Significance Protein kinases represent a critically important family of regulatory enzymes. Their activity can be altered by mutations and binding events distant from the active site. To understand nature these long-distance effects, we used microsecond-timescale molecular dynamic simulation to subdivide prototypical kinase, protein kinase A, into contiguous communities that exhibit internally correlated motions. Surprisingly, most unconventional structural entities were centered around...

Allostery describes altered protein function at one site due to a perturbation another site. One mechanism of allostery involves correlated motions, which can occur even in the absence substantial conformational change. We present novel method, "MutInf", identify statistically significant motions from equilibrium molecular dynamics simulations. Our approach analyzes both backbone and sidechain using internal coordinates account for gear-like twists that take place large changes typical...

There is significant interest in identifying and characterizing allosteric sites enzymes such as protein kinases both for understanding mechanisms well drug discovery. Here, we apply a site-directed technology, disulfide trapping, to interrogate structurally functionally how an site on the Ser/Thr kinase, 3-phosphoinositide-dependent kinase 1 (PDK1)—the PDK1-interacting-fragment (PIF) pocket—is engaged by activating peptide motif downstream substrate (PIFtides) small molecule fragments. By...

Eukaryotic protein kinases (EPKs) constitute a class of allosteric switches that mediate myriad signaling events. It has been postulated EPKs' active and inactive states depend on the structural architecture their hydrophobic cores, organized around two highly conserved spines: C-spine R-spine. How spines orchestrate transition enzyme between catalytically uncommitted committed remains elusive. Using relaxation dispersion nuclear magnetic resonance spectroscopy, we found core catalytic...

Binding free energy calculations predict the potency of compounds to protein binding sites in a physically rigorous manner and see broad application prioritizing synthesis novel drug candidates. Relative (RBFE) have emerged as an industry-standard approach achieve highly accurate rank-order predictions related compounds; however, this requires that ligands share common scaffold mode, restricting methods' domain applicability. This is critical limitation since complex modifications ligands,...

We present a thermodynamical approach to identify changes in macromolecular structure and dynamics response perturbations such as mutations or ligand binding, using an expansion of the Kullback-Leibler Divergence that connects local population shifts torsion angles free energy landscape protein. While is known formula from information theory, novelty power our implementation lies its formal developments, connection thermodynamics, statistical filtering, ease visualization results,...

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 network of inter-converting conformers underlie data remains generally challenging. Here, we combine experiments with simulation visualize networks conformers. We demonstrate our approach apo Pin1-WW domain, for which has revealed a loop in range. sample and cluster free energy...

The expertise of protein kinases lies in their dynamic structure, wherein they are able to modulate cellular signaling by phosphotransferase activity. Only a few hundreds regulate key processes human cells, and play pivotal role health disease. present study dwells on understanding the working kinase-molecular switch as an allosteric network "communities" composed congruently residues that make up kinase core. Girvan-Newman algorithm-based community maps domain cAMP-dependent A allow for...

Eukaryotic protein kinases regulate most cellular functions by phosphorylating targeted substrates through a highly conserved catalytic core. In the active state, core oscillates between open, intermediate, and closed conformations. Currently, intramolecular interactions that state mechanics are not well understood. Here, using cAMP-dependent kinase as representative model coupled with biochemical, biophysical, computational techniques, we define set of electrostatic hydrophobic working...

For disease network intervention, up-regulating enzyme activities is equally as important down-regulating activities. However, the design of activators presents a challenging route for drug discovery. Previous studies have suggested that activating 15-lipoxygenase (15-LOX) promising strategy to intervene arachidonic acid (AA) metabolite and control inflammation. To prove this concept, we used computational approach discover previously unknown allosteric site on 15-LOX. Both inhibitors novel...

Modulating protein activity with small-molecules binding to cryptic pockets offers great opportunities overcome hurdles in drug design. Cryptic sites are atypical proteins that closed the absence of a stabilizing ligand and thus inherently difficult identify. Many studies have proposed methods predict sites. However, general approach prospectively sample open conformations these identify an unbiased manner suitable for structure-based design remains elusive. Here, we describe all-atom,...

A new coarse-grained model of the E. coli cytoplasm is developed by describing proteins as flexible units consisting one or more spheres that follow Brownian dynamics (BD), with hydrodynamic interactions (HI) accounted for a mean-field approach. Extensive BD simulations were performed to calculate diffusion coefficients three different in cellular environment. The results are close agreement experimental previously simulated values, where available. Control without HI showed use essential...

G protein-coupled receptors (GPCRs) modulate diverse cellular signaling pathways and are important drug targets. Despite the availability of high-resolution structures, discovery allosteric modulators remains challenging due to dynamic nature GPCRs in native membranes. We developed a strategy covalently tether fragments adjacent sites enhance their potency enable fragment-based screening cell-based systems. employed genetic code expansion site-specifically introduce noncanonical amino acids...

Allostery describes the functional coupling between sites in biomolecules. Recently, role of changes protein dynamics for allosteric communication has been highlighted. A quantitative and predictive description allostery is fundamental understanding biological processes. Here, we integrate an ensemble-based perturbation approach with analysis biomolecular rigidity flexibility to construct a model dynamic allostery. Our model, by definition, excludes possibility conformational changes,...

The catalytic subunit of protein kinase A (PKA-C) is subject to several post- or cotranslational modifications that regulate its activity both spatially and temporally. Among those, N-myristoylation increases the affinity for membranes might also be implicated in substrate recognition allosteric regulation. Here, we investigated effects on structure, dynamics, conformational equilibrium PKA-C using atomistic molecular dynamics simulations. We found myristoyl group inserts into hydrophobic...

Post-translational modification by the addition of an oxoanion functional group, usually a phosphate group and less commonly sulfate leads to diverse structural consequences in protein systems. Building upon previous studies phosphoserine residue (pSer), we address distinct nature hydrogen bonding interactions phosphotyrosine (pTyr) sulfotyrosine (sTyr) residues. We derive partial charges for these modified residues then study them context molecular dynamics simulation model tripeptides...

Oligomerization of G protein-coupled receptors is a recognized mode regulation receptor activities, with alternate oligomeric states resulting in different signaling functions. The CXCR4 chemokine that post-translationally modified by tyrosine sulfation at three sites on its N-terminus (Y7, Y12, Y21), leading to enhanced affinity for ligand, stromal cell derived factor (SDF-1, also called CXCL12). complex has been implicated cancer metastasis and therapeutic target treatment. Using molecular...

The Janus Kinase 2 (JAK2) plays essential roles in transmitting signals from multiple cytokine receptors, and constitutive activation of JAK2 results hematopoietic disorders oncogenesis. kinase activity is negatively regulated by its pseudokinase domain (JH2), where the gain-of-function mutation V617F that causes myeloproliferative neoplasms resides. In absence a crystal structure full-length JAK2, how JH2 inhibits (JH1), hyperactivates remain elusive. We modeled JH1–JH2 complex using novel...

To better understand the mechanism of long distance allosteric signaling inside protein kinase core using A (PKA) as a prototype, we obtained 5μs molecular dynamics trajectories in different liganded and conformational states Anton supercomputer, providing for first time an opportunity to observe intermediate-timescale transitions study this stable kinase. We used community analysis identify structurally-contiguous groups residues exhibiting correlated motions catalytic domain. This dynamic...

Binding free energy calculations predict the potency of compounds to protein binding sites in a physically rigorous manner and see broad application prioritizing synthesis novel drug candidates. Relative (RBFE) have emerged as an industry standard approach achieve highly accurate rank-order predictions related compounds; however, this requires that ligands share common scaffold mode, restricting methods' domain applicability. This is critical limitation, since complex modifications ligands,...