Crystal Polymorph Search in the NPT Ensemble via a Deposition/Sublimation Alchemical Path.

The formulation of active pharmaceutical ingredients involves discovering stable crystal packing arrangements or polymorphs, each of which has distinct pharmaceutically relevant properties. Traditional experimental screening techniques utilizing various conditions are commonly supplemented with in silico crystal structure prediction (CSP) to inform the crystallization process and mitigate risk. Predictions are often based on advanced classical force fields or quantum mechanical calculations that model the crystal potential energy landscape but do not fully incorporate temperature, pressure, or solution conditions during the search procedure. This study proposes an innovative alchemical path that utilizes an advanced polarizable atomic multipole force field to predict crystal structures based on direct sampling of the NPT ensemble. The use of alchemical (i.e., nonphysical) intermediates, a novel Monte Carlo barostat, and an orthogonal space tempering bias combine to enhance the sampling efficiency of the deposition/sublimation phase transition. The proposed algorithm was applied to 2-((4-(2-(3,4-dichlorophenyl)ethyl)phenyl)amino)benzoic acid (Cambridge Crystallography Database Centre ID: XAFPAY) as a case study to showcase the algorithm. Each experimentally determined polymorph with one molecule in the asymmetric unit was successfully reproduced via approximately 1000 short 1 ns simulations per space group where each simulation was initiated from random rigid body coordinates and unit cell parameters. Utilizing two threads of a recent Intel CPU (a Xeon Gold 6330 CPU at 2.00 GHz), 1 ns of sampling using the polarizable AMOEBA force field can be acquired in 4 h (equating to more than 300 ns/day using all 112 threads/56 cores of a dual CPU node) within the Force Field X software (https://ffx.biochem.uiowa.edu). These results demonstrate a step forward in the rigorous use of the NPT ensemble during the CSP search process and open the door to future algorithms that incorporate solution conditions using continuum solvation methods.

Constant-pH Simulations with the Polarizable Atomic Multipole AMOEBA Force Field.

Accurately predicting protein behavior across diverse pH environments remains a significant challenge in biomolecular simulations. Existing constant-pH molecular dynamics (CpHMD) algorithms are limited to fixed-charge force fields, hindering their application to biomolecular systems described by permanent atomic multipoles or induced dipoles. This work overcomes these limitations by introducing the first polarizable CpHMD algorithm in the context of the Atomic Multipole Optimized Energetics for Biomolecular Applications (AMOEBA) force field. Additionally, our implementation in the open-source Force Field X (FFX) software has the unique ability to handle titration state changes for crystalline systems including flexible support for all 230 space groups. The evaluation of constant-pH molecular dynamics (CpHMD) with the AMOEBA force field was performed on 11 crystalline peptide systems that span the titrating amino acids (Asp, Glu, His, Lys, and Cys). Titration states were correctly predicted for 15 out of the 16 amino acids present in the 11 systems, including for the coordination of Zn2+ by cysteines. The lone exception was for a HIS-ALA peptide where CpHMD predicted both neutral histidine tautomers to be equally populated, whereas the experimental model did not consider multiple conformers and diffraction data are unavailable for rerefinement. This work demonstrates the promise polarizable CpHMD simulations for pKa predictions, the study of biochemical mechanisms such as the catalytic triad of proteases, and for improved protein-ligand binding affinity accuracy in the context of pharmaceutical lead optimization.

Endothelial lipase variant T111I does not alter inhibition by angiopoietin-like proteins.

High levels of HDL-C are correlated with a decreased risk of cardiovascular disease. HDL-C levels are modulated in part by the secreted phospholipase, endothelial lipase (EL), which hydrolyzes the phospholipids of HDL and decreases circulating HDL-C concentrations. A 584C/T polymorphism in LIPG, the gene which encodes EL, was first identified in individuals with increased HDL levels. This polymorphism results in a T111I point mutation the EL protein. The association between this variant, HDL levels, and the risk of coronary artery disease (CAD) in humans has been extensively studied, but the findings have been inconsistent. In this study, we took a biochemical approach, investigating how the T111I variant affected EL activity, structure, and stability. Moreover, we tested whether the T111I variant altered the inhibition of phospholipase activity by angiopoietin-like 3 (ANGPTL3) and angiopoietin-like 4 (ANGPTL4), two known EL inhibitors. We found that neither the stability nor enzymatic activity of EL was altered by the T111I variant. Moreover, we found no difference between wild-type and T111I EL in their ability to be inhibited by ANGPTL proteins. These data suggest that any effect this variant may have on HDL-C levels or cardiovascular disease are not mediated through alterations in these functions.

Germline landscape of RPA1, RPA2 and RPA3 variants in pediatric malignancies: identification of RPA1 as a novel cancer predisposition candidate gene.

Replication Protein A (RPA) is single-strand DNA binding protein that plays a key role in the replication and repair of DNA. RPA is a heterotrimer made of 3 subunits - RPA1, RPA2, and RPA3. Germline pathogenic variants affecting RPA1 were recently described in patients with Telomere Biology Disorders (TBD), also known as dyskeratosis congenita or short telomere syndrome. Premature telomere shortening is a hallmark of TBD and results in bone marrow failure and predisposition to hematologic malignancies. Building on the finding that somatic mutations in RPA subunit genes occur in ~1% of cancers, we hypothesized that germline RPA alterations might be enriched in human cancers. Because germline RPA1 mutations are linked to early onset TBD with predisposition to myelodysplastic syndromes, we interrogated pediatric cancer cohorts to define the prevalence and spectrum of rare/novel and putative damaging germline RPA1, RPA2, and RPA3 variants. In this study of 5,993 children with cancer, 75 (1.25%) harbored heterozygous rare (non-cancer population allele frequency (AF) < 0.1%) variants in the RPA heterotrimer genes, of which 51 cases (0.85%) had ultra-rare (AF < 0.005%) or novel variants. Compared with Genome Aggregation Database (gnomAD) non-cancer controls, there was significant enrichment of ultra-rare and novel RPA1, but not RPA2 or RPA3, germline variants in our cohort (adjusted p-value < 0.05). Taken together, these findings suggest that germline putative damaging variants affecting RPA1 are found in excess in children with cancer, warranting further investigation into the functional role of these variants in oncogenesis.

Endothelial Lipase Variant, T111I, Does Not Alter Inhibition by Angiopoietin-like Proteins.

High levels of HDL-C are correlated with a decreased risk of cardiovascular disease. HDL-C levels are modulated in part by the secreted phospholipase, endothelial lipase (EL), which hydrolyzes the phospholipids of HDL and decreases circulating HDL-C concentrations. A 584C/T polymorphism in LIPG, the gene which encodes EL, was first identified in individuals with increased HDL levels. This polymorphism results in a T111I point mutation the EL protein. The association between this variant, HDL levels, and the risk of coronary artery disease (CAD) in humans has been extensively studied, but the findings have been inconsistent. In this study, we took a biochemical approach, investigating how the T111I variant affected EL activity, structure, and stability. Moreover, we tested whether the T111I variant altered the inhibition of phospholipase activity by angiopoietin-like 3 (ANGPTL3) and angiopoietin-like 4 (ANGPTL4), two known EL inhibitors. We found that neither the stability nor enzymatic activity of EL was altered by the T111I variant. Moreover, we found no difference between wild-type and T111I EL in their ability to be inhibited by ANGPTL proteins. These data suggest that any effect this variant may have on HDL-C levels or cardiovascular disease are not mediated through alterations in these functions.

GJA3 Genetic Variation and Autosomal Dominant Congenital Cataracts and Glaucoma Following Cataract Surgery.

Importance: The p.Asp67Tyr genetic variant in the GJA3 gene is responsible for congenital cataracts in a family with a high incidence of glaucoma following cataract surgery. Objective: To describe the clinical features of a family with a strong association between congenital cataracts and glaucoma following cataract surgery secondary to a genetic variant in the GJA3 gene (NM_021954.4:c.199G>T, p.Asp67Tyr). Design, Setting, and Participants: This was a retrospective, observational, case series, genetic association study from the University of Iowa spanning 61 years. Examined were the ophthalmic records from 1961 through 2022 of the family members of a 4-generation pedigree with autosomal dominant congenital cataracts. Main Outcomes and Measures: Frequency of glaucoma following cataract surgery and postoperative complications among family members with congenital cataract due to the p.Asp67Tyr GJA3 genetic variant. Results: Medical records were available from 11 of 12 family members (7 male [63.6%]) with congenital cataract with a mean (SD) follow-up of 30 (21.7) years (range, 0.2-61 years). Eight of 9 patients with congenital cataracts developed glaucoma, and 8 of 8 patients who had cataract surgery at age 2 years or younger developed glaucoma following cataract surgery. The only family member with congenital cataracts who did not develop glaucoma had delayed cataract surgery until 12 and 21 years of age. Five of 11 family members (45.5%) had retinal detachments after cataract extraction and vitrectomy. No patients developed retinal detachments after prophylactic 360-degree endolaser. Conclusions and Relevance: The GJA3 genetic variant, p.Asp67Tyr, was identified in a 4-generation congenital cataract pedigree from Iowa. This report suggests that patients with congenital cataract due to some GJA3 genetic variants may be at especially high risk for glaucoma following cataract surgery. Retinal detachments after cataract extraction in the first 2 years of life were also common in this family, and prophylactic retinal endolaser may be indicated at the time of surgery.

A generalized Kirkwood implicit solvent for the polarizable AMOEBA protein model.

Computational simulation of biomolecules can provide important insights into protein design, protein-ligand binding interactions, and ab initio biomolecular folding, among other applications. Accurate treatment of the solvent environment is essential in such applications, but the use of explicit solvents can add considerable cost. Implicit treatment of solvent effects using a dielectric continuum model is an attractive alternative to explicit solvation since it is able to describe solvation effects without the inclusion of solvent degrees of freedom. Previously, we described the development and parameterization of implicit solvent models for small molecules. Here, we extend the parameterization of the generalized Kirkwood (GK) implicit solvent model for use with biomolecules described by the AMOEBA force field via the addition of corrections to the calculation of effective radii that account for interstitial spaces that arise within biomolecules. These include element-specific pairwise descreening scale factors, a short-range neck contribution to describe the solvent-excluded space between pairs of nearby atoms, and finally tanh-based rescaling of the overall descreening integral. We then apply the AMOEBA/GK implicit solvent to a set of ten proteins and achieve an average coordinate root mean square deviation for the experimental structures of 2.0 Å across 500 ns simulations. Overall, the continued development of implicit solvent models will help facilitate the simulation of biomolecules on mechanistically relevant timescales.

Assessing variants of uncertain significance implicated in hearing loss using a comprehensive deafness proteome.

Hearing loss is the leading sensory deficit, affecting ~ 5% of the population. It exhibits remarkable heterogeneity across 223 genes with 6328 pathogenic missense variants, making deafness-specific expertise a prerequisite for ascribing phenotypic consequences to genetic variants. Deafness-implicated variants are curated in the Deafness Variation Database (DVD) after classification by a genetic hearing loss expert panel and thorough informatics pipeline. However, seventy percent of the 128,167 missense variants in the DVD are "variants of uncertain significance" (VUS) due to insufficient evidence for classification. Here, we use the deep learning protein prediction algorithm, AlphaFold2, to curate structures for all DVD genes. We refine these structures with global optimization and the AMOEBA force field and use DDGun3D to predict folding free energy differences (∆∆GFold) for all DVD missense variants. We find that 5772 VUSs have a large, destabilizing ∆∆GFold that is consistent with pathogenic variants. When also filtered for CADD scores (> 25.7), we determine 3456 VUSs are likely pathogenic at a probability of 99.0%. Of the 224 genes in the DVD, 166 genes (74%) exhibit one or more missense variants predicted to cause a pathogenic change in protein folding stability. The VUSs prioritized here affect 119 patients (~ 3% of cases) sequenced by the OtoSCOPE targeted panel. Approximately half of these patients previously received an inconclusive report, and reclassification of these VUSs as pathogenic provides a new genetic diagnosis for six patients.

Assessing Variants of Uncertain Significance Implicated in Hearing Loss Using a Comprehensive Deafness Proteome.

Hearing loss is the leading sensory deficit, affecting ~ 5% of the population. It exhibits remarkable heterogeneity across 223 genes with 6,328 pathogenic missense variants, making deafness-specific expertise a prerequisite for ascribing phenotypic consequences to genetic variants. Deafness-implicated variants are curated in the Deafness Variation Database (DVD) after classification by a genetic hearing loss expert panel and thorough informatics pipeline. However, seventy percent of the 128,167 missense variants in the DVD are "variants of uncertain significance" (VUS) due to insufficient evidence for classification. Here, we use the deep learning protein prediction algorithm, AlphaFold2, to curate structures for all DVD genes. We refine these structures with global optimization and the AMOEBA force field and use DDGun3D to predict folding free energy differences (∆∆G Fold ) for all DVD missense variants. We find that 5,772 VUSs have a large, destabilizing ∆∆G Fold that is consistent with pathogenic variants. When also filtered for CADD scores (> 25.7), we determine 3,456 VUSs are likely pathogenic at a probability of 99.0%. These VUSs affect 119 patients (~ 3% of cases) sequenced by the OtoSCOPE targeted panel. Approximately half of these patients previously received an inconclusive report, and reclassification of these VUSs as pathogenic provides a new genetic diagnosis for six patients.

Progressive alignment of crystals: reproducible and efficient assessment of crystal structure similarity.

During in silico crystal structure prediction of organic molecules, millions of candidate structures are often generated. These candidates must be compared to remove duplicates prior to further analysis (e.g. optimization with electronic structure methods) and ultimately compared with structures determined experimentally. The agreement of predicted and experimental structures forms the basis of evaluating the results from the Cambridge Crystallographic Data Centre (CCDC) blind assessment of crystal structure prediction, which further motivates the pursuit of rigorous alignments. Evaluating crystal structure packings using coordinate root-mean-square deviation (RMSD) for N molecules (or N asymmetric units) in a reproducible manner requires metrics to describe the shape of the compared molecular clusters to account for alternative approaches used to prioritize selection of molecules. Described here is a flexible algorithm called Progressive Alignment of Crystals (PAC) to evaluate crystal packing similarity using coordinate RMSD and introducing the radius of gyration (R g) as a metric to quantify the shape of the superimposed clusters. It is shown that the absence of metrics to describe cluster shape adds ambiguity to the results of the CCDC blind assessments because it is not possible to determine whether the superposition algorithm has prioritized tightly packed molecular clusters (i.e. to minimize R g) or prioritized reduced RMSD (i.e. via possibly elongated clusters with relatively larger R g). For example, it is shown that when the PAC algorithm described here uses single linkage to prioritize molecules for inclusion in the superimposed clusters, the results are nearly identical to those calculated by the widely used program COMPACK. However, the lower R g values obtained by the use of average linkage are favored for molecule prioritization because the resulting RMSDs more equally reflect the importance of packing along each dimension. It is shown that the PAC algorithm is faster than COMPACK when using a single process and its utility for biomolecular crystals is demonstrated. Finally, parallel scaling up to 64 processes in the open-source code Force Field X is presented.

Whole-genome sequencing reveals de-novo mutations associated with nonsyndromic cleft lip/palate.

The majority (85%) of nonsyndromic cleft lip with or without cleft palate (nsCL/P) cases occur sporadically, suggesting a role for de novo mutations (DNMs) in the etiology of nsCL/P. To identify high impact protein-altering DNMs that contribute to the risk of nsCL/P, we conducted whole-genome sequencing (WGS) analyses in 130 African case-parent trios (affected probands and unaffected parents). We identified 162 high confidence protein-altering DNMs some of which are based on available evidence, contribute to the risk of nsCL/P. These include novel protein-truncating DNMs in the ACTL6A, ARHGAP10, MINK1, TMEM5 and TTN genes; as well as missense variants in ACAN, DHRS3, DLX6, EPHB2, FKBP10, KMT2D, RECQL4, SEMA3C, SEMA4D, SHH, TP63, and TULP4. Many of these protein-altering DNMs were predicted to be pathogenic. Analysis using mouse transcriptomics data showed that some of these genes are expressed during the development of primary and secondary palate. Gene-set enrichment analysis of the protein-altering DNMs identified palatal development and neural crest migration among the few processes that were significantly enriched. These processes are directly involved in the etiopathogenesis of clefting. The analysis of the coding sequence in the WGS data provides more evidence of the opportunity for novel findings in the African genome.

In Silico and In Vivo Analysis of Amino Acid Substitutions That Cause Laminopathies.

Mutations in the LMNA gene cause diseases called laminopathies. LMNA encodes lamins A and C, intermediate filaments with multiple roles at the nuclear envelope. LMNA mutations are frequently single base changes that cause diverse disease phenotypes affecting muscles, nerves, and fat. Disease-associated amino acid substitutions were mapped in silico onto three-dimensional structures of lamin A/C, revealing no apparent genotype-phenotype connections. In silico analyses revealed that seven of nine predicted partner protein binding pockets in the Ig-like fold domain correspond to sites of disease-associated amino acid substitutions. Different amino acid substitutions at the same position within lamin A/C cause distinct diseases, raising the question of whether the nature of the amino acid replacement or genetic background differences contribute to disease phenotypes. Substitutions at R249 in the rod domain cause muscular dystrophies with varying severity. To address this variability, we modeled R249Q and R249W in Drosophila Lamin C, an orthologue of LMNA. Larval body wall muscles expressing mutant Lamin C caused abnormal nuclear morphology and premature death. When expressed in indirect flight muscles, R249W caused a greater number of adults with wing posturing defects than R249Q, consistent with observations that R249W and R249Q cause distinct muscular dystrophies, with R249W more severe. In this case, the nature of the amino acid replacement appears to dictate muscle disease severity. Together, our findings illustrate the utility of Drosophila for predicting muscle disease severity and pathogenicity of variants of unknown significance.

Implicit Solvents for the Polarizable Atomic Multipole AMOEBA Force Field.

Computational protein design, ab initio protein/RNA folding, and protein-ligand screening can be too computationally demanding for explicit treatment of solvent. For these applications, implicit solvent offers a compelling alternative, which we describe here for the polarizable atomic multipole AMOEBA force field based on three treatments of continuum electrostatics: numerical solutions to the nonlinear and linearized versions of the Poisson-Boltzmann equation (PBE), the domain-decomposition conductor-like screening model (ddCOSMO) approximation to the PBE, and the analytic generalized Kirkwood (GK) approximation. The continuum electrostatics models are combined with a nonpolar estimator based on novel cavitation and dispersion terms. Electrostatic model parameters are numerically optimized using a least-squares style target function based on a library of 103 small-molecule solvation free energy differences. Mean signed errors for the adaptive Poisson-Boltzmann solver (APBS), ddCOSMO, and GK models are 0.05, 0.00, and 0.00 kcal/mol, respectively, while the mean unsigned errors are 0.70, 0.63, and 0.58 kcal/mol, respectively. Validation of the electrostatic response of the resulting implicit solvents, which are available in the Tinker (or Tinker-HP), OpenMM, and Force Field X software packages, is based on comparisons to explicit solvent simulations for a series of proteins and nucleic acids. Overall, the emergence of performative implicit solvent models for polarizable force fields opens the door to their use for folding and design applications.

Nanophthalmos patient with a THR518MET mutation in MYRF, a case report.

BACKGROUND: Nanophthalmos has a significant genetic background and disease-causing mutations have been recently been reported in the myelin regulatory factor (MYRF) gene. We report clinical features in a patient with nanophthalmos and a Thr518Met MYRF mutation. CASE PRESENTATION: A three-year-old male was discovered to have nanophthalmos after first presenting to the emergency department for a frontal headache, eye pain, emesis, and lethargy. Imaging studies (CT and MRI) were negative except for increased posterior fossa cerebrospinal fluid. Subsequent examinations revealed nanophthalmos (short axial eye lengths 18.1 mm OD and 18.3 mm OS), microcornea, and a large crystalline lens. Peripheral chorioretinal pigment abnormalities were also observed. He experienced episodes of marked ocular hypertension (53 mmHg OD and 60 mmHg) likely due to intermittent angle closure precipitated by nanophthalmos. The ocular hypertension was responsive to topical medicines. Genetic analysis of known nanophthalmos genes MFRP and TMEM98 were negative, while a novel mutation, Thr518Met was detected in MYRF. The Thr518Met mutation was absent from 362 matched normal controls and was extremely rare in a large population database, allele frequency of 0.000024. The Thr518Met mutation altered a highly conserved amino acid in the MYRF protein and three of four algorithms suggested that this mutation is likely pathogenic. Finally, molecular modeling showed that the Thr518Met mutation is damaging to MYRF structure. Together these data suggest that the Thr518Met mutation causes nanophthalmos. CONCLUSIONS: Nanophthalmos may present at an early age with features of angle closure glaucoma and a Thr518Met mutation in MYRF was detected in a patient with nanophthalmos. Prevalence data, homology data, mutation analysis data, and protein modeling data suggest that this variant is pathogenic and may expand the phenotypic range of syndromic nanophthalmos caused by MYRF mutations to include central nervous system abnormalities (increased posterior fossa cerebrospinal fluid).

Novel Intragenic PAX6 Deletion in a Pedigree with Aniridia, Morbid Obesity, and Diabetes.

Purpose: Aniridia is a rare congenital eye disease, characterized by a constellation of symptoms including hypoplastic irides, foveal hypoplasia, early cataract, corneal stem cell deficiency, and glaucoma. Large chromosomal deletions spanning the PAX6 gene cause WAGR syndrome (Wilms tumor, aniridia, genitourinary anomalies, and intellectual disability [formerly called mental retardation]). We describe clinical and genetic studies of a three-generation pedigree with aniridia along with additional systemic conditions (morbid obesity, diabetes) suggesting the possibility of a contiguous-gene syndrome like WAGR.Methods: Clinical records were obtained and DNA was prepared from blood samples from three of the four patients and tested for mutations in the coding sequences of the PAX6 gene. The index patient also had cardiomyopathy and was tested for known cardiomyopathy genetic mutations using a next-generation DNA sequencing assay.Results: We discovered a novel intragenic PAX6 mutation, a 16 bp heterozygous deletion c.203delCCAGGGCAATCGGTGG, with Sanger sequencing that is the likely cause of autosomal dominant aniridia in this pedigree. This PAX6 deletion causes a frameshift in predicted protein translation and a subsequent premature termination, p.Pro68Leufs*6. The PAX6 deletion was detected in all three available family members with aniridia, the index patient, his mother, and his maternal aunt but was not observed in the Exome Aggregation Consortium (ExAC) database. Targeted sequencing of known cardiomyopathy genes in the index patient identified a second mutation, a 1.7 Mp deletion that spans the MYBPC3 gene.Conclusions: We report a pedigree with aniridia and other systemic abnormalities that were initially suspicious for a contiguous-gene syndrome like WAGR. However, genetic analysis of the pedigree revealed two independent genetic abnormalities on chromosome 11p: 1) a novel PAX6 mutation, and 2) a large chromosome deletion spanning MYBPC3, a known cardiomyopathy gene. It is unclear if morbid obesity and type II diabetes mellitus have a related genetic cause.

Characterization of a TP53 Somatic Variant of Unknown Function From an Ovarian Cancer Patient Using Organoid Culture and Computational Modeling.

In our proof-of-concept study of 1 patient with stage IIIC carcinosarcoma of the ovary, we discovered a rare mutation in the tumor suppressor, TP53, that results in the deletion of N131. Immunofluorescence imaging of the organoid culture revealed hyperstaining of p53 protein. Computational modeling suggests this residue is important for maintaining protein conformation. Drug screening identified the combination of a proteasome inhibitor with a histone deacetylase inhibitor as the most effective treatment. These data provide evidence for the successful culture of a patient tumor and analysis of drug response ex vivo.

Scalable Indirect Free Energy Method Applied to Divalent Cation-Metalloprotein Binding.

Many biological processes are based on molecular recognition between highly charged molecules such as nucleic acids, inorganic ions, charged amino acids, etc. For such cases, it has been demonstrated that molecular simulations with fixed partial charges often fail to achieve experimental accuracy. Although incorporation of more advanced electrostatic models (such as multipoles, mutual polarization, etc.) can significantly improve simulation accuracy, it increases computational expense by a factor of 5-20×. Indirect free energy (IFE) methods can mitigate this cost by modeling intermediate states at fixed-charge resolution. For example, an efficient "reference" model such as a pairwise Amber, CHARMM, or OPLS-AA force field can be used to derive an initial estimate, followed by thermodynamic corrections to a more advanced "target" potential such as the polarizable AMOEBA model. Unfortunately, all currently described IFE methods encounter difficulties reweighting more than ∼50 atoms between resolutions due to extensive scaling of both the magnitude of the thermodynamic corrections and their statistical uncertainty. We present an approach called "simultaneous bookending" (SB) that is fundamentally different from existing IFE methods based on a tunable sampling approximation, which permits scaling to thousands of atoms. SB is demonstrated on the relative binding affinity of Mg2+/Ca2+ to a set of metalloproteins with up to 2972 atoms, finding no statistically significant difference between direct AMOEBA results and those from correcting Amber to AMOEBA. The ability to change the resolution of thousands of atoms during reweighting suggests the approach may be applicable in the future to protein-protein binding affinities or nucleic acid thermodynamics.

Structural Insights into Hearing Loss Genetics from Polarizable Protein Repacking.

Hearing loss is associated with ∼8100 mutations in 152 genes, and within the coding regions of these genes are over 60,000 missense variants. The majority of these variants are classified as "variants of uncertain significance" to reflect our inability to ascribe a phenotypic effect to the observed amino acid change. A promising source of pathogenicity information is biophysical simulation, although input protein structures often contain defects because of limitations in experimental data and/or only distant homology to a template. Here, we combine the polarizable atomic multipole optimized energetics for biomolecular applications force field, many-body optimization theory, and graphical processing unit acceleration to repack all deafness-associated proteins and thereby improve average structure MolProbity score from 2.2 to 1.0. We then used these optimized wild-type models to create over 60,000 structures for missense variants in the Deafness Variation Database, which are being incorporated into the Deafness Variation Database to inform deafness pathogenicity prediction. Finally, this work demonstrates that advanced polarizable atomic multipole force fields are efficient enough to repack the entire human proteome.

Tinker 8: Software Tools for Molecular Design.

The Tinker software, currently released as version 8, is a modular molecular mechanics and dynamics package written primarily in a standard, easily portable dialect of Fortran 95 with OpenMP extensions. It supports a wide variety of force fields, including polarizable models such as the Atomic Multipole Optimized Energetics for Biomolecular Applications (AMOEBA) force field. The package runs on Linux, macOS, and Windows systems. In addition to canonical Tinker, there are branches, Tinker-HP and Tinker-OpenMM, designed for use on message passing interface (MPI) parallel distributed memory supercomputers and state-of-the-art graphical processing units (GPUs), respectively. The Tinker suite also includes a tightly integrated Java-based graphical user interface called Force Field Explorer (FFE), which provides molecular visualization capabilities as well as the ability to launch and control Tinker calculations.

Genomic Landscape and Mutational Signatures of Deafness-Associated Genes.

The classification of genetic variants represents a major challenge in the post-genome era by virtue of their extraordinary number and the complexities associated with ascribing a clinical impact, especially for disorders exhibiting exceptional phenotypic, genetic, and allelic heterogeneity. To address this challenge for hearing loss, we have developed the Deafness Variation Database (DVD), a comprehensive, open-access resource that integrates all available genetic, genomic, and clinical data together with expert curation to generate a single classification for each variant in 152 genes implicated in syndromic and non-syndromic deafness. We evaluate 876,139 variants and classify them as pathogenic or likely pathogenic (more than 8,100 variants), benign or likely benign (more than 172,000 variants), or of uncertain significance (more than 695,000 variants); 1,270 variants are re-categorized based on expert curation and in 300 instances, the change is of medical significance and impacts clinical care. We show that more than 96% of coding variants are rare and novel and that pathogenicity is driven by minor allele frequency thresholds, variant effect, and protein domain. The mutational landscape we define shows complex gene-specific variability, making an understanding of these nuances foundational for improved accuracy in variant interpretation in order to enhance clinical decision making and improve our understanding of deafness biology.