Modeling Conformational Ensembles of Slow Functional Motions in Pin1-WW

Protein-protein interactions are often mediated by flexible loops that experience conformational dynamics on the microsecond to millisecond time scales. NMR relaxation studies can map these dynamics. However, defining the network of inter-converting conformers that underlie the relaxation data remains generally challenging. Here, we combine NMR relaxation experiments with simulation to visualize networks of inter-converting conformers. We demonstrate our approach with the apo Pin1-WW domain, for which NMR has revealed conformational dynamics of a flexible loop in the millisecond range. We sample and cluster the free energy landscape using Markov State Models (MSM) with major and minor exchange states with high correlation with the NMR relaxation data and low NOE violations. These MSM are hierarchical ensembles of slowly interconverting, metastable macrostates and rapidly interconverting microstates. We found a low population state that consists primarily of holo-like conformations and is a “hub” visited by most pathways between macrostates. These results suggest that conformational equilibria between holo-like and alternative conformers pre-exist in the intrinsic dynamics of apo Pin1-WW. Analysis using MutInf, a mutual information method for quantifying correlated motions, reveals that WW dynamics not only play a role in substrate recognition, but also may help couple the substrate binding site on the WW domain to the one on the catalytic domain. Our work represents an important step towards building networks of inter-converting conformational states and is generally applicable

Substrate and Inhibitor-induced Dimerization and Cooperativity in Caspase-1 but Not Caspase-3*

Caspases are intracellular cysteine-class proteases with aspartate specificity that is critical for driving processes as diverse as the innate immune response and apoptosis, exemplified by caspase-1 and caspase-3, respectively. Interestingly, caspase-1 cleaves far fewer cellular substrates than caspase-3 and also shows strong positive cooperativity between the two active sites of the homodimer, unlike caspase-3. Biophysical and kinetic studies here present a molecular basis for this difference. Analytical ultracentrifugation experiments show that mature caspase-1 exists predominantly as a monomer under physiological concentrations that undergoes dimerization in the presence of substrate; specifically, substrate binding shifts the KD for dimerization by 20-fold. We have created a hemi-active site-labeled dimer of caspase-1, where one site is blocked with the covalent active site inhibitor, benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone. This hemi-labeled enzyme is about 9-fold more active than the apo-dimer of caspase-1. These studies suggest that substrate not only drives dimerization but also, once bound to one site in the dimer, promotes an active conformation in the other monomer. Steady-state kinetic analysis and modeling independently support this model, where binding of one substrate molecule not only increases substrate binding in preformed dimers but also drives the formation of heterodimers. Thus, the cooperativity in caspase-1 is driven both by substrate-induced dimerization as well as substrate-induced activation. Substrate-induced dimerization and activation seen in caspase-1 and not in caspase-3 may reflect their biological roles. Whereas caspase-1 cleaves a dramatically smaller number of cellular substrates that need to be concentrated near inflammasomes, caspase-3 is a constitutively active dimer that cleaves many more substrates located diffusely throughout the cell

Cosolvent-enhanced Sampling and Unbiased Identification of Cryptic Pockets Suitable for Structure-based Drug Design

Modulating protein activity with small molecules binding to cryptic pockets offers great opportunities to overcome hurdles in drug design. Cryptic sites are atypical binding sites in proteins that are closed in the absence of a stabilizing ligand and are thus inherently difficult to identify. Many studies have proposed methods to predict cryptic sites. However, a general approach to prospectively sample open conformations of these sites and to identify cryptic pockets in an unbiased manner suitable for structure-based drug design remains elusive. Here, we describe an all-atom, explicit cosolvent, molecular dynamics (MD) simulations-based workflow to sample the open states of cryptic sites and identify opened pockets, in a manner that does not require a priori knowledge about these sites. Furthermore, the workflow relies on a target-independent parameterization that only distinguishes between binding pockets for peptides or small-molecules. We validated our approach on a diverse test set of seven proteins with crystallographically determined cryptic sites. The known cryptic sites were found among the three highest-ranked predicted cryptic sites, and an open site conformation was sampled and selected for most of the systems. Crystallographic ligand poses were well reproduced by docking into these identified open conformations for five of the systems. When the fully open state could not be reproduced, we were still able to predict the location of the cryptic site, or identify other cryptic sites that could be retrospectively validated with knowledge of the protein target. These characteristics render our approach valuable for investigating novel protein targets without any prior information

Dynamic architecture of a protein kinase

Protein kinases are dynamically regulated signaling proteins that act as switches in the cell by phosphorylating target proteins. To establish a framework for analyzing linkages between structure, function, dynamics, and allostery in protein kinases, we carried out multiple microsecond-scale molecular-dynamics simulations of protein kinase A (PKA), an exemplar active kinase. We identified residue-residue correlated motions based on the concept of mutual information and used the Girvan-Newman method to partition PKA into structurally contiguous "communities." Most of these communities included 40-60 residues and were associated with a particular protein kinase function or a regulatory mechanism, and well-known motifs based on sequence and secondary structure were often split into different communities. The observed community maps were sensitive to the presence of different ligands and provide a new framework for interpreting long-distance allosteric coupling. Communication between different communities was also in agreement with the previously defined architecture of the protein kinase core based on the "hydrophobic spine" network. This finding gives us confidence in suggesting that community analyses can be used for other protein kinases and will provide an efficient tool for structural biologists. The communities also allow us to think about allosteric consequences of mutations that are linked to disease

Hydrogen Bond Strengths in Phosphorylated and Sulfated Amino Acid Residues

<div><p>Post-translational modification by the addition of an oxoanion functional group, usually a phosphate group and less commonly a sulfate group, leads to diverse structural and functional consequences in protein systems. Building upon previous studies of the phosphoserine residue (pSer), we address the distinct nature of hydrogen bonding interactions in phosphotyrosine (pTyr) and sulfotyrosine (sTyr) residues. We derive partial charges for these modified residues and then study them in the context of molecular dynamics simulation of model tripeptides and sulfated protein complexes, potentials of mean force for interacting residue pairs, and a survey of the interactions of modified residues among experimental protein structures. Overall, our findings show that for pTyr, bidentate interactions with Arg are particularly dominant, as has been previously demonstrated for pSer. sTyr interactions with Arg are significantly weaker, even as compared to the same interactions made by the Glu residue. Our work sheds light on the distinct nature of these modified tyrosine residues, and provides a physical-chemical foundation for future studies with the goal of understanding their roles in systems of biological interest.</p> </div

Ab initio modeling and experimental assessment of Janus Kinase 2 (JAK2) kinase-pseudokinase complex structure.

The Janus Kinase 2 (JAK2) plays essential roles in transmitting signals from multiple cytokine receptors, and constitutive activation of JAK2 results in hematopoietic disorders and oncogenesis. JAK2 kinase activity is negatively regulated by its pseudokinase domain (JH2), where the gain-of-function mutation V617F that causes myeloproliferative neoplasms resides. In the absence of a crystal structure of full-length JAK2, how JH2 inhibits the kinase domain (JH1), and how V617F hyperactivates JAK2 remain elusive. We modeled the JAK2 JH1-JH2 complex structure using a novel informatics-guided protein-protein docking strategy. A detailed JAK2 JH2-mediated auto-inhibition mechanism is proposed, where JH2 traps the activation loop of JH1 in an inactive conformation and blocks the movement of kinase αC helix through critical hydrophobic contacts and extensive electrostatic interactions. These stabilizing interactions are less favorable in JAK2-V617F. Notably, several predicted binding interfacial residues in JH2 were confirmed to hyperactivate JAK2 kinase activity in site-directed mutagenesis and BaF3/EpoR cell transformation studies. Although there may exist other JH2-mediated mechanisms to control JH1, our JH1-JH2 structural model represents a verifiable working hypothesis for further experimental studies to elucidate the role of JH2 in regulating JAK2 in both normal and pathological settings

Mutation of a kinase allosteric node uncouples dynamics linked to phosphotransfer

The expertise of protein kinases lies in their dynamic structure, wherein they are able to modulate cellular signaling by their phosphotransferase activity. Only a few hundreds of protein kinases regulate key processes in human cells, and protein kinases play a pivotal role in health and disease. The present study dwells on understanding the working of the protein kinase-molecular switch as an allosteric network of "communities" composed of congruently dynamic residues that make up the protein kinase core. Girvan-Newman algorithm-based community maps of the kinase domain of cAMP-dependent protein kinase A allow for a molecular explanation for the role of protein conformational entropy in its catalytic cycle. The community map of a mutant, Y204A, is analyzed vis-à-vis the wild-type protein to study the perturbations in its dynamic profile such that it interferes with transfer of the γ-phosphate to a protein substrate. Conventional biochemical measurements are used to ascertain the effect of these dynamic perturbations on the kinetic profiles of both proteins. These studies pave the way for understanding how mutations far from the kinase active site can alter its dynamic properties and catalytic function even when major structural perturbations are not obvious from static crystal structures

Implicit Solvent Potentials of Mean Force for representative residue pairs.

<p>Plots show interaction energy vs. distance, at distance intervals of 0.25 Å, for a pair of residues in a given orientation. Distance refers to the P-C distance between the phosphate atom and the terminal carbon on Arg (coplanar), or the N-O distance (collinear).</p