Search

The recruitment of the protein spleen tyrosine kinase (Syk) to membrane-bound immune receptors is an essential step in initiating an immune response mediated through the activated receptors. Syk recognizes intracellular phosphorylated regions of membrane receptors known as immunoreceptor tyrosine-based activation motifs (ITAMs) defined by a sequence with two tyrosine (Y) amino acids separated by a certain spacing of six to eight residues: YXX(I/L)X 6-8 YXX(I/L). Syk with doubly phosphorylated ITAM is high-affinity and negatively regulated when Syk itself becomes phosphorylated. While the role of Syk in immune signaling is well characterized, recent information affords new functionality to Syk related to cytoplasmic processes, including the clearance of stress granules and P-bodies, both formed by liquid-liquid phase separation. Little to nothing is known about the molecular interactions involving Syk in these cytoplasmic processes. Given the essential role of receptor ITAMs in recruiting and localizing Syk for immune signaling, we explore here the possibility of a similar localization mechanism occurring for cytoplasmic processes by searching sequences of proteins related to Syk cytoplasmic function for regions that resemble receptor ITAMs. Protein sequence databases were generated from a Syk-dependent phosphoproteome and from genes related to P-bodies. A search of these databases for ITAM-like sequences yielded 102 unique hits, and 33 of these were synthesized and tested experimentally for binding to Syk tandem SH2 domains. The equilibrium dissociation constants were 0.1-50 μM for 28 peptides, and binding was negatively regulated by phosphorylation for many peptides. These results identify cytoplasmic proteins with potential for regulating the localization of Syk in a phosphorylation-dependent manner to nonmembrane cellular regions.

Since its inception nearly a half century ago, CHARMM has been playing a central role in computational biochemistry and biophysics. Commensurate with the developments in experimental research and advances in computer hardware, the range of methods and applicability of CHARMM have also grown. This review summarizes major developments that occurred after 2009 when the last review of CHARMM was published. They include the following: new faster simulation engines, accessible user interfaces for convenient workflows, and a vast array of simulation and analysis methods that encompass quantum mechanical, atomistic, and coarse-grained levels, as well as extensive coverage of force fields. In addition to providing the current snapshot of the CHARMM development, this review may serve as a starting point for exploring relevant theories and computational methods for tackling contemporary and emerging problems in biomolecular systems. CHARMM is freely available for academic and nonprofit research at https://academiccharmm.org/program.

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 investigate 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 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., (© 2024. The Author(s).)

The relevance of motions in biological macromolecules has been clear since the early structural analyses of proteins by X-ray crystallography. Computer simulations have been applied to provide a deeper understanding of the dynamics of biological macromolecules since 1976, and are now a standard tool in many labs working on the structure and function of biomolecules. In this mini-review we highlight some areas of current interest and active development for simulations, in particular all-atom molecular dynamics simulations., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Charles L. Brooks III reports financial support was provided by NIH grant GM130587. Alexander D MacKerell Jr reports financial support was provided by NIH grant GM131710. Carol B. Post reports financial support was provided by NIH grant RO1GM039478., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

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.

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., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

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., (© 2019 The Protein Society.)

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.

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 IC 50' 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.

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.

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 K d ) 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 K d ). 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., (© 2018 The Protein Society.)

t 1 noise appears as random or semi-random spurious streaks along the indirect t 1 (F1) dimension of a 2D or nD NMR spectrum. It can significantly downgrade spectral quality, especially for spectra with strong diagonal signals such as NOESY, because useful and weak cross-peaks can be easily buried under t 1 noise. One of the significant contributing factors to t 1 noise is unwanted and semi-random F2 signal modulation during t 1 acquisition. As such, t 1 noise from different acquisitions is unlikely to correlate with each other strongly. In the case of NOESY, co-addition of multiple spectra significantly reduces t 1 noise compared with conventional acquisition with the same amount of total acquisition time and resolution., (Copyright © 2017 Elsevier Inc. All rights reserved.)

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.

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.

Proteins with a modular architecture of multiple domains connected by linkers often exhibit diversity in the relative positions of domains, while the domain tertiary structure remains unchanged. The biological function of these modular proteins, or the regulation of their activity, depends on the variation in domain orientation and separation. Accordingly, careful characterization of interdomain motion and correlated fluctuations of multidomain systems is relevant for understanding the functional behavior of modular proteins. Molecular dynamics (MD) simulations provides a powerful approach to study these motions in atomic detail. Nevertheless, the common procedure for analyzing fluctuations from MD simulations after rigid-body alignment fails for multidomain proteins; it greatly overestimates correlated positional fluctuations in the presence of relative domain motion. We show here that expressing the atomic motions of a multidomain protein as a combination of displacement within the domain reference frame and motion of the relative domains correctly separates the internal motions to allow a useful description of correlated fluctuations. We illustrate the methodology of separating the domain fluctuations and local fluctuations by application to the tandem SH2 domains of human Syk protein kinase and by characterizing an effect of phosphorylation on the dynamics. Correlated motions are assessed from a distance covariance rather than the more common vector-coordinate covariance. The approach makes it possible to calculate the proper correlations in fluctuations internal to a domain as well as between domains.

Three implicit solvent models, namely GBMVII, FACTS, and SCPISM, were evaluated for their abilities to emulate an explicit solvent environment by comparing the simulated conformational ensembles, dynamics, and electrostatic interactions of the Src SH2 domain and the Lyn kinase domain. This assessment in terms of structural features in folded proteins expands upon the use of hydration energy as a metric for comparison. All-against-all rms coordinate deviation, average positional fluctuations, and ion-pair distance distribution were used to compare the implicit solvent models with the TIP3P explicit solvent model. Our study shows that the Src SH2 domains solvated with TIP3P, GBMVII, and FACTS sample similar global conformations. Additionally, the Src SH2 ion-pair distance distributions of solvent-exposed side chains corresponding to TIP3P, GBMVII, and FACTS do not differ substantially, indicating that GBMVII and FACTS are capable of modeling these electrostatic interactions. The ion-pair distance distributions of SCPISM are distinct from others, demonstrating that these electrostatic interactions are not adequately reproduced with the SCPISM model. On the other hand, for the Lyn kinase domain, a non-globular protein with bilobal structure and a large concavity on the surface, implicit solvent does not accurately model solvation to faithfully reproduce partially buried electrostatic interactions and lobe-lobe conformations. Our work reveals that local structure and dynamics of small, globular proteins are modeled well using FACTS and GBMVII. Nonetheless, global conformations and electrostatic interactions in concavities of multi-lobal proteins resulting from simulations with implicit solvent models do not match those obtained from explicit water simulations., (© 2015 The Protein Society.)

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., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The potential for reliably predicting relative binding enthalpies, ΔΔ E , from a direct method utilizing molecular dynamics is examined for a system of three phosphotyrosyl peptides binding to a protein receptor, the Src SH2 domain. The binding enthalpies were calculated from the potential energy differences between the bound and the unbound end-states of each peptide from equilibrium simulations in explicit water. The statistical uncertainties in the ensemble-mean energy values from multiple, independent simulations were obtained using a bootstrap method. Simulations were initiated with different starting coordinates as well as different velocities. Statistical uncertainties in ΔΔ E are 2 to 3 kcal/mol based on calculations from 40, 10 ns trajectories for each system (three SH2-peptide complexes or unbound peptides). Uncertainties in relative component energies, comprising solute-solute, solute-solvent and solvent-solvent interactions, are considerably larger. Energy values were estimated from an unweighted ensemble averaging of multiple trajectories with the a priori assumption that all trajectories are equally likely. Distributions in energy-rmsd space indicate that the trajectories sample the same basin and the difference in mean energy values between trajectories is due to sampling of alternative local regions of this superbasin. The direct estimate of relative binding enthalpies is concluded to be a reasonable approach for well-ordered systems with ΔΔ E values greater than ∼3 kcal/mol, although the approach would benefit from future work to determine properly distributed starting points that would enable efficient sampling of conformational space using multiple trajectories.

The Syk protein-tyrosine kinase can have multiple effects on cancer cells, acting in some as a tumor suppressor by inhibiting motility and in others as a tumor promoter by enhancing survival. Phosphoproteomic analyses identified PKA as a Syk-specific substrate. Syk catalyzes the phosphorylation of the catalytic subunit of PKA (PKAc) both in vitro and in cells on Tyr-330. Tyr-330 lies within the adenosine-binding motif in the C-terminal tail of PKAc within a cluster of acidic amino acids (DDYEEEE), which is a characteristic of Syk substrates. The phosphorylation of PKAc on Tyr-330 by Syk strongly inhibits its catalytic activity. Molecular dynamics simulations suggest that this additional negative charge prevents the C-terminal tail from interacting with the substrate and the nucleotide-binding site to stabilize the closed conformation of PKAc, thus preventing catalysis from occurring. Phosphoproteomic analyses and Western blotting studies indicate that Tyr-330 can be phosphorylated in a Syk-dependent manner in MCF7 breast cancer cells and DT40 B cells. The phosphorylation of a downstream substrate of PKAc, cAMP-responsive element-binding protein (CREB), is inhibited in cells expressing Syk but can be rescued by a selective inhibitor of Syk. Modulation of CREB activity alters the expression of the CREB-regulated gene BCL2 and modulates cellular responses to genotoxic agents. Thus, PKA is a novel substrate of Syk, and its phosphorylation on Tyr-330 inhibits its participation in downstream signaling pathways.

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., (Copyright © 2012 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

We apply the adaptive biasing potential (ABP) method to optimize the principal curve defining a conformational transition between two known end states and to subsequently compute the one-dimensional potential of mean force as a function of arc length along the principal curve. This approach allows the use of the ABP method in a collective variable space of arbitrary dimension and offers several advantages over line-search methods. First, configurations are neither generated along an initial path for the transition nor equilibrated during evolution of the path. Second, and most importantly, the powerful sampling provided by the ABP serves to accelerate the dynamics during the optimization and computation of the free energy. Finally, the free energy is formulated as a potential of mean force that captures changes in the reaction channel along the principal curve, in contrast to the free energy profile evaluated from the local free-energy gradient in restrained path optimization methods. We first demonstrate the ABP formulation of path optimization using a two-dimensional potential surface and then with a more complex system of Src protein tyrosine kinase. The method is shown to be efficient and robust in the case of rugged, free-energy landscapes.

We asses the ability of a distance correlation coefficient (DiCC), calculated from distance covariance, for detecting long-range concerted motion in proteins. We establish a set of criteria for ideal correlation coefficient values based on the coefficient of determination in multi-dimension, R 2 . We compare in detail DiCC and conventional coefficients against these criteria. We demonstrate that in contrast to conventional correlation coefficients, which capture long-distance correlation adequately only with certain restrictions in multi-dimension, DiCC reflects appropriate correlation in both one- and multi-dimension. Finally we demonstrate the usefulness of DiCC for assessing long-distance correlated fluctuation in protein dynamics.

One mechanism of regulating the catalytic activity of protein kinases is through conformational transitions. Despite great diversity in the structural changes involved in the transitions, a certain set of changes within the kinase domain (KD) has been observed for many kinases including Src and CDK2. We investigated this conformational transition computationally to identify the topological features that are energetically critical to the transition. Results from both molecular dynamics sampling and transition path optimization highlight the displacement of the αC helix as the major energy barrier, mediating the switch of the KD between the active and down-regulated states. The critical role of the αC helix is noteworthy by providing a rationale for a number of activation and deactivation mechanisms known to occur in cells. We find that kinases with the αC helix displacement exist throughout the kinome, suggesting that this feature may have emerged early in evolution.

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.

A large number of viral capsids, as well as other macromolecular assemblies, have icosahedral structure or structures with other rotational symmetries. This symmetry can be exploited during molecular dynamics (MD) to model in effect the full viral capsid using only a subset of primary atoms plus copies of image atoms generated from rotational symmetry boundary conditions (RSBC). A pure rotational symmetry operation results in both primary and image atoms at short range, and within nonbonded interaction distance of each other, so that nonbonded interactions can not be specified by the minimum image convention and explicit treatment of image atoms is required. As such an unavoidable consequence of RSBC is that the enumeration of nonbonded interactions in regions surrounding certain rotational axes must include both a primary atom and its copied image atom, thereby imposing microscopic symmetry for some forces. We examined the possibility of artifacts arising from this imposed microscopic symmetry of RSBC using two simulation systems: a water shell and human rhinovirus 14 (HRV14) capsid with explicit water. The primary unit was a pentamer of the icosahedron, which has the advantage of direct comparison of icosahedrally equivalent spatial regions, for example regions near a 2-fold symmetry axis with imposed symmetry and a 2-fold axis without imposed symmetry. Analysis of structural and dynamic properties of water molecules and protein atoms found similar behavior near symmetry axes with imposed symmetry and where the minimum image convention fails compared with that in other regions in the simulation system, even though an excluded volume effect was detected for water molecules near the axes with imposed symmetry. These results validate the use of RSBC for icosahedral viral capsids or other rotationally symmetric systems.

Bridging integrator 1 (BIN1) is a nucleocytoplasmic adaptor protein with tumor suppressor properties. The protein interacts with and inhibits the c-MYC transcription factor through the BIN1 MYC-binding domain (MBD). However, in vitro colony formation assays have clearly demonstrated that the MBD is not essential for BIN1-mediated growth arrest. We hypothesized that BIN1 contains a MYC-independent effector domain (MID) for cancer suppression. Because a functionally unique domain frequently contains a distinct structure, the human full-length BIN1 protein was subjected to limited trypsin digestion and the digested peptides were analyzed with Edman sequencing and mass spectrometry. We identified a trypsin-resistant peptide that corresponds to amino acids 146-268 of BIN1. It encompassed part of the BAR region, a putative effector region of BIN1. Computational analysis predicted that the peptide is very likely to exhibit coiled-coil motifs, implying a potential role for this region in sustaining the BIN1 structure and function. Like MBD-deleted BIN1, the trypsin-resistant peptide of BIN1 was predominantly present in the cytoplasm and was sufficient to inhibit cancer growth, regardless of dysregulated c-MYC activity. Our results suggest that the coiled-coil BIN1 BAR peptide encodes a novel BIN1 MID domain, through which BIN1 acts as a MYC-independent cancer suppressor., (Copyright © 2011 Wiley-Liss, Inc.)

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.

Activated Syk, an essential tyrosine kinase in B cell signaling, interacts with Vav guanine nucleotide exchange factors and regulates Vav activity through tyrosine phosphorylation. The Vav SH2 domain binds Syk linker B by an unusual recognition of two closely spaced Syk tyrosines: Y342 and Y346. The binding affinity is highest when both Y342 and Y346 are phosphorylated. An investigation in B cells of the dependence of Vav phosphorylation and NFAT activation on phosphorylation of Y342 and Y346 finds that cellular response levels match the relative binding affinities of the Vav1 SH2 domain for singly and doubly phosphorylated linker B peptides. This key result suggests that the uncommon recognition determinant of these two closely spaced tyrosines is a limiting factor in signaling. Interestingly, differences in affinities for binding singly and doubly phosphorylated peptides are reflected in the on rate, not the off rate. Such a control mechanism would be highly effective for regulating binding among competing Syk binding partners. The nuclear magnetic resonance (NMR) structure of Vav1 SH2 in complex with a doubly phosphorylated linker B peptide reveals diverse conformations associated with the unusual SH2 recognition of two phosphotyrosines. NMR relaxation indicates compensatory changes in loop fluctuations upon binding, with implications for nonphosphotyrosine interactions of Vav1 SH2.

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.

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.

CHARMM (Chemistry at HARvard Molecular Mechanics) is a highly versatile and widely used molecular simulation program. It has been developed over the last three decades with a primary focus on molecules of biological interest, including proteins, peptides, lipids, nucleic acids, carbohydrates, and small molecule ligands, as they occur in solution, crystals, and membrane environments. For the study of such systems, the program provides a large suite of computational tools that include numerous conformational and path sampling methods, free energy estimators, molecular minimization, dynamics, and analysis techniques, and model-building capabilities. The CHARMM program is applicable to problems involving a much broader class of many-particle systems. Calculations with CHARMM can be performed using a number of different energy functions and models, from mixed quantum mechanical-molecular mechanical force fields, to all-atom classical potential energy functions with explicit solvent and various boundary conditions, to implicit solvent and membrane models. The program has been ported to numerous platforms in both serial and parallel architectures. This article provides an overview of the program as it exists today with an emphasis on developments since the publication of the original CHARMM article in 1983., (Copyright 2009 Wiley Periodicals, Inc.)

The high mortality and financial burden associated with prostate cancer can be partly attributed to a lack of sensitive screening methods for detection and staging of the disease. Guided by in silico docking studies using the crystal structure of PSMA, we designed and synthesized a series of PSMA-targeted (99m)Tc-chelate complexes for imaging PSMA-expressing human prostate cancer cells (LNCaP cell line). Of the six targeted radioimaging agents synthesized, three were found to bind LNCaP cells with low nanomolar affinity. Moreover, the same three PSMA-targeted imaging agents were shown to localize primarily to LNCaP tumor xenografts in nu/nu mice, with an average of 9.8 +/- 2.4% injected dose/g tissue accumulating in the tumor and only 0.11% injected dose/g tissue retained in the muscle at 4 h postinjection. Collectively, these high affinity, PSMA-specific radioimaging agents demonstrate significant potential for use in localizing prostate cancer masses, monitoring response to therapy, detecting prostate cancer recurrence following surgery, and selecting patients for subsequent PSMA-targeted chemotherapy.

Targeted, steered, and biased molecular dynamics (MD) are widely used methods for studying transition processes of biomolecules. They share the common feature of adding external perturbations along a conformational progress variable to guide the transition in a predefined direction in conformational space, yet differ in how these perturbations are applied. In the present paper, we report a comparison of these three methods on generating transition paths for two different processes: the unfolding of the B domain of protein A and a conformational transition of the catalytic domain of a Src kinase Lyn. Transition pathways were calculated with different simulation parameters including the choice of progress variable and the simulation length or biasing force constant. A comparison of the generated paths based on structural similarity finds that the three perturbation MD methods generate similar transition paths for a given progress variable in most cases. On the other hand, the path depends more strongly on the choice of progress variable used to move the system between the initial and final states. Potentials of mean force (PMF) were calculated starting from unfolding trajectories to estimate the relative probabilities of the paths. A lower PMF was found for the lowest biasing force constant with BMD.

Infection by the mosquito-borne dengue virus causes dengue fever and the sometimes fatal dengue hemorrhagic fever. The increasing number of dengue infections per year suggests that the virus is becoming more virulent and its transmission is expanding. Nevertheless, no effective treatment for dengue infection currently exists. In a search for antiviral agents effective against dengue virus, we investigated the potential of targeting a structural protein site rather than an enzymatic one. Using this approach, we now report the discovery of a small molecule ligand that inhibits viral growth. Our results also provide the first evidence that the binding site, a pocket located at the hinge between domains 1 and 2 of the envelope protein (E protein) on the virus surface, is a valid target for antiviral therapy. Ligand candidates were identified from libraries of approximately 142,000 compounds using a computational high-throughput screening protocol targeting this pocket of the E protein. Cell-based assays were conducted on 23 top-ranked compounds. Among four with good antiviral activity profiles, the compound P02 was found to inhibit viral reproduction at micromolar concentrations. Using saturation transfer difference NMR spectroscopy, we also show that the compound binds virus and competes for binding E protein with the known ligand N-octyl-beta-D-glucoside. Together, the results are consistent with an inhibition mechanism against maturation or host-cell entry mediated by ligand binding to the E-protein pocket. P02 is a promising lead compound for future development of an effective treatment against dengue virus and related flaviviruses.

The regulatory mechanism of Src tyrosine kinases includes conformational activation by a change in the catalytic domain tertiary structure and in domain-domain contacts between the catalytic domain and the SH2/SH3 regulatory domains. The kinase is activated when tyrosine phosphorylation occurs on the activation loop, but without phosphorylation of the C-terminal tail. Activation also occurs by allostery when contacts between the catalytic domain (CD) and the regulatory SH3 and SH2 domains are released as a result of exogenous protein binding. The aim of this work is to examine the proposed role of an electrostatic network in the conformational transition and to elucidate the molecular mechanism for long-range, allosteric conformational activation by using a combination of experimental enzyme kinetics and nonequilibrium molecular dynamics simulations. Salt dependence of the induction phase is observed in kinetic assays and supports the role of an electrostatic network in the transition. In addition, simulations provide evidence that allosteric activation involves a concerted motion coupling highly conserved residues, and spanning several nanometers from the catalytic site to the regulatory domain interface to communicate between the CD and the regulatory domains.

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.

Flavivirus envelope proteins (E proteins) have been shown to play a pivotal role in virus assembly, morphogenesis, and infection of host cells. Inhibition of flavivirus infection of a host cell by means of a small molecule envelope protein antagonist is an attractive strategy for the development of antiviral agents. Virtual screening of the NCI chemical database using the dengue virus envelope protein structure revealed several hypothetical hit compounds. Bioassay results identified a class of thiazole compounds with antiviral potency in cell-based assays. Modification of these lead compounds led to a series of analogues with improved antiviral activity and decreased cytotoxicity. The most active compounds 11 and 36 were effective in the low micromolar concentration range in a cellular assay system.

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.

The experimental determination of protein compressibility reflects both the protein intrinsic compressibility and the difference between the compressibility of water in the protein hydration shell and bulk water. We use molecular dynamics simulations to explore the dependence of the isothermal compressibility of the hydration shell surrounding globular proteins on differential contributions from charged, polar, and apolar protein-water interfaces. The compressibility of water in the protein hydration shell is accounted for by a linear combination of contributions from charged, polar, and apolar solvent-accessible surfaces. The results provide a formula for the deconvolution of experimental data into intrinsic and hydration contributions when a protein of known structure is investigated. The physical basis for the model is the variation in water density shown by the surface-specific radial distribution functions of water molecules around globular proteins. The compressibility of water hydrating charged atoms is lower than bulk water compressibility, the compressibility of water hydrating apolar atoms is somewhat larger than bulk water compressibility, and the compressibility of water around polar atoms is about the same as the compressibility of bulk water. We also assess whether hydration water compressibility determined from small compound data can be used to estimate the compressibility of hydration water surrounding proteins. The results, based on an analysis from four dipeptide solutions, indicate that small compound data cannot be used directly to estimate the compressibility of hydration water surrounding proteins.

B-Myc is an endogenous, N-terminal homologue of transcription factor c-Myc that lacks the C-terminal DNA binding and protein dimerization domain of c-Myc. Clinical mutations in the c-Myc N-terminal region, and the subsequent misregulation of Myc, are implicated in the development of numerous human cancers. Myc functions to both activate and repress transcription by associating with multiple binding partners. We investigated the structural and dynamical properties of B-Myc, free or associated with the transactivation inhibitor, MM-1, and the activator, TBP, using NMR spectroscopy. B-Myc has no persistent tertiary structure, yet regions corresponding to Myc homology boxes 1 and 2 (MBI and MBII, respectively) have molten globule-like characteristics. B-Myc binds to MM-1 in a specific manner without becoming highly structured. The local regions of B-Myc involved in binding differ for MM-1 and TBP, and regions not identified by mutagenesis are found to be involved in MM-1 binding. The results provide new insights into Myc N-terminal protein-protein interactions. We propose a model for Myc regulation through differential involvement of MBI and MBII in the binding of Myc interacting proteins.

Src tyrosine kinases are essential in numerous cell signaling pathways, and improper functioning of these enzymes has been implicated in many diseases. The activity of Src kinases is regulated by conformational activation, which involves several structural changes within the catalytic domain (CD): the orientation of two lobes of CD; rearrangement of the activation loop (A-loop); and movement of an alpha-helix (alphaC), which is located at the interface between the two lobes, into or away from the catalytic cleft. Conformational activation was investigated using biased molecular dynamics to explore the transition pathway between the active and the down-regulated conformation of CD for the Src-kinase family member Lyn kinase, and to gain insight into the interdependence of these changes. Lobe opening is observed to be a facile motion, whereas movement of the A-loop motion is more complex requiring secondary structure changes as well as communication with alphaC. A key result is that the conformational transition involves a switch in an electrostatic network of six polar residues between the active and the down-regulated conformations. The exchange between interactions links the three main motions of the CD. Kinetic experiments that would demonstrate the contribution of the switched electrostatic network to the enzyme mechanism are proposed. Possible implications for regulation conferred by interdomain interactions are also discussed.

Human glutaredoxin (GRx), also known as thioltransferase, is a 12 kDa thiol-disulfide oxidoreductase that is highly selective for reduction of glutathione-containing mixed disulfides. The apparent pK(a) for the active site Cys22 residue is approximately 3.5. Previously we observed that the catalytic enhancement by glutaredoxin could be ascribed fully to the difference between the pK(a) of its Cys22 thiol moiety and the pK(a) of the product thiol, each acting as a leaving group in the enzymatic and nonenzymatic reactions, respectively [Srinivasan et al. (1997), Biochemistry 36, 3199-3206]. Continuum electrostatic calculations suggest that the low pK(a) of Cys22 results primarily from stabilization of the thiolate anion by a specific ion-pairing with the positively charged Lys19 residue, although hydrogen bonding interactions with Thr21 also appear to contribute. Variants of Lys19 were considered to further assess the predicted role of Lys19 on the pK(a) of Cys22. The variants K19Q and K19L were generated by molecular modeling, and the pK(a) value for Cys22 was calculated for each variant. For K19Q, the predicted Cys22 pK(a) is 7.3, while the predicted value is 8.3 for K19L. The effects of the mutations on the interaction energy between the adducted glutathionyl moiety and GRx were roughly estimated from the van der Waals and electrostatic energies between the glutathionyl moiety and proximal protein residues in a mixed disulfide adduct of GRx and glutathione, i.e., the GRx-SSG intermediate. The values for the K19 mutants differed by only a small amount compared to those for the wild type enzyme intermediate. Together, the computational analysis predicted that the mutant enzymes would have markedly reduced catalytic rates while retaining the glutathionyl specificity displayed by the wild type enzyme. Accordingly, we constructed and characterized the K19L and K19Q mutants of two forms of the GRx enzyme. Each of the mutants retained glutathionyl specificity as predicted and displayed diminution in activity, but the decreases in activity were not to the extent predicted by the theoretical calculations. Changes in the respective Cys22-thiol pK(a) values of the mutant enzymes, as shown by pH profiles for iodoacetamide inactivation of the respective enzymes, clearly revealed that the K19-C22 ion pair cannot fully account for the low pK(a) of the Cys22 thiol. Additional contributions to stabilization of the Cys22 thiolate are likely donated by Thr21 and the N-terminal partial positive charge of the neighboring alpha-helix.

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.

WIN antiviral compounds bind human rhinovirus, as well as enterovirus and parechovirus, in an internal cavity located within the viral protein capsid. Access to the buried pocket necessitates deviation from the average viral protein structure identified by crystallography. We investigated the dissociation of WIN 52084 from the pocket in human rhinovirus 14 by using an adiabatic, biased molecular dynamics simulation method. Multiple dissociation trajectories are used to characterize the pathway. WIN 52084 exits between the polypeptide chain near the ends of betaC and betaH in a series of steps. Small, transient packing defects in the protein are sufficient for dissociation. A number of torsion-angle transitions of the antiviral compound are involved, which suggests that flexibility in antiviral compounds is important for binding. It is interesting to note that dissociation is associated with an increase in the conformational fluctuations of residues never in direct contact with WIN 52084 over the course of dissociation. These residues are N-terminal residues in the viral proteins VP3 and VP4 and are located in the interior of the capsid near the icosahedral 5-fold axis. The observed changes in dynamics may be relevant to structural changes associated with virion uncoating and its inhibition by antiviral compounds.

A late stage in assembly of alphaviruses within infected cells is thought to be directed by interactions between the nucleocapsid and the cytoplasmic domain of the E2 protein, a component of the viral E1/E2 glycoprotein complex that is embedded in the plasma membrane. Recognition between the nucleocapsid protein and the E2 protein was explored in solution using NMR spectroscopy, as well as in binding assays using a model phospholipid membrane system that incorporated a variety of Sindbis virus E2 cytoplasmic domain (cdE2) and capsid protein constructs. In these binding assays, synthetic cdE2 peptides were reconstituted into phospholipid vesicles to simulate the presentation of cdE2 on the inner leaflet of the plasma membrane. Results from these binding assays showed a direct interaction between a peptide containing the C-terminal 16 amino acids of the cdE2 sequence and a Sindbis virus capsid protein construct containing amino acids 19-264. Additional experiments that probed the sequence specificity of this cdE2-capsid interaction are also described. Further binding assays demonstrated an interaction between the 19-264 capsid protein and artificial vesicles containing neutral or negatively charged phospholipids, while capsid protein constructs with N-terminal truncations displayed either little or no affinity for such vesicles. The membrane-binding property of the capsid protein suggests that the membrane may play an active role in alphavirus assembly. The results are consistent with an assembly process involving an initial membrane association, whereby an association with E2 glycoprotein further enhances capsid binding to facilitate membrane envelopment of the nucleocapsid for budding. Collectively, these experiments elucidate certain requirements for the binding of Sindbis virus capsid protein to the cytoplasmic domain of the E2 glycoprotein, a critical event in the alphavirus maturation pathway.

After engagement of the B cell receptor for antigen, the Syk protein-tyrosine kinase becomes phosphorylated on multiple tyrosines, some of which serve as docking sites for downstream effectors with SH2 or other phosphotyrosine binding domains. The most frequently identified binding partner for catalytically active Syk identified in a yeast two-hybrid screen was the p85 regulatory subunit of phosphoinositide 3-kinase. The C-terminal SH2 domain of p85 was sufficient for mediating an interaction with tyrosine-phosphorylated Syk. Interestingly, this domain interacted with Syk at phosphotyrosine 317, a site phosphorylated in trans by the Src family kinase, Lyn, and identified previously as a binding site for c-Cbl. This site interacted preferentially with the p85 C-terminal SH2 domain compared with the c-Cbl tyrosine kinase binding domain. Molecular modeling studies showed a good fit between the p85 SH2 domain and a peptide containing phosphotyrosine 317. Tyr-317 was found to be essential for Syk to support phagocytosis mediated by FcgammaRIIA receptors expressed in a heterologous system. These studies establish a new type of p85 binding site that can exist on proteins that serve as substrates for Src family kinases and provide a molecular explanation for observations on direct interactions between Syk and phosphoinositide 3-kinase.