Iron(II) triggered conformational changes in Escherichia coli fur upon DNA binding: a study using molecular modeling

In order to identify the Fur dimerization domain, a three-dimensional structure of the ferric uptake regulation protein from Escherichia coli (Fur EC) was determined using homology modeling and energy minimization. The Fur monomer consists of turn- helix -turn motif on the N-terminal domain, followed by another helix-turn-helix-turn motif, and two beta-strands separated by a turn which forms the wing. The C-terminal domain, separated by a long coil from the N-terminal, and consisting of two anti parallel beta strands, and a turn-helix-turn-helix-turn motif. Residues in central domain were found to aid the dimer formation, residues 45-70 as evident in the calculated distances; this region is rich in hydrophobic residues. Most interactions occur between residues Val55, Leu53, Gln52, Glu49 and Tyr56 with closest contacts occurring at residues 49-56. These residues are part of an alpha-helix (alpha(4)) near the N-terminal. Upon raising the Fe(2+) concentration the binding of Fur dimer to DNA was enhanced, this was evident when, the Fur EC dimer was docked onto DNA "iron box" (it was found to bind the AT-rich region) and upon addition of Fe(2+) the helices near the N-terminal bound to the major groove of the DNA. Addition of high Fe(2+) concentration triggered further conformational changes in the Fur dimer as was measured by distances between the two subunits, Fe(2+) mediated the Fur binding to DNA by attaching itself to the DNA. At the same time DNA changed conformation as was evident in the distortion in the backbone and the shrinking of major groove distance from 11.4 to 9.3A. Two major Fe(2+) sites were observed on the C-terminal domain: site 1, the traditional Zn site, the cavity contains the residues Cys92, Cys95, Asp137, Asp141, Arg139, Glu 140, His 145 and His 143 at distances range from 1.3 to 2.2A. Site 2 enclave consists of His71, Ile50, Asn72, Gly97, Asp105 and Ala109 at very close proximity to Fe(2+). The closest contacts between Fur dimer and DNA at the AT-rich region were at residues Ala11, Gly12, Leu13, Pro18 and Arg19 mostly hydrophobic residues near the N-terminal domain. Close contacts repeated at His87, His88 and Arg112, and a third region near the C-terminal at Asn137, Arg 139, Glu140, Asn141, His143, Asn141 and His145. Fur dimer has three major contact regions with DNA, the first on the N-terminal domain, a second smaller region at His87, His88 and Arg112 mediated by Fe(2+) ions, and a third region on the C-terminal domain consisting mainly of hydrophobic contacts and mediated by Fe(2+) ions at high concentration.     

Molecular modeling study of CodX reveals importance of N-terminal and C-terminal domain in the CodWX complex structure of Bacillus subtilis

In Bacillus subtilis, CodW peptidase and CodX ATPase function together as a distinctive ATP-dependent protease called CodWX, which participates in protein degradation and regulates cell division. The molecular structure of CodX and the assembly structure of CodW-CodX have not yet been resolved. Here we present the first three-dimensional structure of CodX N-terminal (N) and C-terminal (C) domain including possible structure of intermediate (I) domain based on the crystal structure of homologous Escherichia coli HslU ATPase. Moreover, the biologically relevant CodWX (W(6)W(6)X(6)) octadecamer complex structure was constructed using the recently identified CodW-HslU hybrid crystal structure. Molecular dynamics (MD) simulation shows a reasonably stable structure of modeled CodWX and explicit behavior of key segments in CodX N and C domain: nucleotide binding residues, GYVG pore motif and CodW-CodX interface. Predicted structure of the possible I domain is flexible in nature with highly coiled hydrophobic region (M153-M206) that could favor substrate binding and entry. Electrostatic surface potential observation unveiled charge complementarity based CodW-CodX interaction pattern could be a possible native interaction pattern in the interface of CodWX. CodX GYVG pore motif structural features, flexible nature of glycine (G92 and G95) residues and aromatic ring conformation preserved Y93 indicated that it may follow the similar mode during the proteolysis mechanism as in the HslU closed state. This molecular modeling study uncovers the significance of CodX N and C domain in CodWX complex and provides possible explanations which would be helpful to understand the CodWX-dependent proteolysis mechanism of B. subtilis.     

In silico identification and computational analysis of the nucleotide binding site in the C-terminal domain of Hsp90

Hsp90 contains two distinct Nucleotide Binding Sites (NBS), in its N-terminal domain (NTD) and C-terminal domain (CTD), respectively. The NTD site belongs to the GHKL super-family of ATPases and has been the subject of extensive characterization. However, a structure of the nucleotide-bound form of CTD is still unavailable. In this study molecular modeling was employed to incorporate experimental data using partial constructs of the CTD, from work published by many research groups, onto existing structural models of its apo- form. Our attempts to locate potential nucleotide ligand-binding sites or cavities yielded one major candidate—a structurally unconventional site—exhibiting the requisite shape and volume for accommodation of tri-phosphate nucleotides. Its structure was refined by molecular dynamics (MD)-based techniques. We reproducibly docked the Mg²⁺ complexed form of ATP, GTP, CTP, TTP and UTP to this putative NBS. These docking simulations and calculated ligand-binding scores are in general agreement with published data about experimentally measured binding to the CTD. The overall pattern of interactions between residues lining the site and docked nucleotides is conserved and broadly similar to that of other nucleotide-binding sites. Our docking simulations suggest that nucleotide binding stabilizes the only structurally labile region, thereby providing a rationale for the increased resistance to thermal denaturation and proteolysis. The docked nucleotides do not intrude onto the surface of residues involved in dimerization or chaperoning. Our molecular modeling permitted recognition of larger structural changes in the nucleotide-bound CTD dimer, including stabilization of helix-2 in both chains and intra- and inter- chain interactions between three residues (I613, Q617, R620).     

The three-dimensional model of Dictyostelium discoideum racE based on the human rhoA-GDP crystal structure

The three-dimensional structure of racE was modeled using several homologous small G proteins, and the best model obtained using the human rhoA as modeling template is reported. The three-dimensional fold of the racE model is remarkably similar to the cellular form of human ras p21 crystal structure. Its secondary structure consists of six alpha-helices, six beta-strands and three 3(10) helices. The model retains its secondary structure after a 300 K, 300 ps molecular dynamics (MD) simulation. Important domains of the protein include its effector loop (residues 34-46), the insertion domain (residues 121-136), and the polybasic motif (between 210 and 220) not modeled in the current structure. The effector loop is inherently flexible and the structure docked with GDP exhibits the effector loop moving significantly closer to the nucleotide binding pocket, forming a tighter complex with the bound GDP. The mobility of the effector loop is conferred by a single residue 'hinge' point at residue 34Asp, also allowing the Switch I region, immediately preceding the effector loop, to be equally mobile. In comparison, the Switch II region shows average mobility. The insertion domain is highly flexible, with the insertion taking the form of a helical domain, with several charged residues forming a complex charged interface over the entire insertion region. While the GDP moiety is loosely held in the active site, the metal cation is extensively co-ordinated. The critical residue 38Thr exhibits high mobility, and is seen interacting directly with the metal ion at a distance of 2.64 A, and indirectly via an intervening water molecule. 64Gln, a key residue involved in GTP hydrolysis in ras, is seen facing the beta-phosphate group and the metal ion. Certain residues (i.e. 51Asn, 38Thr and 65Glu) exhibit unique characteristics and these residues, together with 158Val, may play important roles in the maintenance of the protein's integrity and function. There is strong consensus of secondary structural elements between models generated using various templates, such as h-rac1, h-rhoA and h-cdc42 bound to RhoGDI, all sharing only 50-55% sequence identity with racE, which suggests that this model is in all probability an accurate prediction of the true tertiary structure of racE.     

Quantum mechanical investigations on the role of C-terminal residue in influencing the structural features of dipeptides containing N-terminal proline

This study investigates the influence of the side chain moiety of C-terminal residue on the structural and molecular properties of seven dipeptides having proline at their N-terminal positions. The C-terminal component of the dipeptides is varied with seven different combinations viz. Ala, Leu, Asp, Thr, Asn, Arg and Sec. The calculations are carried out using B3LYP/6-311++G(d,p) level of theory in gas and implicit aqueous phase. Effects of explicit aqueous environment on the dipeptide structures are also investigated for two systems. The results furnished by this DFT study provide valuable information regarding the role of the side chain groups of C-terminal residues in determining the structural features of the amide planes, values of the ψ and ф dihedrals, geometry about the α-carbon atoms, theoretical IR spectra as well as the number and type of intramolecular H-bond interactions existing in the dipeptides, and extend a fine corroboration to the earlier theoretical and experimental observations. In aqueous phase the dipeptide geometries exhibit larger values of total dipole moments, greater HOMO–LUMO energy gaps and enhanced thermodynamic stability than those in gas phase. The explicit water molecules are found to modify the geometrical parameters related to the amide planes and vibrational spectra of the dipeptides.     

Homology modeling of the DNA-binding domain of human Smad5: a molecular model for inhibitor design

Members of the Smad protein family function as signal transducers of the transforming growth factor (TGF-beta) superfamily proteins. The human Smad5 protein, a signal transducer downstream of TGF-beta/BMP receptors, is composed of N-terminal DNA binding domain (MH1) and C-terminal protein-protein interaction domain (MH2) connected together by a linker motif. We used homology-modeling techniques to generate a reliable molecular model of the Smad5 MH1 domain based on the crystal structure of Smad3 MH1 domain. Our study presents the structural features of a BMP-regulated, R-Smad subfamily member (consisting of Smad1, Smad5 and Smad8) for the first time. This model provides a structural basis for explaining both functional similarities and differences between Smad3 and Smad5. Also, the structural model of this molecular target would be useful for structure-based inhibitor design because of its high accuracy. The results of our study provide important insights into understanding the structure-function relationship of the members of the Smad protein family and can serve to guide future genetic and biochemical experiments in this area.     

Simulation of coupled folding and binding of an intrinsically disordered protein in explicit solvent with metadynamics

The C-terminal domain of measles virus nucleoprotein is an intrinsically disordered protein that could bind to the X domain (XD) of phosphoprotein P to exert its physiological function. Experiments reveal that the minimal binding unit is a 21-residue α-helical molecular recognition element (α-MoRE-MeV), which adopts a fully helical conformation upon binding to XD. Due to currently limited computing power, direct simulation of this coupled folding and binding process with atomic force field in explicit solvent cannot be achieved. In this work, two advanced sampling methods, metadynamics and parallel tempering, are combined to characterize the free energy surface of this process and investigate the underlying mechanism. Starting from an unbound and partially folded state of α-MoRE-MeV, multiple folding and binding events are observed during the simulation and the energy landscape was well estimated. The results demonstrate that the isolated α-MoRE-MeV resembles a molten globule and rapidly interconverts between random coil and multiple partially helical states in solution. The coupled folding and binding process occurs through the induced fit mechanism, with the residual helical conformations providing the initial binding sites. Upon binding, α-MoRE-MeV can easily fold into helical conformation without obvious energy barriers. Two mechanisms, namely, the system tending to adopt the structure in which the free energy of isolated α-MoRE-MeV is the minimum, and the binding energy of α-MoRE-MeV to its partner protein XD tending to the minimum, jointly dominate the coupled folding and binding process. With the advanced sampling approach, more IDP systems could be simulated and common mechanisms concerning the coupled folding and binding process could be investigated in the future.     

Flexibility of the murine prion protein and its Asp178Asn mutant investigated by molecular dynamics simulations.

Inherited forms of transmissible spongiform encephalopathy, e.g. familial Creutzfeldt-Jakob disease, Gerstmann-Str?ussler-Scheinker syndrome and fatal familial insomnia, segregate with specific point mutations of the prion protein. It has been proposed that the pathologically relevant Asp178Asn (D178N) mutation might destabilize the structure of the prion protein because of the loss of the Arg164-Asp178 salt bridge. Molecular dynamics simulations of the structured C-terminal domain of the murine prion protein and the D178N mutant were performed to investigate this hypothesis. The D178N mutant did not deviate from the NMR conformation more than the wild type on the nanosecond time scale of the simulations. In agreement with CD spectroscopy experiments, no major structural rearrangement could be observed for the D178N mutant, apart from the N-terminal elongation of helix 2. The region of structure around the disulfide bridge deviated the least from the NMR conformation and showed the smallest fluctuations in all simulations in agreement with hydrogen exchange data of the wild type prion protein. Large deviations and flexibility were observed in the segments which are ill-defined in the NMR conformation. Moreover, helix 1 showed an increased degree of mobility, especially at its N-terminal region. The dynamic behavior of the D178N mutant and its minor deviation from the folded conformation suggest that the salt bridge between Arg164 and Asp178 might not be crucial for the stability of the prion protein.     

A DNA Computing-based Genetic Program for In Vitro Protein Evolution via Constrained Pseudomodule Shuffling

An in vitro domainal shuffling strategy for protein evolution was proposed in (J. Kolkman and W. Stemmer, Nat. Biotech. 19 (423) 2001). Due to backhybridization, however this method appears unlikely to be an efficient means of iteratively generating massive libraries of combinatorially shuffled genes. Recombination at the domain level (30–300 residues) also appears too coarse to support the evolution of proteins with substantially new folds. In this work, the module (10–25 residues long) and pseudomodule are adopted as the fundamental units of protein structure. Each protein is modelled as an N to C-terminal tour of a digraph composed of pseudomodules. An in vitro method based on PNA-mediated Whiplash PCR (PWPCR), RNA-protein fusion, and restriction-based recombination, XWPCR is then presented for evolving proteins with a high affinity for a given motif, subject to the constraint that each corresponds to a walk on the pseudomodule digraph of interest. Simulations predict that PWPCR is an efficient method of producing massive, shuffled gene libraries encoding for proteins as long as roughly 600 residues.     

Structural and dynamic insights into the C-terminal extension of cysteine proteinase B from Leishmania amazonensis

Cysteine proteinase B (CPB) is a significant virulence factor for Leishmania infections. Upon processing from its zymogen form, it happens a release of the immunomodulatory CPB C-terminal extension (cyspep) into the cytoplasm of the macrophage. Epitopes derived from this fragment were shown to influence the proportion of lymphocytes CD8+ upon infection, favoring the parasite escaping from the host́s immune system. At present, there is no available structural data of cyspep, which impairs a proper understanding of its biological functions. Here, we attempted to build molecular models for this fragment and subsequently evaluate their stabilities in aqueous solution from molecular dynamics simulations analysis. Characterization of our models obtained with distinct techniques (comparative modeling, threading, and ab initio) indicates a prevalence of β−sheets in agreement with consensus secondary structure predictions. Simulation data supported this finding since the formation of new strands, along with a rapid disruption of helical content, were observed. Overall, this study provides a rationalization of epitope mapping data and an improved understanding of cyspep antigenicity.     

Molecular dynamics simulations of isoleucine-release pathway in GAF domain of N-CodY from Bacillus Subtilis

The GAF domain located in the N-terminal motifs of CodY (N-CodY) is responsible for increasing the affinity of CodY to its target sites on DNA by its interaction with the branched chain amino acids (BCAAs) involving isoleucine, leucine and valine. The study of the interaction of GAF domain with isoleucine gains much attention in recent years, but the mechanism of isoleucine release still remains unclear. In this paper, a conventional molecular dynamics (MD) and force probe molecular dynamics (FPMD) simulations have been performed with the aim to understand how the isoleucine ligand escapes from the GAF domain of N-CodY from Bacillus subtilis. The MD results reveal that the ligand release is a gradual process, which is accompanied by the movement of the loop between β3 and β4. During the periods of ligand escaping from the bottom to the top of binding pocket, isoleucine forms hydrogen bonds one after another with series of residues, such as ARG61, THR96, PHE98, VAL100, GLU101 and ASN102, under the mediation of hydrophobic contacts. The FPMD results show that the easiest way to pull ligand out of the cavity is along x direction (i.e. the direction is opposite to MET62).     

Structural insights into the interaction between molluscan hemocyanins and phenolic substrates: An in silico study using docking and molecular dynamics

Hemocyanin is a multimeric type-3 copper containing oxygen carrier protein that exhibits phenoloxidase-like activity and is found in selected species of arthropoda and mollusca. The phenoloxidase activity in the molluscan hemocyanins can be triggered by the proteolytic removal of the C-terminal β-rich sandwich domain of the protein or by the treatment with chemical agents like SDS, both of which enable active site access to the phenolic substrates. The mechanism by which SDS treatment enhances active site access to the substrates is however not well understood in molluscan hemocyanins. Here, using a combination of in silico molecular dynamics (MD) and docking studies on the crystal structure of Octopus dofleini hemocyanin (PDB code:1JS8), we demonstrate that the C-terminal β-domain of the protein plays a crucial role in regulating active site access to bulky phenolic substrates. Furthermore, MD simulation of hemocyanin in SDS revealed displacement of β-domain, enhanced active site access and a resulting increase in binding affinity for substrates. These observations were further validated by enzyme kinetics experiments.     

Effects of different solvents on the conformations of apoptotic cytochrome c: Structural insights from molecular dynamics simulation

Cytochrome c (cyt-c) upon binding with cardiolipin acquires peroxidase activity and is strictly connected to the pathogenesis of many human diseases including neurodegenerative and cardiovascular diseases. Interaction of cyt-c with cardiolipin mimics partial unfolding/conformational changes of cyt-c in different solvent environments. Dynamic pictures of these conformational changes of cyt-c are crucial in understanding their physiological roles in mitochondrial functions. Therefore, atomistic molecular dynamics (MD) simulations have been carried out to investigate the effect of different solvents (water, urea/water, MeOH and DMSO) on the structure and conformations of apoptotic cyt-c (Fe³⁺). Our study demonstrates that the structural changes in the protein are solvent dependent. The structural differences are observed majorly on the β-sheets and α-helical conformations and the degree of their perturbation are specific to the solvent. Although a complete loss of β-sheets (0%) is observed in MeOH and DMSO, by contrast, well preserved β-sheets (3.84%) are observed in water and urea/water. A significant decrease in the α-helical contents is observed in MeOH (41.34%) and water (42.46%), a negligible alteration in DMSO (44.25%) and well preserved α-helical (45.19%) contents in urea/water. The distances between the residues critical for electron transfer are decreased considerably for DMSO. Further, the reduction in residue flexibility and the conformational space indicate that the collective motions of cyt-c are reduced when compared to other cosolvents. Essential dynamics analysis implies that the overall motions of cyt-c in water, MeOH and urea/water are involved in three to four eigenvectors and in first eigenvector in DMSO. Overall, we believe that MD simulations of cyt-c in different solvents can provide a detailed microscopic understanding of the physiological roles, electron transport and peroxidase function in the early events of apoptosis which are hard to probe experiments.     

Studying the protein-protein interactions in the postsynaptic density by means of immunoabsorption and chemical crosslinking

Postsynaptic densities (PSDs), isolated from porcine cerebral cortices, are large disk-shaped aggregates consisting of hundreds of different proteins. To study the protein-protein interactions in such complex supramolecules, we developed a procedure to break up the PSD's overall structure, while preserving some interactions between individual proteins. Using the resulting PSD sample and an indirect immunoabsorption procedure, PSD-95 was isolated along with the α- and β-subunits of calcium calmodulin-dependent protein kinase II (CaMKIIα and CaMKIIβ), α-tubulin, β-tubulin, and Chapsyn110. Similarly, CaMKIIα was isolated along with CaMKIIβ, α-tubulin, β-tubulin, and small amounts of PSD-95. The proteins isolated from PSDs treated with a cleavable bifunctional crosslinking reagent were further subjected to diagonal gel electrophoresis analysis, and the results indicated that CaMKIIα resides next to α-tubulin in the PSD. Overall, the results obtained here suggest that within the PSD, large aggregates of CaMKIIα, CaMKIIβ, α-tubulin, and β-tubulin may occur that indirectly associate with PSD-95 and Chapsyn110. Such a protein organization would allow interactions with F-actin in the cytoplasm and with proteins, such as N-methyl-D-aspartate receptors, which reside on the postsynaptic membrane. Furthermore, it would facilitate binding to proteins such as the various microtubule-associated proteins that reside in the core region of the PSD.     

LGTBase: LARGE-like GlcNAc Transferase Database

Information technology has greatly speeded up the discovery in biomedical research. However, most of the modern biomedical scientists are not familiar with the new technology. To overcome the difficulty, an intelligent information system is needed to help scientists in designing and conducting research projects. Previously, we have designed a static knowledge management system for the LARGE-like glycosyltransferase, a novel GlcNAc transferase involved in human diseases such as muscular dystrophy and human meningioma, by integrating data from public databases. According the characteristics of protein structure of LARGE protein family members include an N-terminal cytoplasmic domain, a transmembrane region, a coiled-coil motif and a putative catalytic domain with the conserved aspartate-any residue-aspartate motif and a conserved protein structural domain. In this paper, we have described an intelligent information system for the LARGE-like GlcNAc Transferase Database, by setting up an automatic updating databank for genes of the LARGE family and by integrating several bioinformatics tools which can identify characteristics structural domains of the protein family.     

Structural insights into a high affinity nanobody:antigen complex by homology modelling

Porphyromonas gingivalis is a major periodontitis-causing pathogens. P. gingivalis secrete a cysteine protease termed RgpB, which is specific for Arg-Xaa bonds in substrates. Recently, a nanobody-based assay was used to demonstrate that RgpB could represent a novel diagnostic target, thereby simplifying. P. gingivalis detection. The nanobody, VHH7, had a high binding affinity and was specific for RgpB, when tested towards the highly identical RgpA.In this study a homology model of VHH7 was build. The complementarity determining regions (CDR) comprising the paratope residues responsible for RgpB binding were identified and used as input to the docking. Furthermore, residues likely involved in the RgpB epitope was identified based upon RgpB:RgpA alignment and analysis of residue surface accessibility. CDR residues and putitative RgpB epitope residues were used as input to an information-driven flexible docking approach using the HADDOCK server. Analysis of the VHH7:RgpB model demonstrated that the epitope was found in the immunoglobulin-like domain and residue pairs located at the molecular paratope:epitope interface important for complex stability was identified.Collectively, the VHH7 homology model and VHH7:RgpB docking supplies knowledge of the residues involved in the high affinity interaction. This information could prove valuable in the design of an antibody-drug conjugate for specific RgpB targeting.     

Insights into the folding pathway of the Engrailed Homeodomain protein using replica exchange molecular dynamics simulations

Protein folding studies were carried out by performing microsecond time scale simulations on the ultrafast/fast folding protein Engrailed Homeodomain (EnHD) from Drosophila melanogaster. It is a three-helix bundle protein consisting of 54 residues (PDB ID: 1ENH). The positions of the helices are 8-20 (Helix I), 26-36 (Helix II) and 40-53 (Helix III). The second and third helices together form a Helix-Turn-Helix (HTH) motif which belongs to the family of DNA binding proteins. The molecular dynamics (MD) simulations were performed using replica exchange molecular dynamics (REMD). REMD is a method that involves simulating a protein at different temperatures and performing exchanges at regular time intervals. These exchanges were accepted or rejected based on the Metropolis criterion. REMD was performed using the AMBER FF03 force field with the generalised Born solvation model for the temperature range 286-373 K involving 30 replicas. The extended conformation of the protein was used as the starting structure. A simulation of 600 ns per replica was performed resulting in an overall simulation time of 18 μs. The protein was seen to fold close to the native state with backbone root mean square deviation (RMSD) of 3.16 ?. In this low RMSD structure, the Helix I was partially formed with a backbone RMSD of 3.37 ? while HTH motif had an RMSD of 1.81 ?. Analysis suggests that EnHD folds to its native structure via an intermediate in which the HTH motif is formed. The secondary structure development occurs first followed by tertiary packing. The results were in good agreement with the experimental findings.     

Locally Implicit Time Integration Strategies in a Discontinuous Galerkin Method for Maxwell’s Equations

An attractive feature of discontinuous Galerkin (DG) spatial discretization is the possibility of using locally refined space grids to handle geometrical details. However, locally refined meshes lead to severe stability constraints on explicit integration methods to numerically solve a time-dependent partial differential equation. If the region of refinement is small relative to the computational domain, the time step size restriction can be overcome by blending an implicit and an explicit scheme where only the solution variables living at fine elements are treated implicitly. The downside of this approach is having to solve a linear system per time step. But due to the assumed small region of refinement relative to the computational domain, the overhead will also be small while the solution can be advanced in time with step sizes determined by the coarse elements. In this paper, we present two locally implicit time integration methods for solving the time-domain Maxwell equations spatially discretized with a DG method. Numerical experiments for two-dimensional problems illustrate the theory and the usefulness of the implicit–explicit approaches in presence of local refinements.     

Unravelling the structural interactions between PKR kinase domain and its small molecule inhibitors using computational approaches

The RNA-dependent protein kinase (PKR), an eIF2α kinase plays an important role in anti-viral response, apoptosis and cell survival. It is also implicated to play a role in several cancers, metabolic and neurodegenerative disorders. A few ATP competitive inhibitors of the PKR have been reported in the literature with promising results in vitro and in vivo. The aim of this study was to unravel the structural interactions between these inhibitors and the PKR kinase domain using molecular simulations and docking. Our study reveals that the reported inhibitors bind in the adenine pocket and form hydrogen bonds with the hinge region and vdW interactions with non-polar residues in the binding site. The most potent inhibitor has several favorable interactions with the binding site and induces the P-loop to fold inward, creating a significant hydrophobic enclosure for itself. The computed binding free energies of these inhibitors are in accord with experimental data (IC₅₀). Strategies to design potent and selective PKR inhibitors are discussed to overcome the reported promiscuity.