Rfree Values Research Articles

FDA Approved Niraparib-based Virtual Screening and Molecular Docking Studies to Discover Novel 6NRF Protein Modulator for Ovarian Cancer

The abstract summarizes the findings of a comprehensive analysis of chemical compounds, focusing on their potential therapeutic relevance in ovarian cancer treatment. Utilizing data from the ChEMBL database, pharmacophore-based screening identified compounds with promising activity against ovarian cancer, with notable catching scores ranging from 0.556 to 1.000. Crystallographic refinement metrics highlighted improvements in structural accuracy and quality following PDB-REDO refinement, with reductions in R and R-free values and significant enhancements in bond length RMS Z-scores. Molecular docking studies revealed predicted binding affinities ranging from -9.6 to -8.4, indicating strong interactions with target proteins. ADME analysis of niraparib, a lead compound, demonstrated favorable physicochemical properties and pharmacokinetic profiles, with Lipinski, Ghose, Veber, and Egan’s rules for drug-likeness and bioavailability met. Toxicity prediction models identified potential organ toxicity endpoints, with neurotoxicity, respiratory toxicity, and immunotoxicity showing active involvement. These findings underscore the complexity of structure-activity relationships and the importance of predictive models in guiding drug discovery efforts and ensuring safety profiles. Overall, this study provides valuable insights into the landscape of PARP inhibitors and their potential in advancing personalized medicine approaches for ovarian cancer therapy.

Repurposing Existing FDA-Approved Medications by Virtual Screening for Combatting Pseudomonas aeruginosa Infections

Pseudomonas aeruginosa, a notorious pathogen, poses significant challenges due to its resistance to multiple antibiotics. This study aims to identify potential FDA-approved drugs that could be repurposed to combat P. aeruginosa infections through virtual screening. The target protein, DNA gyrase B 24kDa ATPase subdomain (PDB ID: 7PTF), was refined using PDBREDO, improving geometric parameters despite a slight increase in R and R-free values. The virtual screening revealed several promising candidates with high binding affinities, including acetyldigitoxin (-9.9), digoxin (-9.5), digitoxin (-9.4), posaconazole (-9.3), venetoclax (-8.8), and itraconazole (-8.7). These top hits, primarily comprising cardiac glycosides and antifungal agents, exhibited large molecular weights, numerous hydrogen bond donors and acceptors, and significant molecular flexibility. The findings suggest potential repurposing opportunities for these drugs in treating P. aeruginosa infections, warranting further in-vitro and in-vivo investigations to validate their antimicrobial efficacy.

Deciphering the crystal structure of a novel nanobody against the NEIL1 DNA glycosylase.

Nanobodies (VHHs) are single-domain antibodies with three antigenic CDR regions and are used in diverse scientific applications. Here, an ∼14 kDa nanobody (A5) specific for the endonuclease VIII (Nei)-like 1 or NEIL1 DNA glycosylase involved in the first step of the base-excision repair pathway was crystallized and its structure was determined to 2.1 Å resolution. The crystals posed challenges due to potential twinning and anisotropic diffraction. Despite inconclusive twinning indicators, reprocessing in an orthorhombic setting and molecular replacement in space group P21212 enabled the successful modeling of 96% of residues in the asymmetric unit, with final Rwork and Rfree values of 0.199 and 0.229, respectively.

Repurposing Entacapone, a Catechol-O-Methyl Transferase Inhibitor, for Alcohol Use Disorder: A Combined Morgan Fingerprint and Docking Approach

This study investigates the potential of repurposing Entacapone, a known catechol-O-methyl transferase (COMT) inhibitor, for the treatment of Alcohol Use Disorder (AUD) using an integrated approach that combines Morgan Fingerprint analysis and molecular docking studies. Entacapone, originally approved for managing Parkinson’s disease, was identified as a candidate through ligand-based virtual screening on the DrugRep platform. The screening revealed a strong binding affinity of Entacapone to aldehyde dehydrogenase (ALDH), with a docking score of -8.3, suggesting its potential to inhibit this enzyme, similar to the mechanism of action seen with Disulfiram, a commonly used AUD medication. Molecular docking studies further highlighted key interactions between Entacapone and critical residues in ALDH, providing a structural basis for its repurposing in AUD therapy. Validation metrics from PDB-REDO showed notable improvements in model quality, with reductions in the R-free value from 0.2772 to 0.2490, and enhancements in bond angle RMS Zscores and Ramachandran plot normality, further supporting the robustness of the docking results. This study underscores the feasibility of using Entacapone as a potential therapeutic agent for AUD and highlights the utility of combining ligand-based screening with molecular docking in drug repurposing. The findings pave the way for further experimental validation and potential clinical applications for AUD treatment, especially in patients seeking alternative therapeutic options.

Insight into the structural basis of the dual inhibitory mode of Lima bean (Phaseolus lunatus) serine protease inhibitor.

Bovine pancreatic trypsin was crystallized, in-complex with Lima bean trypsin inhibitor (LBTI) (Phaseolus lunatus L.), in the form of a ternary complex. LBTI is a Bowman-Birk-type bifunctional serine protease inhibitor, which has two independent inhibitory loops. Both of the loops can inhibit trypsin, however, only the hydrophobic loop is specific for inhibiting chymotrypsin. The structure of trypsin incomplex with the LBTI has been solved and refined at 2.25 Å resolution, in the space group P41, with Rwork /Rfree values of 18.1/23.3. The two binding sites of LBTI differ in only two amino acids. Lysine and leucine are the key residues of the two different binding loops positioned at the P1, and involved in binding the S1 binding site of trypsin. The asymmetric unit cell contains two molecules of trypsin and one molecule of LBTI. The key interactions include hydrogen bonds between LBTI and active site residues of trypsin. The 3D structure of the enzyme-inhibitor complex provided details insight into the trypsin inhibition by LBTI. To the best of our knowledge, this is the first report on the structure of trypsin incomplex with LBTI.

Modeling a unit cell: crystallographic refinement procedure using the biomolecular MD simulation platform Amber.

A procedure has been developed for the refinement of crystallographic protein structures based on the biomolecular simulation program Amber. The procedure constructs a model representing a crystal unit cell, which generally contains multiple protein molecules and is fully hydrated with TIP3P water. Periodic boundary conditions are applied to the cell in order to emulate the crystal lattice. The refinement is conducted in the form of a specially designed short molecular-dynamics run controlled by the Amber ff14SB force field and the maximum-likelihood potential that encodes the structure-factor-based restraints. The new Amber-based refinement procedure has been tested on a set of 84 protein structures. In most cases, the new procedure led to appreciably lower R free values compared with those reported in the original PDB depositions or obtained by means of the industry-standard phenix.refine program. In particular, the new method has the edge in refining low-accuracy scrambled models. It has also been successful in refining a number of molecular-replacement models, including one with an r.m.s.d. of 2.15 Å. In addition, Amber-refined structures consistently show superior MolProbity scores. The new approach offers a highly realistic representation of protein-protein interactions in the crystal, as well as of protein-water interactions. It also offers a realistic representation of protein crystal dynamics (akin to ensemble-refinement schemes). Importantly, the method fully utilizes the information from the available diffraction data, while relying on state-of-the-art molecular-dynamics modeling to assist with those elements of the structure that do not diffract well (for example mobile loops or side chains). Finally, it should be noted that the protocol employs no tunable parameters, and the calculations can be conducted in a matter of several hours on desktop computers equipped with graphical processing units or using a designated web service.

Exploring ligand dynamics in protein crystal structures with ensemble refinement.

Understanding the dynamics of ligands bound to proteins is an important task in medicinal chemistry and drug design. However, the dominant technique for determining protein-ligand structures, X-ray crystallography, does not fully account for dynamics and cannot accurately describe the movements of ligands in protein binding sites. In this article, an alternative method, ensemble refinement, is used on six protein-ligand complexes with the aim of understanding the conformational diversity of ligands in protein crystal structures. The results show that ensemble refinement sometimes indicates that the flexibility of parts of the ligand and some protein side chains is larger than that which can be described by a single conformation and atomic displacement parameters. However, since the electron-density maps are comparable and Rfree values are slightly increased, the original crystal structure is still a better model from a statistical point of view. On the other hand, it is shown that molecular-dynamics simulations and automatic generation of alternative conformations in crystallographic refinement confirm that the flexibility of these groups is larger than is observed in standard refinement. Moreover, the flexible groups in ensemble refinement coincide with groups that give high atomic displacement parameters or non-unity occupancy if optimized in standard refinement. Therefore, the conformational diversity indicated by ensemble refinement seems to be qualitatively correct, indicating that ensemble refinement can be an important complement to standard crystallographic refinement as a tool to discover which parts of crystal structures may show extensive flexibility and therefore are poorly described by a single conformation. However, the diversity of the ensembles is often exaggerated (probably partly owing to the rather poor force field employed) and the ensembles should not be trusted in detail.

XRRpred: accurate predictor of crystal structure quality from protein sequence.

X-ray crystallography was used to produce nearly 90% of protein structures. These efforts were supported by numerous sequence-based tools that accurately predict crystallizable proteins. However, protein structures vary widely in their quality, typically measured with resolution and R-free. This impacts the ability to use these structures for some applications including rational drug design and molecular docking and motivates development of methods that accurately predict structure quality from sequence. We introduce XRRpred, the first predictor of the resolution and R-free values from protein sequences. XRRpred relies on original sequence profiles, hand-crafted features, empirically selected and parametrized regressors and modern resampling techniques. Using an independent test dataset, we show that XRRpred provides accurate predictions of resolution and R-free. We demonstrate that XRRpred's predictions correctly model relationship between the resolution and R-free and reproduce structure quality relations between structural classes of proteins. We also show that XRRpred significantly outperforms indirect alternative ways to predict the structure quality that include predictors of crystallization propensity and an alignment-based approach. XRRpred is available as a convenient webserver that allows batch predictions and offers informative visualization of the results. http://biomine.cs.vcu.edu/servers/XRRPred/. Supplementary data are available at Bioinformatics online.

Crystal structure and characterization of nucleoside diphosphate kinase from Vibrio cholerae

Nucleoside diphosphate kinases (NDK) are ubiquitous enzymes that catalyse the transfer of the γ phosphate from nucleoside triphosphates (NTPs) to nucleoside diphosphate (NDPs), to maintain appropriate NTP levels in cells. NDKs are associated with signal transduction, cell development, proliferation, differentiation, tumor metastasis, apoptosis and motility. The critical role of NDK in bacterial virulence renders it a potential drug target. The present manuscript reports crystal structure and functional characterization of Vibrio cholerae NDK (VNDK). The 16 kDa VNDK was crystallized in a solution containing 30% PEG 4000, 100 mM Tris-HCl pH 8.5 and 200 mM sodium acetate in orthorhombic space group P212121 with unit cell parameters a = 48.37, b = 71.21, c = 89.14 Å, α = β = γ = 90° with 2 molecules in asymmetric unit. The crystal structure was solved by molecular replacement and refined to crystallographic Rfactor and Rfree values of 22.8% and 25.8% respectively. VNDK exists as both dimer and tetramer in solution as confirmed by size exclusion chromatography, glutaraldehyde crosslinking and small angle X-ray scattering while the crystal structure appears to be a dimer. The biophysical characterization states that VNDK has kinase and DNase activity with maximum stability at pH 8–9 and temperature up to 40 °C. VNDK shows elevated thermolability as compared to other NDK and shows preferential binding with GTP rationalized using computational studies.

Crystal Structure of Peptidyl-tRNA Hydrolase from <i>Acinetobacter baumannii</i> at 1.00 Å Resolution

The essential process of protein biosynthesis in the cell often gets stalled due to the premature abortion of the translation process and generates a byproduct of peptidyl-tRNA molecules. This defect is corrected by peptidyl-tRNA hydrolase (Pth) by hydrolyzing peptidyl-tRNA to yield tRNA and peptides. In order to understand the mechanism of catalytic action and detailed stereochemical features of the substrate binding site, the structure of Pth has been determined at 1.00 A resolution. The Pth enzyme from Acinetobacter baumannii (AbPth) was cloned, expressed, purified and crystallized. The structure was refined to Rcryst and Rfree values of 0.145 and 0.157 respectively. The electron densities were observed for many hydrogen atoms in the structure. In AbPth, the residues, Asn12, His22, Asn70, Asp95 and Asn116 are involved in the catalytic process. The structure determination revealed that His22 Nᵟ1 forms a hydrogen bond with Asp95 Oᵟ2 while His22 Ne2 is hydrogen bonded to Asn116 Nᵟ2. In this case, the side chain of Asn116 adopts a conformation with  value of 65°. Upon ligand binding, Asn116 adopts a different conformation with  value of -70⁰. In the present structure, the conformation of Tyr68 is observed in the disallowed region of Ramachandran’s plot with φ, ѱ values of 80⁰, 150⁰. However, it is observed that Tyr68 adopts both disallowed and allowed conformations in Pth enzymes indicating a structural flexibility. The structure determination also revealed multiple conformations of the side chains of a number of amino acid residues.

Structures of the SARS-CoV-2 nucleocapsid and their perspectives for drug design.

COVID-19, caused by SARS-CoV-2, has resulted in severe and unprecedented economic and social disruptions in the world. Nucleocapsid (N) protein, which is the major structural component of the virion and is involved in viral replication, assembly and immune regulation, plays key roles in the viral life cycle. Here, we solved the crystal structures of the N- and C-terminal domains (N-NTD and N-CTD) of SARS-CoV-2 N protein, at 1.8 and 1.5Å resolution, respectively. Both structures show conserved features from other CoV N proteins. The binding sites targeted by small molecules against HCoV-OC43 and MERS-CoV, which inhibit viral infection by blocking the RNA-binding activity or normal oligomerization of N protein, are relatively conserved in our structure, indicating N protein is a promising drug target. In addition, certain areas of N-NTD and N-CTD display distinct charge distribution patterns in SARS-CoV-2, which may alter the RNA-binding modes. The specific antigenic characteristics are critical for developing specific immune-based rapid diagnostic tests. Our structural information can aid in the discovery and development of antiviral inhibitors against SARS-CoV-2 in the future.

Local structural plasticity of the Staphylococcus aureus evasion protein EapH1 enables engagement with multiple neutrophil serine proteases

Members of the EAP family of Staphylococcus aureus immune evasion proteins potently inhibit the neutrophil serine proteases (NSPs) neutrophil elastase, cathepsin-G, and proteinase-3. Previously, we determined a 1.8 Å resolution crystal structure of the EAP family member EapH1 bound to neutrophil elastase. This structure revealed that EapH1 blocks access to the enzyme's active site by forming a noncovalent complex with this host protease. To determine how EapH1 inhibits other NSPs, we studied here the effects of EapH1 on cathepsin-G. We found that EapH1 inhibits cathepsin-G with a Ki of 9.8 ± 4.7 nm Although this Ki value is ∼466-fold weaker than the Ki for EapH1 inhibition of neutrophil elastase, the time dependence of inhibition was maintained. To define the physical basis for EapH1's inhibition of cathepsin-G, we crystallized EapH1 bound to this protease, solved the structure at 1.6 Å resolution, and refined the model to Rwork and Rfree values of 17.4% and 20.9%, respectively. This structure revealed a protease-binding mode for EapH1 with cathepsin-G that was globally similar to that seen in the previously determined EapH1-neutrophil elastase structure. The nature of the intermolecular interactions formed by EapH1 with cathepsin-G differed considerably from that with neutrophil elastase, however, with far greater contributions from the inhibitor backbone in the cathepsin-G-bound form. Together, these results reveal that EapH1's ability to form high-affinity interactions with multiple NSP targets is due to its remarkable level of local structural plasticity.

Using cryo-electron microscopy maps for X-ray structure determination of homologues.

The combination of cryo-electron microscopy (cryo-EM) and X-ray crystallography reflects an important trend in structural biology. In a previously published study, a hybrid method for the determination of X-ray structures using initial phases provided by the corresponding parts of cryo-EM maps was presented. However, if the target structure of X-ray crystallography is not identical but homologous to the corresponding molecular model of the cryo-EM map, then the decrease in the accuracy of the starting phases makes the whole process more difficult. Here, a modified hybrid method is presented to handle such cases. The whole process includes three steps: cryo-EM map replacement, phase extension by NCS averaging and dual-space iterative model building. When the resolution gap between the cryo-EM and X-ray crystallographic data is large and the sequence identity is low, an intermediate stage of model building is necessary. Six test cases have been studied with sequence identity between the corresponding molecules in the cryo-EM and X-ray structures ranging from 34 to 52% and with sequence similarity ranging from 86 to 91%. This hybrid method consistently produced models with reasonable Rwork and Rfree values which agree well with the previously determined X-ray structures for all test cases, thus indicating the general applicability of the method for X-ray structure determination of homologues using cryo-EM maps as a starting point.

Noncovalent structure of SENP1 in complex with SUMO2.

SUMOylation is a post-translational modification in which a small ubiquitin-like molecule (SUMO) is appended to substrate proteins and is known to influence myriads of biological processes. A delicate interplay between several families of SUMOylation proteins and their substrates ensures the proper level of SUMOylation required for normal cell function. Among the SUMO proteins, SUMO2 is known to form mono-SUMOylated proteins and engage in poly-SUMO chain formation, while sentrin-specific protease 1 (SENP1) is a key enzyme in regulating both events. Determination of the SENP1-SUMO2 interaction is therefore necessary to better understand SUMOylation. In this regard, the current paper reports the noncovalent structure of SENP1 in complex with SUMO2, which was refined to a resolution of 2.62 Å with R and Rfree values of 22.92% and 27.66%, respectively. The structure shows that SENP1-SUMO2 complex formation is driven largely by polar interactions and limited hydrophobic contacts. The essential C-terminal motif (QQTGG) of SUMO2 is stabilized by a number of specific bonding interactions that enable it to protrude into the catalytic triad of SENP1 and provide the arrangement necessary for maturation of SUMO and deSUMOylation activity. Overall, the structure shows a number of structural details that pinpoint the basis of SENP1-SUMO2 complex formation.

Evaluation of models determined by neutron diffraction and proposed improvements to their validation and deposition.

The Protein Data Bank (PDB) contains a growing number of models that have been determined using neutron diffraction or a hybrid method that combines X-ray and neutron diffraction. The advantage of neutron diffraction experiments is that the positions of all atoms can be determined, including H atoms, which are hardly detectable by X-ray diffraction. This allows the determination of protonation states and the assignment of H atoms to water molecules. Because neutrons are scattered differently by hydrogen and its isotope deuterium, neutron diffraction in combination with H/D exchange can provide information on accessibility, dynamics and chemical lability. In this study, the deposited data, models and model-to-data fit for all PDB entries that used neutron diffraction as the source of experimental data have been analysed. In many cases, the reported Rwork and Rfree values were not reproducible. In such cases, the model and data files were analysed to identify the reasons for this mismatch. The issues responsible for the discrepancies are summarized and explained. The analysis unveiled limitations to the annotation, deposition and validation of models and data, and a lack of community-wide accepted standards for the description of neutron models and data, as well as deficiencies in current model refinement tools. Most of the issues identified concern the handling of H atoms. Since the primary use of neutron macromolecular crystallography is to locate and directly visualize H atoms, it is important to address these issues, so that the deposited neutron models allow the retrieval of the maximum amount of information with the smallest effort of manual intervention. A path forward to improving the annotation, validation and deposition of neutron models and hybrid X-ray and neutron models is suggested.

Discovery of novel inhibitors for Leishmania nucleoside diphosphatase kinase (NDK) based on its structural and functional characterization.

Nucleoside diphosphate kinases (NDKs) are ubiquitous enzymes that catalyze the transfer of the γ-phosphate moiety from an NTP donor to an NDP acceptor, crucial for maintaining the cellular level of nucleoside triphosphates (NTPs). The inability of trypanosomatids to synthesize purines de novo and their dependence on the salvage pathway makes NDK an attractive target to develop drugs for the diseases they cause. Here we report the discovery of novel inhibitors for Leishmania NDK based on the structural and functional characterization of purified recombinant NDK from Leishmania amazonensis. Recombinant LaNDK possesses auto-phosphorylation, phosphotransferase and kinase activities with Histidine 117 playing an essential role. LaNDK crystals were grown by hanging drop vapour diffusion method in a solution containing 18% PEG-MME 500, 100mM Bis-Tris propane pH 6.0 and 50mM MgCl2. It belongs to the hexagonal space group P6322 with unit cell parameters a = b = 115.18, c = 62.18Å and α = β = 90°, γ = 120°. The structure solved by molecular replacement methods was refined to crystallographic R-factor and Rfree values of 22.54 and 26.52%, respectively. Molecular docking and dynamics simulation-based virtual screening identified putative binding compounds. Protein inhibition studies of selected hits identified five inhibitors effective at micromolar concentrations. One of the compounds showed ~45% inhibition of Leishmania promastigotes proliferation. Analysis of inhibitor-NDK complexes reveals the mode of their binding, facilitating design of new compounds for optimization of activities as drugs against leishmaniasis.

Polarizable Amoeba Force Field Metadynamics with Minimization Predicts Missing Protein Loops

Computational methods developed to find the global free energy minimum of amino acid sequences are increasingly successful, but limitations in both accuracy and efficiency remain. Optimization algorithms are typically focused on proteins of modest size (i.e. of approximately 100 residues) and utilize potential energy functions based on fixed charged force fields, statistical or knowledge based potentials, and/or potentials incorporating experimental data. Although the aforementioned methods are widely used, known limitations include 1) search protocols that are inefficient or not deterministic due to rough energy landscapes characterized by large energy barriers between multiple minima and 2) use of a target function whose global minimum does not correspond to the actual free energy minimum. To overcome the first limitation, this work describes a global optimization approach based on metadynamics to drive the search of conformational space toward unexplored regions by adding a time-dependent bias to the objective function. To overcome the second limitation, a hybrid objective function is defined as the sum of the polarizable AMOEBA polarizable force field and an experimental X-ray crystallography target. As metadynamics drives the search, periodic quenching via local minimization is used to access structure quality via evaluation of Rwork. Thus, the overall method is called AMOEBA Metadynamics with Minimization (AMM), and is suitable for optimization of side-chains, ligand binding poses, protein loops or even protein complexes. Here we focus on characterizing the ability of AMM to elucidate the structural details of missing protein loops, which are often excluded from experimental X-ray crystallography structures due to conformational heterogeneity and/or limitations in the resolution of the data. We first show that the correlation between experimental data and AMOEBA structural minima is stronger than that for OPLS-AA/L (i.e. a fixed charge force field). Next, missing protein loops are optimized using 5 nsec of sampling for both AMM and simulated annealing with OPLS-AA/L. The AMM procedure provides more accurate structures in terms of both experimental (i.e. lower Rfree values) and structural metrics (i.e. MolProbity). In addition to providing more accurate loop conformations, AMM converged faster than the simulated annealing protocol. Overall, this work suggests that AMM is well-suited to refine or predict the coordinates of missing amino acid residues and/or protein loops due to both the increased accuracy of the target function relative to OPLS-AA/L and more rapid convergence of the metadynamics driven search compared to simulation annealing.

Production in Pichia pastoris, antifungal activity and crystal structure of a class I chitinase from cowpea (Vigna unguiculata): Insights into sugar binding mode and hydrolytic action

A cowpea class I chitinase (VuChiI) was expressed in the methylotrophic yeast P. pastoris. The recombinant protein was secreted into the culture medium and purified by affinity chromatography on a chitin matrix. The purified chitinase migrated on SDS-polyacrylamide gel electrophoresis as two closely-related bands with apparent molecular masses of 34 and 37 kDa. The identity of these bands as VuChiI was demonstrated by mass spectrometry analysis of tryptic peptides and N-terminal amino acid sequencing. The recombinant chitinase was able to hydrolyze colloidal chitin but did not exhibit enzymatic activity toward synthetic substrates. The highest hydrolytic activity of the cowpea chitinase toward colloidal chitin was observed at pH 5.0. Furthermore, most VuChiI activity (approximately 92%) was retained after heating to 50 °C for 30 min, whereas treatment with 5 mM Cu2+ caused a reduction of 67% in the enzyme's chitinolytic activity. The recombinant protein had antifungal activity as revealed by its ability to inhibit the spore germination and mycelial growth of Penicillium herquei. The three-dimensional structure of VuChiI was resolved at a resolution of 1.55 Å by molecular replacement. The refined model had 245 amino acid residues and 381 water molecules, and the final R-factor and Rfree values were 14.78 and 17.22%, respectively. The catalytic domain of VuChiI adopts an α-helix-rich fold, stabilized by 3 disulfide bridges and possessing a wide catalytic cleft. Analysis of the crystallographic model and molecular docking calculations using chito-oligosaccharides provided evidences about the VuChiI residues involved in sugar binding and catalysis, and a possible mechanism of antifungal action is suggested.

Dead-End Elimination with a Polarizable Force Field Repacks PCNA Structures

A balance of van der Waals, electrostatic, and hydrophobic forces drive the folding and packing of protein side chains. Although such interactions between residues are often approximated as being pairwise additive, in reality, higher-order many-body contributions that depend on environment drive hydrophobic collapse and cooperative electrostatics. Beginning from dead-end elimination, we derive the first algorithm, to our knowledge, capable of deterministic global repacking of side chains compatible with many-body energy functions. The approach is applied to seven PCNA x-ray crystallographic data sets with resolutions 2.5–3.8 Å (mean 3.0 Å) using an open-source software. While PDB_REDO models average an Rfree value of 29.5% and MOLPROBITY score of 2.71 Å (77th percentile), dead-end elimination with the polarizable AMOEBA force field lowered Rfree by 2.8–26.7% and improved mean MOLPROBITY score to atomic resolution at 1.25 Å (100th percentile). For structural biology applications that depend on side-chain repacking, including x-ray refinement, homology modeling, and protein design, the accuracy limitations of pairwise additivity can now be eliminated via polarizable or quantum mechanical potentials.

A Universal Stress Protein (USP) in Mycobacteria Binds cAMP

Mycobacteria are endowed with rich and diverse machinery for the synthesis, utilization, and degradation of cAMP. The actions of cyclic nucleotides are generally mediated by binding of cAMP to conserved and well characterized cyclic nucleotide binding domains or structurally distinct cGMP-specific and -regulated cyclic nucleotide phosphodiesterase, adenylyl cyclase, and E. coli transcription factor FhlA (GAF) domain-containing proteins. Proteins with cyclic nucleotide binding and GAF domains can be identified in the genome of mycobacterial species, and some of them have been characterized. Here, we show that a significant fraction of intracellular cAMP is bound to protein in mycobacterial species, and by using affinity chromatography techniques, we identify specific universal stress proteins (USP) as abundantly expressed cAMP-binding proteins in slow growing as well as fast growing mycobacteria. We have characterized the biochemical and thermodynamic parameters for binding of cAMP, and we show that these USPs bind cAMP with a higher affinity than ATP, an established ligand for other USPs. We determined the structure of the USP MSMEG_3811 bound to cAMP, and we confirmed through structure-guided mutagenesis, the residues important for cAMP binding. This family of USPs is conserved in all mycobacteria, and we suggest that they serve as "sinks" for cAMP, making this second messenger available for downstream effectors as and when ATP levels are altered in the cell.