motions and structural features along the path includes details of the switched electrostatic network found to underpin the transition. The free energy barrier along the path results from rotation of a helix, αC, that is tightly correlated with motions in the activation loop (A-loop) as well as distal regions in the C-lobe. Path profiles of the reduced variables clearly demonstrate the strongly correlated motions. The exchange of electrostatic interactions among residues in the network is key to these interdependent motions. In addition, the increased resolution from an all-atom model in defining the path shows multiple components for the A-loop motion and that different parts of the A-loop contribute throughout the length of the path.

Application of a Substrate-Mediated Selection with c-Src Tyrosine Kinase to a DNA-Encoded Chemical Library.

As aberrant activity of protein kinases is observed in many disease states, these enzymes are common targets for therapeutics and detection of activity levels. The development of non-natural protein kinase substrates offers an approach to protein substrate competitive inhibitors, a class of kinase inhibitors with promise for improved specificity. Also, kinase activity detection approaches would benefit from substrates with improved activity and specificity. Here, we apply a substrate-mediated selection to a peptidomimetic DNA-encoded chemical library for enrichment of molecules that can be phosphorylated by the protein tyrosine kinase, c-Src. Several substrates were identified and characterized for activity. A lead compound (SrcDEL10) showed both the ability to serve as a substrate and to promote ATP hydrolysis by the kinase. In inhibition assays, compounds displayed IC50's ranging from of 8-100 µM. NMR analysis of SrcDEL10 bound to the c-Src:ATP complex was conducted to characterize the binding mode. An ester derivative of the lead compound demonstrated cellular activity with inhibition of Src-dependent signaling in cell culture. Together, the results show the potential for substrate-mediated selections of DNA-encoded libraries to discover molecules with functions other than simple protein binding and offer a new discovery method for development of synthetic tyrosine kinase substrates.

A minimization principle for transition paths of maximum flux for collective variables.

Considered is the construction of transition paths of conformational changes for proteins and other macromolecules, using methods that do not require the generation of dynamics trajectories. Special attention is given to the use of a reduced set of collective variables for describing such paths. A favored way to define transition paths is to seek channels through the transition state having cross sections with a high reactive flux (density of last hitting points of reactive trajectories). Given here is a formula for reactive flux that is independent of the parameterization of "collective variable space." This formula is needed for the principal curve of the reactive flux (as in the revised finite temperature string method) and for the maximum flux transition (MaxFlux) path. Additionally, a resistance functional is derived for narrow tubes, which when minimized yields a MaxFlux path. A strategy for minimization is outlined in the spirit of the string method. Finally, alternative approaches based on determining trajectories of high probability are considered, and it is observed that they yield paths that depend on the parameterization of collective variable space, except in the case of zero temperature, where such a path coincides with a MaxFlux path.

Insights into the allosteric regulation of Syk association with receptor ITAM, a multi-state equilibrium.

The phosphorylation of interdomain A (IA), a linker region between tandem SH2 domains of Syk tyrosine kinase, regulates the binding affinity for association of Syk with doubly-phosphorylated ITAM regions of the B cell receptor. The mechanism of this allosteric regulation has been suggested to be a switch from the high-affinity bifunctional binding, mediated through both SH2 domains binding two phosphotyrosine residues of ITAM, to a substantially lower-affinity binding of only one SH2 domain. IA phosphorylation triggers the switch by inducing disorder in IA and weakening the SH2-SH2 interaction. The postulated switch to a single-SH2-domain binding mode is examined using NMR to monitor site-specific binding to each SH2 domain of Syk variants engineered to have IA regions that differ in conformational flexibility. The combined analysis of titration curves and NMR line-shapes provides sufficient information to determine the energetics of inter-molecular binding at each SH2 site along with an intra-molecular binding or isomerization step. A less favorable isomerization equilibrium associated with the changes in the SH2-SH2 conformational ensemble and IA flexibility accounts for the inhibition of Syk association with membrane ITAM regions when IA is phosphorylated, and refutes the proposed switch to single-SH2-domain binding. Syk localizes in the cell through its SH2 interactions, and this basis for allosteric regulation of ITAM association proposes for the first time a phosphorylation-dependent model to regulate Syk binding to alternate receptors and other signaling proteins that differ either in the number of residues separating ITAM phosphotyrosines or by having only one phosphotyrosine, a half ITAM.

Effects of impaired membrane interactions on α-synuclein aggregation and neurotoxicity.

The post-mortem brains of individuals with Parkinson's disease (PD) and other synucleinopathy disorders are characterized by the presence of aggregated forms of the presynaptic protein α-synuclein (aSyn). Understanding the molecular mechanism of aSyn aggregation is essential for the development of neuroprotective strategies to treat these diseases. In this study, we examined how interactions between aSyn and phospholipid vesicles influence the protein's aggregation and toxicity to dopaminergic neurons. Two-dimensional NMR data revealed that two familial aSyn mutants, A30P and G51D, populated an exposed, membrane-bound conformer in which the central hydrophobic region was dissociated from the bilayer to a greater extent than in the case of wild-type aSyn. A30P and G51D had a greater propensity to undergo membrane-induced aggregation and elicited greater toxicity to primary dopaminergic neurons compared to the wild-type protein. In contrast, the non-familial aSyn mutant A29E exhibited a weak propensity to aggregate in the presence of phospholipid vesicles or to elicit neurotoxicity, despite adopting a relatively exposed membrane-bound conformation. Our findings suggest that the aggregation of exposed, membrane-bound aSyn conformers plays a key role in the protein's neurotoxicity in PD and other synucleinopathy disorders.

Long-distance correlations of rhinovirus capsid dynamics contribute to uncoating and antiviral activity.

Human rhinovirus (HRV) and other members of the enterovirus genus bind small-molecule antiviral compounds in a cavity buried within the viral capsid protein VP1. These compounds block the release of the viral protein VP4 and RNA from inside the capsid during the uncoating process. In addition, the antiviral compounds prevent "breathing" motions, the transient externalization of the N-terminal regions of VP1 and VP4 from the inside of intact viral capsid. The site for externalization of VP1/VP4 or release of RNA is likely between protomers, distant to the binding cavity for antiviral compounds. Molecular dynamics simulations were conducted to explore how the antiviral compound, WIN 52084, alters properties of the HRV 14 capsid through long-distance effect. We developed an approach to analyze capsid dynamics in terms of correlated radial motion and the shortest paths of correlated motions. In the absence of WIN, correlated radial motion is observed between residues separated by as much as 85 Å, a remarkably long distance. The most frequently populated path segments of the network were localized near the fivefold symmetry axis and included those connecting the N termini of VP1 and VP4 with other regions, in particular near twofold symmetry axes, of the capsid. The results provide evidence that the virus capsid exhibits concerted long-range dynamics, which have not been previously recognized. Moreover, the presence of WIN destroys this radial correlation network, suggesting that the underlying motions contribute to a mechanistic basis for the initial steps of VP1 and VP4 externalization and uncoating.

Evolutionary sequence and structural basis for the distinct conformational landscapes of Tyr and Ser/Thr kinases.

Protein kinases are molecular machines with rich sequence variation that distinguishes the two main evolutionary branches - tyrosine kinases (TKs) from serine/threonine kinases (STKs). Using a sequence co-variation Potts statistical energy model we previously concluded that TK catalytic domains are more likely than STKs to adopt an inactive conformation with the activation loop in an autoinhibitory "folded" conformation, due to intrinsic sequence effects. Here we investigated the structural basis for this phenomenon by integrating the sequence-based model with structure-based molecular dynamics (MD) to determine the effects of mutations on the free energy difference between active and inactive conformations, using a novel thermodynamic cycle involving many (n=108) protein-mutation free energy perturbation (FEP) simulations in the active and inactive conformations. The sequence and structure-based results are consistent and support the hypothesis that the inactive conformation "DFG-out Activation Loop Folded", is a functional regulatory state that has been stabilized in TKs relative to STKs over the course of their evolution via the accumulation of residue substitutions in the activation loop and catalytic loop that facilitate distinct substrate binding modes in trans and additional modes of regulation in cis for TKs.

Prediction of protein relative enthalpic stability from molecular dynamics simulations of the folded and unfolded states.

For proteins of known structure, the relative enthalpic stability with respect to wild-type, ΔΔH(U), can be estimated by direct computation of the folded and unfolded state energies. We propose a model by which the change in stability upon mutation can be predicted from all-atom molecular dynamics simulations for the folded state and a peptide-based model for the unfolded state. The unfolding enthalpies are expressed in terms of environmental and hydration-solvent reorganization contributions that readily allow a residue-specific analysis of ΔΔH(U). The method is applied to estimate the relative enthalpic stability of variants with buried charged groups in T4 lysozyme. The predicted relative stabilities are in good agreement with experimental data. Environmental factors are observed to contribute more than hydration to the overall ΔΔH(U). The residue-specific analysis finds that the effects of burying charge are both localized and long-range. The enthalpy for hydration-solvent reorganization varies considerably among different amino-acid types, but because the variant folded state structures are similar to those of the wild-type, the hydration-solvent reorganization contribution to ΔΔH(U) is localized at the mutation site, in contrast to environmental contributions. Overall, mutation of apolar and polar amino acids to charged amino acids are destabilizing, but the reasons are complex and differ from site to site.

Domain cooperativity in multidomain proteins: what can we learn from molecular alignment in anisotropic media?

Many proteins have modular design with multiple globular domains connected via flexible linkers. As a simple model of such system, we study a tandem construct consisting of two identical SH3 domains and a variable-length Gly/Ser linker. When the linker is short, this construct represents a dumbbell-shaped molecule with limited amount of domain-domain mobility. Due to its elongated shape, this molecule efficiently aligns in steric alignment media. As the length of the linker increases, the two domains become effectively uncoupled and begin to behave as independent entities. Consequently, their degree of alignment drops, approaching that found in the (near-spherical) isolated SH3 domains. To model the dependence of alignment parameters on the length of the interdomain linker, we have generated in silico a series of conformational ensembles representing SH3 tandems with different linker length. These ensembles were subsequently used as input for alignment prediction software PALES. The predicted alignment tensors were compared with the results of experimental measurements using a series of tandem-SH3 samples in PEG/hexanol alignment media. This comparison broadly confirmed the expected trends. At the same time, it has been found that the isolated SH3 domain aligns much stronger than expected. This finding can be attributed to complex morphology of the PEG/hexanol media and/or to weak site-specific interactions between the protein and the media. In the latter case, there are strong indications that electrostatic interactions may play a role. The fact that PEG/hexanol does not behave as a simple steric media should serve as a caution for studies that use PALES as a quantitative prediction tool (especially for disordered proteins). Further progress in this area depends on our ability to accurately model the anisotropic media and its site-specific interactions with protein molecules. Once this ability is improved, it should be possible to use the alignment parameters as a measure of domain-domain cooperativity, thus identifying the situations where two domains transiently interact with each other or become coupled through a partially structured linker.

Tyr130 phosphorylation triggers Syk release from antigen receptor by long-distance conformational uncoupling.

The Syk protein-tyrosine kinase plays a major role in signaling through the B cell receptor for antigen (BCR). Syk binds the receptor via its tandem pair of SH2 domains interacting with a doubly phosphorylated immunoreceptor tyrosine-based activation motif (dp-ITAM) of the BCR complex. Upon phosphorylation of Tyr-130, which lies between the two SH2 domains distant to the phosphotyrosine binding sites, Syk dissociates from the receptor. To understand the structural basis for this dissociation, we investigated the structural and dynamic characteristics of the wild type tandem SH2 region (tSH2) and a variant tandem SH2 region (tSH2(pm)) with Tyr-130 substituted by Glu to permanently introduce a negative charge at this position. NMR heteronuclear relaxation experiments, residual dipolar coupling measurements and analytical ultracentrifugation revealed substantial differences in the hydrodynamic behavior of tSH2 and tSH2(pm). Although the two SH2 domains in tSH2 are tightly associated, the two domains in tSH2(pm) are partly uncoupled and tumble in solution with a faster correlation time. In addition, the equilibrium dissociation constant for the binding of tSH2(pm) to dp-ITAM (1.8 microM) is significantly higher than that for the interaction between dp-ITAM and tSH2 but is close to that for a singly tyrosine-phosphorylated peptide binding to a single SH2 domain. Experimental data and hydrodynamic calculations both suggest a loss of domain-domain contacts and change in relative orientation upon the introduction of a negative charge on residue 130. A long-distance structural mechanism by which the phosphorylation of Y130 negatively regulates the interaction of Syk with immune receptors is proposed.

NMR relaxation studies of an RNA-binding segment of the rous sarcoma virus gag polyprotein in free and bound states: a model for autoinhibition of assembly.

Assembly of retrovirus particles is promoted by interaction of the Gag polyprotein with RNA. Nonspecific RNA association with the nucleocapsid domain (NC) of Gag induces the dimerization of Gag through protein-protein contacts in the capsid domain (CA), followed by higher order assembly to form the immature virus particle. NMR relaxation studies were conducted to investigate the initial steps of Rous sarcoma virus (RSV) assembly by examining the association with nucleic acid of a fragment of Gag comprising the C-terminal domain of CA (CTD) postulated to mediate Gag dimerization, the spacer region between CA and NC (SP), and NC. This fragment, CTD-SP-NC (residues 394-577), spans the critical SP region and allows assessment of this key Gag-nucleic acid interaction in the context of the Gag polyprotein rather than the isolated domains. Main-chain amide relaxation of CTD-SP-NC was measured in the absence and presence of (GT)(4), an 8-mer DNA oligonucleotide that binds tightly to the polyprotein but is too short to promote Gag dimerization. The results show that the CTD and NC domains tumble independently. In contrast, the two zinc finger domains within NC are rotationally coupled in both the unbound and bound states, even though only the first zinc finger appears to make direct contact with (GT)(4). In addition, the NMR data indicate that SP and flanking residues undergo a conformational exchange process that is slowed in the presence of (GT)(4). This region around SP where relaxation is strongly affected by (GT)(4) binding is nearly identical to the assembly domain defined previously by mutagenesis studies. Other changes in relaxation induced by (GT)(4) implicate conformational perturbations of helices 1 and 4 in CTD. On the basis of the combined data, we propose a model for the promotion of Gag dimerization by RNA association in which NC-RNA binding disrupts an assembly inhibitory, intramolecular interaction involving SP and CTD. Disruption of this intramolecular interaction is proposed to enhance the accessibility of the Gag dimer contact surface and release the assembly domain to promote intermolecular oligomerization.

Constraining binding hot spots: NMR and molecular dynamics simulations provide a structural explanation for enthalpy-entropy compensation in SH2-ligand binding.

NMR spectroscopy and molecular dynamics (MD) simulations were used to probe the structure and dynamics of complexes of three phosphotyrosine-derived peptides with the Src SH2 domain in an effort to uncover a structural explanation for enthalpy-entropy compensation observed in the binding thermodynamics. The series of phosphotyrosine peptide derivatives comprises the natural pYEEI Src SH2 ligand, a constrained mimic, in which the phosphotyrosine (pY) residue is preorganized in the bound conformation for the purpose of gaining an entropic advantage to binding, and a flexible analogue of the constrained mimic. The expected gain in binding entropy of the constrained mimic was realized; however, a balancing loss in binding enthalpy was also observed that could not be rationalized from the crystallographic structures. We examined protein dynamics to evaluate whether the observed enthalpic penalty might be the result of effects arising from altered motions in the complex. (15)N-relaxation studies and positional fluctuations from molecular dynamics indicate that the main-chain dynamics of the protein show little variation among the three complexes. Root mean squared (rms) coordinate deviations vary by less than 1.5 A for all non-hydrogen atoms for the crystal structures and in the ensemble average structures calculated from the simulations. In contrast to this striking similarity in the structures and dynamics, there are a number of large chemical shift differences from residues across the binding interface, but particularly from key Src SH2 residues that interact with pY, the "hot spot" residue, which contributes about one-half of the binding free energy. Rank-order correlations between chemical shifts and ligand binding enthalpy for several pY-binding residues, coupled with available mutagenesis and calorimetric data, suggest that subtle structural perturbations (<1 A) from the conformational constraint of the pY residue sufficiently alter the geometry of enthalpically critical interactions in the binding pocket to cause the loss of binding enthalpy, leading to the observed enthalpy-entropy compensation. We find no evidence to support the premise that enthalpy-entropy compensation is an inherent property and conclude that preorganization of Src SH2 ligand residues involved in binding hot spots may eventuate in suboptimal interactions with the domain. We propose that introducing constraints elsewhere in the ligand could minimize enthalpy-entropy compensation effects. The results illustrate the utility of the NMR chemical shift to highlight small, but energetically significant, perturbations in structure that might otherwise go unnoticed in an apparently rigid protein.

Substrate binding to Src: A new perspective on tyrosine kinase substrate recognition from NMR and molecular dynamics.

Most signal transduction pathways in humans are regulated by protein kinases through phosphorylation of their protein substrates. Typical eukaryotic protein kinases are of two major types: those that phosphorylate-specific sequences containing tyrosine (~90 kinases) and those that phosphorylate either serine or threonine (~395 kinases). The highly conserved catalytic domain of protein kinases comprises a smaller N lobe and a larger C lobe separated by a cleft region lined by the activation loop. Prior studies find that protein tyrosine kinases recognize peptide substrates by binding the polypeptide chain along the C-lobe on one side of the activation loop, while serine/threonine kinases bind their substrates in the cleft and on the side of the activation loop opposite to that of the tyrosine kinases. Substrate binding structural studies have been limited to four families of the tyrosine kinase group, and did not include Src tyrosine kinases. We examined peptide-substrate binding to Src using paramagnetic-relaxation-enhancement NMR combined with molecular dynamics simulations. The results suggest Src tyrosine kinase can bind substrate positioning residues C-terminal to the phosphoacceptor residue in an orientation similar to serine/threonine kinases, and unlike other tyrosine kinases. Mutagenesis corroborates this new perspective on tyrosine kinase substrate recognition. Rather than an evolutionary split between tyrosine and serine/threonine kinases, a change in substrate recognition may have occurred within the TK group of the human kinome. Protein tyrosine kinases have long been therapeutic targets, but many marketed drugs have deleterious off-target effects. More accurate knowledge of substrate interactions of tyrosine kinases has the potential for improving drug selectivity.

NmrLineGuru: Standalone and User-Friendly GUIs for Fast 1D NMR Lineshape Simulation and Analysis of Multi-State Equilibrium Binding Models.

The ability of high-resolution NMR spectroscopy to readout the response of molecular interactions at multiple atomic sites presents a unique capability to define thermodynamic equilibrium constants and kinetic rate constants for complex, multiple-step biological interactions. Nonetheless, the extraction of the relevant equilibrium binding and rate constants requires the appropriate analysis of not only a readout that follows the equilibrium concentrations of typical binding titration curves, but also the lineshapes of NMR spectra. To best take advantage of NMR data for characterizing molecular interactions, we developed NmrLineGuru, a software tool with a user-friendly graphical user interface (GUI) to model two-state, three-state, and four-state binding processes. Application of NmrLineGuru is through stand-alone GUIs, with no dependency on other software and no scripted input. NMR spectra can be fitted or simulated starting with user-specified input parameters and a chosen kinetic model. The ability to both simulate and fit NMR spectra provides the user the opportunity to not only determine the binding parameters that best reproduce the measured NMR spectra for the selected kinetic model, but to also query the possibility that alternative models agree with the data. NmrLineGuru is shown to provide an accurate, quantitative analysis of complex molecular interactions.

Contribution of charged groups to the enthalpic stabilization of the folded states of globular proteins.

In this paper we use the results from all-atom molecular dynamics (MD) simulations of proteins and peptides to assess the individual contribution of charged atomic groups to the enthalpic stability of the native state of globular proteins and investigate how the distribution of charged atomic groups in terms of solvent accessibility relates to protein enthalpic stability. The contributions of charged groups is calculated using a comparison of nonbonded interaction energy terms from equilibrium simulations of charged amino acid dipeptides in water (the "unfolded state") and charged amino acids in globular proteins (the "folded state"). Contrary to expectation, the analysis shows that many buried, charged atomic groups contribute favorably to protein enthalpic stability. The strongest enthalpic contributions favoring the folded state come from the carboxylate (COO(-)) groups of either Glu or Asp. The contributions from Arg guanidinium groups are generally somewhat stabilizing, while N(+)(3) groups from Lys contribute little toward stabilizing the folded state. The average enthalpic gain due to the transfer of a methyl group in an apolar amino acid from solution to the protein interior is described for comparison. Notably, charged groups that are less exposed to solvent contribute more favorably to protein native-state enthalpic stability than charged groups that are solvent exposed. While solvent reorganization/release has favorable contributions to folding for all charged atomic groups, the variation in folded state stability among proteins comes mainly from the change in the nonbonded interaction energy of charged groups between the unfolded and folded states. A key outcome is that the calculated enthalpic stabilization is found to be inversely proportional to the excess charge density on the surface, in support of an hypothesis proposed previously.

Protein Conformational Transitions from All-Atom Adaptively Biased Path Optimization.

Simulation methods are valuable for elucidating protein conformational transitions between functionally diverse states given that transition pathways are difficult to capture experimentally. Nonetheless, specific computational algorithms are required because of the high free energy barriers between these different protein conformational states. Adaptively biased path optimization (ABPO) is an unrestrained, transition-path optimization method that works in a reduced-variable space to construct an adaptive biasing potential to aid convergence. ABPO was previously applied using a coarse-grained Go̅-model to study conformational activation of Lyn, a Src family tyrosine kinase. How effectively ABPO can be applied at the higher resolution of an all-atom model to explore protein conformational transitions is not yet known. Here, we report the all-atom conformational transition paths of three protein systems constructed using the ABPO methodology. Two systems, triose phosphate isomerase and dihydrofolate reductase, undergo local flipping of a short loop that promotes ligand binding. The third system, estrogen receptor α ligand binding domain, has a helix that adopts different conformations when the protein is bound to an agonist or an antagonist. For each protein, distance-based or torsion-angle reduced variables were identified from unbiased trajectories. ABPO was computed in this reduced variable space to obtain the transition path between the two states. The all-atom ABPO is shown to successfully converge an optimal transition path for each of the three systems.

Entropic allostery dominates the phosphorylation-dependent regulation of Syk tyrosine kinase release from immunoreceptor tyrosine-based activation motifs.

Spleen tyrosine kinase (Syk) is an essential player in immune signaling through its ability to couple multiple classes of membrane immunoreceptors to intracellular signaling pathways. Ligand binding leads to the recruitment of Syk to a phosphorylated cytoplasmic region of the receptors called ITAM. Syk binds to ITAM with high-affinity (nanomolar Kd ) via its tandem pair of SH2 domains. The affinity between Syk and ITAM is allosterically regulated by phosphorylation at Y130 in a linker connecting the tandem SH2 domains; when Y130 is phosphorylated, the binding affinity decreases (micromolar Kd ). Previous equilibrium binding studies attribute the increase in the binding free energy to an intra-molecular binding (isomerization) step of the tandem SH2 and ITAM, but a physical basis for the increased free energy is unknown. Here, we provide evidence that Y130 phosphorylation imposes an entropy penalty to isomerization, but surprisingly, has negligible effect on the SH2 binding interactions with ITAM and thus on the binding enthalpy. An analysis of NMR chemical shift differences characterized conformational effects of ITAM binding, and binding thermodynamics were measured from isothermal titration calorimetry. Together the data support a previously unknown mechanism for the basis of regulating protein-protein interactions through protein phosphorylation. The decreased affinity for Syk association with immune receptor ITAMs by Y130 phosphorylation is an allosteric mechanism driven by an increased entropy penalty, likely contributed by conformational disorder in the SH2-SH2 inter-domain structure, while SH2-ITAM binding contacts are not affected, and binding enthalpy is unchanged.

Structural basis for the requirement of two phosphotyrosine residues in signaling mediated by Syk tyrosine kinase.

The protein-tyrosine kinase Syk couples immune recognition receptors to multiple signal transduction pathways, including the mobilization of calcium and the activation of NFAT. The ability of Syk to regulate signaling is influenced by its phosphorylation on tyrosine residues within the linker B region. The phosphorylation of both Y342 and Y346 is necessary for optimal signaling from the B cell receptor for antigen. The SH2 domains of multiple signaling proteins share the ability to bind this doubly phosphorylated site. The NMR structure of the C-terminal SH2 domain of PLCgamma (PLCC) bound to a doubly phosphorylated Syk peptide reveals a novel mode of phosphotyrosine recognition. PLCC undergoes extensive conformational changes upon binding to form a second phosphotyrosine-binding pocket in which pY346 is largely desolvated and stabilized through electrostatic interactions. The formation of the second binding pocket is distinct from other modes of phosphotyrosine recognition in SH2-protein association. The dependence of signaling on simultaneous phosphorylation of these two tyrosine residues offers a new mechanism to fine-tune the cellular response to external stimulation.