Modeling Immunity with Rosetta: Methods for Antibody and Antigen Design.

Structure-based antibody and antigen design has advanced greatly in recent years, due not only to the increasing availability of experimentally determined structures but also to improved computational methods for both prediction and design. Constant improvements in performance within the Rosetta software suite for biomolecular modeling have given rise to a greater breadth of structure prediction, including docking and design application cases for antibody and antigen modeling. Here, we present an overview of current protocols for antibody and antigen modeling using Rosetta and exemplify those by detailed tutorials originally developed for a Rosetta workshop at Vanderbilt University. These tutorials cover antibody structure prediction, docking, and design and antigen design strategies, including the addition of glycans in Rosetta. We expect that these materials will allow novice users to apply Rosetta in their own projects for modeling antibodies and antigens.

Specificity and affinity of the N-terminal residues in staphylocoagulase in binding to prothrombin.

In Staphylococcus aureus-caused endocarditis, the pathogen secretes staphylocoagulase (SC), thereby activating human prothrombin (ProT) and evading immune clearance. A previous structural comparison of the SC(1-325) fragment bound to thrombin and its inactive precursor prethrombin 2 has indicated that SC activates ProT by inserting its N-terminal dipeptide Ile1-Val2 into the ProT Ile16 pocket, forming a salt bridge with ProT's Asp194, thereby stabilizing the active conformation. We hypothesized that these N-terminal SC residues modulate ProT binding and activation. Here, we generated labeled SC(1-246) as a probe for competitively defining the affinities of N-terminal SC(1-246) variants preselected by modeling. Using ProT(R155Q,R271Q,R284Q) (ProTQQQ), a variant refractory to prothrombinase- or thrombin-mediated cleavage, we observed variant affinities between ∼1 and 650 nm and activation potencies ranging from 1.8-fold that of WT SC(1-246) to complete loss of function. Substrate binding to ProTQQQ caused allosteric tightening of the affinity of most SC(1-246) variants, consistent with zymogen activation through occupation of the specificity pocket. Conservative changes at positions 1 and 2 were well-tolerated, with Val1-Val2, Ile1-Ala2, and Leu1-Val2 variants exhibiting ProTQQQ affinity and activation potency comparable with WT SC(1-246). Weaker binding variants typically had reduced activation rates, although at near-saturating ProTQQQ levels, several variants exhibited limiting rates similar to or higher than that of WT SC(1-246). The Ile16 pocket in ProTQQQ appears to favor nonpolar, nonaromatic residues at SC positions 1 and 2. Our results suggest that SC variants other than WT Ile1-Val2-Thr3 might emerge with similar ProT-activating efficiency.

Three-dimensional spatial analysis of missense variants in RTEL1 identifies pathogenic variants in patients with Familial Interstitial Pneumonia.

BACKGROUND: Next-generation sequencing of individuals with genetic diseases often detects candidate rare variants in numerous genes, but determining which are causal remains challenging. We hypothesized that the spatial distribution of missense variants in protein structures contains information about function and pathogenicity that can help prioritize variants of unknown significance (VUS) and elucidate the structural mechanisms leading to disease. RESULTS: To illustrate this approach in a clinical application, we analyzed 13 candidate missense variants in regulator of telomere elongation helicase 1 (RTEL1) identified in patients with Familial Interstitial Pneumonia (FIP). We curated pathogenic and neutral RTEL1 variants from the literature and public databases. We then used homology modeling to construct a 3D structural model of RTEL1 and mapped known variants into this structure. We next developed a pathogenicity prediction algorithm based on proximity to known disease causing and neutral variants and evaluated its performance with leave-one-out cross-validation. We further validated our predictions with segregation analyses, telomere lengths, and mutagenesis data from the homologous XPD protein. Our algorithm for classifying RTEL1 VUS based on spatial proximity to pathogenic and neutral variation accurately distinguished 7 known pathogenic from 29 neutral variants (ROC AUC = 0.85) in the N-terminal domains of RTEL1. Pathogenic proximity scores were also significantly correlated with effects on ATPase activity (Pearson r = -0.65, p = 0.0004) in XPD, a related helicase. Applying the algorithm to 13 VUS identified from sequencing of RTEL1 from patients predicted five out of six disease-segregating VUS to be pathogenic. We provide structural hypotheses regarding how these mutations may disrupt RTEL1 ATPase and helicase function. CONCLUSIONS: Spatial analysis of missense variation accurately classified candidate VUS in RTEL1 and suggests how such variants cause disease. Incorporating spatial proximity analyses into other pathogenicity prediction tools may improve accuracy for other genes and genetic diseases.

Pore Polarity and Charge Determine Differential Block of Kir1.1 and Kir7.1 Potassium Channels by Small-Molecule Inhibitor VU590.

VU590 was the first publicly disclosed, submicromolar-affinity (IC50 = 0.2 µM), small-molecule inhibitor of the inward rectifier potassium (Kir) channel and diuretic target, Kir1.1. VU590 also inhibits Kir7.1 (IC50 ∼ 8 µM), and has been used to reveal new roles for Kir7.1 in regulation of myometrial contractility and melanocortin signaling. Here, we employed molecular modeling, mutagenesis, and patch clamp electrophysiology to elucidate the molecular mechanisms underlying VU590 inhibition of Kir1.1 and Kir7.1. Block of both channels is voltage- and K+-dependent, suggesting the VU590 binding site is located within the pore. Mutagenesis analysis in Kir1.1 revealed that asparagine 171 (N171) is the only pore-lining residue required for high-affinity block, and that substituting negatively charged residues (N171D, N171E) at this position dramatically weakens block. In contrast, substituting a negatively charged residue at the equivalent position in Kir7.1 enhances block by VU590, suggesting the VU590 binding mode is different. Interestingly, mutations of threonine 153 (T153) in Kir7.1 that reduce constrained polarity at this site (T153C, T153V, T153S) make wild-type and binding-site mutants (E149Q, A150S) more sensitive to block by VU590. The Kir7.1-T153C mutation enhances block by the structurally unrelated inhibitor VU714 but not by a higher-affinity analog ML418, suggesting that the polar side chain of T153 creates a barrier to low-affinity ligands that interact with E149 and A150. Reverse mutations in Kir1.1 suggest that this mechanism is conserved in other Kir channels. This study reveals a previously unappreciated role of membrane pore polarity in determination of Kir channel inhibitor pharmacology.

Protocols for Molecular Modeling with Rosetta3 and RosettaScripts.

Previously, we published an article providing an overview of the Rosetta suite of biomacromolecular modeling software and a series of step-by-step tutorials [Kaufmann, K. W., et al. (2010) Biochemistry 49, 2987-2998]. The overwhelming positive response to this publication we received motivates us to here share the next iteration of these tutorials that feature de novo folding, comparative modeling, loop construction, protein docking, small molecule docking, and protein design. This updated and expanded set of tutorials is needed, as since 2010 Rosetta has been fully redesigned into an object-oriented protein modeling program Rosetta3. Notable improvements include a substantially improved energy function, an XML-like language termed "RosettaScripts" for flexibly specifying modeling task, new analysis tools, the addition of the TopologyBroker to control conformational sampling, and support for multiple templates in comparative modeling. Rosetta's ability to model systems with symmetric proteins, membrane proteins, noncanonical amino acids, and RNA has also been greatly expanded and improved.

Genetic analysis of the localization of APOBEC3F to human immunodeficiency virus type 1 virion cores.

UNLABELLED: Members of the APOBEC3 family of cytidine deaminases vary in their proportions of a virion-incorporated enzyme that is localized to mature retrovirus cores. We reported previously that APOBEC3F (A3F) was highly localized into mature human immunodeficiency virus type 1 (HIV-1) cores and identified that L306 in the C-terminal cytidine deaminase (CD) domain contributed to its core localization (C. Song, L. Sutton, M. Johnson, R. D'Aquila, J. Donahue, J Biol Chem 287:16965-16974, 2012, http://dx.doi.org/10.1074/jbc.M111.310839). We have now determined an additional genetic determinant(s) for A3F localization to HIV-1 cores. We found that one pair of leucines in each of A3F's C-terminal and N-terminal CD domains jointly determined the degree of localization of A3F into HIV-1 virion cores. These are A3F L306/L368 (C-terminal domain) and A3F L122/L184 (N-terminal domain). Alterations to one of these specific leucine residues in either of the two A3F CD domains (A3F L368A, L122A, and L184A) decreased core localization and diminished HIV restriction without changing virion packaging. Furthermore, double mutants in these leucine residues in each of A3F's two CD domains (A3F L368A plus L184A or A3F L368A plus L122A) still were packaged into virions but completely lost core localization and anti-HIV activity. HIV virion core localization of A3F is genetically separable from its virion packaging, and anti-HIV activity requires some core localization. IMPORTANCE: Specific leucine-leucine interactions are identified as necessary for A3F's core localization and anti-HIV activity but not for its packaging into virions. Understanding these signals may lead to novel strategies to enhance core localization that may augment effects of A3F against HIV and perhaps of other A3s against retroviruses, parvoviruses, and hepatitis B virus.

Computational and functional analyses of a small-molecule binding site in ROMK.

The renal outer medullary potassium channel (ROMK, or Kir1.1, encoded by KCNJ1) critically regulates renal tubule electrolyte and water transport and hence blood volume and pressure. The discovery of loss-of-function mutations in KCNJ1 underlying renal salt and water wasting and lower blood pressure has sparked interest in developing new classes of antihypertensive diuretics targeting ROMK. The recent development of nanomolar-affinity small-molecule inhibitors of ROMK creates opportunities for exploring the chemical and physical basis of ligand-channel interactions required for selective ROMK inhibition. We previously reported that the bis-nitro-phenyl ROMK inhibitor VU591 exhibits voltage-dependent knock-off at hyperpolarizing potentials, suggesting that the binding site is located within the ion-conduction pore. In this study, comparative molecular modeling and in silico ligand docking were used to interrogate the full-length ROMK pore for energetically favorable VU591 binding sites. Cluster analysis of 2498 low-energy poses resulting from 9900 Monte Carlo docking trajectories on each of 10 conformationally distinct ROMK comparative homology models identified two putative binding sites in the transmembrane pore that were subsequently tested for a role in VU591-dependent inhibition using site-directed mutagenesis and patch-clamp electrophysiology. Introduction of mutations into the lower site had no effect on the sensitivity of the channel to VU591. In contrast, mutations of Val(168) or Asn(171) in the upper site, which are unique to ROMK within the Kir channel family, led to a dramatic reduction in VU591 sensitivity. This study highlights the utility of computational modeling for defining ligand-ROMK interactions and proposes a mechanism for inhibition of ROMK.

Integrative analysis of pathogenic variants in glucose-6-phosphatase based on an AlphaFold2 model.

Mediating the terminal reaction of gluconeogenesis and glycogenolysis, the integral membrane protein glucose-6-phosphate catalytic subunit 1 (G6PC1) regulates hepatic glucose production by catalyzing hydrolysis of glucose-6-phosphate (G6P) within the lumen of the endoplasmic reticulum. Consistent with its vital contribution to glucose homeostasis, inactivating mutations in G6PC1 causes glycogen storage disease (GSD) type 1a characterized by hepatomegaly and severe hypoglycemia. Despite its physiological importance, the structural basis of G6P binding to G6PC1 and the molecular disruptions induced by missense mutations within the active site that give rise to GSD type 1a are unknown. In this study, we determine the atomic interactions governing G6P binding as well as explore the perturbations imposed by disease-linked missense variants by subjecting an AlphaFold2 G6PC1 structural model to molecular dynamics simulations and in silico predictions of thermodynamic stability validated with robust in vitro and in situ biochemical assays. We identify a collection of side chains, including conserved residues from the signature phosphatidic acid phosphatase motif, that contribute to a hydrogen bonding and van der Waals network stabilizing G6P in the active site. The introduction of GSD type 1a mutations modified the thermodynamic landscape, altered side chain packing and substrate-binding interactions, and induced trapping of catalytic intermediates. Our results, which corroborate the high quality of the AF2 model as a guide for experimental design and to interpret outcomes, not only confirm the active-site structural organization but also identify previously unobserved mechanistic contributions of catalytic and noncatalytic side chains.

Loop-loop interactions regulate KaiA-stimulated KaiC phosphorylation in the cyanobacterial KaiABC circadian clock.

The Synechococcus elongatus KaiA, KaiB, and KaiC proteins in the presence of ATP generate a post-translational oscillator that runs in a temperature-compensated manner with a period of 24 h. KaiA dimer stimulates phosphorylation of KaiC hexamer at two sites per subunit, T432 and S431, and KaiB dimers antagonize KaiA action and induce KaiC subunit exchange. Neither the mechanism of KaiA-stimulated KaiC phosphorylation nor that of KaiB-mediated KaiC dephosphorylation is understood in detail at present. We demonstrate here that the A422V KaiC mutant sheds light on the former mechanism. It was previously reported that A422V is less sensitive to dark pulse-induced phase resetting and has a reduced amplitude of the KaiC phosphorylation rhythm in vivo. A422 maps to a loop (422-loop) that continues toward the phosphorylation sites. By pulling on the C-terminal peptide of KaiC (A-loop), KaiA removes restraints from the adjacent 422-loop whose increased flexibility indirectly promotes kinase activity. We found in the crystal structure that A422V KaiC lacks phosphorylation at S431 and exhibits a subtle, local conformational change relative to wild-type KaiC. Molecular dynamics simulations indicate higher mobility of the 422-loop in the absence of the A-loop and mobility differences in other areas associated with phosphorylation activity between wild-type and mutant KaiCs. The A-loop-422-loop relay that informs KaiC phosphorylation sites of KaiA dimer binding propagates to loops from neighboring KaiC subunits, thus providing support for a concerted allosteric mechanism of phosphorylation.

Molecular mechanisms of catalytic inhibition for active site mutations in glucose-6-phosphatase catalytic subunit 1 linked to glycogen storage disease.

Mediating the terminal reaction of gluconeogenesis and glycogenolysis, the integral membrane protein G6PC1 regulates hepatic glucose production by catalyzing hydrolysis of glucose-6-phosphate (G6P) within the lumen of the endoplasmic reticulum. Consistent with its vital contribution to glucose homeostasis, inactivating mutations in G6PC1 cause glycogen storage disease (GSD) type 1a characterized by hepatomegaly and severe hypoglycemia. Despite its physiological importance, the structural basis of G6P binding to G6PC1 and the molecular disruptions induced by missense mutations within the active site that give rise to GSD type 1a are unknown. Exploiting a computational model of G6PC1 derived from the groundbreaking structure prediction algorithm AlphaFold2 (AF2), we combine molecular dynamics (MD) simulations and computational predictions of thermodynamic stability with a robust in vitro screening platform to define the atomic interactions governing G6P binding as well as explore the energetic perturbations imposed by disease-linked variants. We identify a collection of side chains, including conserved residues from the signature phosphatidic acid phosphatase motif, that contribute to a hydrogen bonding and van der Waals network stabilizing G6P in the active site. Introduction of GSD type 1a mutations into the G6PC1 sequence elicits changes in G6P binding energy, thermostability and structural properties, suggesting multiple pathways of catalytic impairment. Our results, which corroborate the high quality of the AF2 model as a guide for experimental design and to interpret outcomes, not only confirm active site structural organization but also suggest novel mechanistic contributions of catalytic and non-catalytic side chains.

Second-site suppressors of HIV-1 capsid mutations: restoration of intracellular activities without correction of intrinsic capsid stability defects.

BACKGROUND: Disassembly of the viral capsid following penetration into the cytoplasm, or uncoating, is a poorly understood stage of retrovirus infection. Based on previous studies of HIV-1 CA mutants exhibiting altered capsid stability, we concluded that formation of a capsid of optimal intrinsic stability is crucial for HIV-1 infection. RESULTS: To further examine the connection between HIV-1 capsid stability and infectivity, we isolated second-site suppressors of HIV-1 mutants exhibiting unstable (P38A) or hyperstable (E45A) capsids. We identified the respective suppressor mutations, T216I and R132T, which restored virus replication in a human T cell line and markedly enhanced the fitness of the original mutants as revealed in single-cycle infection assays. Analysis of the corresponding purified N-terminal domain CA proteins by NMR spectroscopy demonstrated that the E45A and R132T mutations induced structural changes that are localized to the regions of the mutations, while the P38A mutation resulted in changes extending to neighboring regions in space. Unexpectedly, neither suppressor mutation corrected the intrinsic viral capsid stability defect associated with the respective original mutation. Nonetheless, the R132T mutation rescued the selective infectivity impairment exhibited by the E45A mutant in aphidicolin-arrested cells, and the double mutant regained sensitivity to the small molecule inhibitor PF74. The T216I mutation rescued the impaired ability of the P38A mutant virus to abrogate restriction by TRIMCyp and TRIM5α. CONCLUSIONS: The second-site suppressor mutations in CA that we have identified rescue virus infection without correcting the intrinsic capsid stability defects associated with the P38A and E45A mutations. The suppressors also restored wild type virus function in several cell-based assays. We propose that while proper HIV-1 uncoating in target cells is dependent on the intrinsic stability of the viral capsid, the effects of stability-altering mutations can be mitigated by additional mutations that affect interactions with host factors in target cells or the consequences of these interactions. The ability of mutations at other CA surfaces to compensate for effects at the NTD-NTD interface further indicates that uncoating in target cells is controlled by multiple intersubunit interfaces in the viral capsid.

A novel, likely pathogenic variant in UBTF-related neurodegeneration with brain atrophy is associated with a severe divergent neurodevelopmental phenotype.

BACKGROUND: A de novo, pathogenic, missense variant in UBTF, c.628G>A p.Glu210Lys, has been described as the cause of an emerging neurodegenerative disorder, Childhood-Onset Neurodegeneration with Brain Atrophy (CONDBA). The p.Glu210Lys alteration yields a positively charged stretch of three lysine residues. Functional studies confirmed this change results in a stronger interaction with negatively charged DNA and gain-of-function activity when compared to the wild-type sequence. The CONDBA phenotype reported in association with p.Glu210Lys consists of normal early-neurodevelopment followed by progressive motor, cognitive, and behavioral regression in early-to-middle childhood. METHODS AND RESULTS: The current proband presented at 9 months of age with baseline developmental delay and more extensive neuroradiological findings, including pontine hypoplasia, thalamic volume loss and signal abnormality, and hypomyelination. Like the recurrent CONDBA p.Glu210Lys variant, this novel variant, c.608A>G p.(Gln203Arg) lies within the highly conserved second HMG-box homology domain and involves the replacement of the wild-type residue with a positively charged residue, arginine. Computational structural modeling demonstrates that this amino acid substitution potentiates the interaction between UBTF and DNA, likely resulting in a gain-of-function effect for the UBTF protein, UBF. CONCLUSION: Here we present a new divergent phenotype associated with a novel, likely pathogenic, missense variant at a different position in the UBTF gene, c.608A>G p.(Gln203Arg).

Personalized structural biology reveals the molecular mechanisms underlying heterogeneous epileptic phenotypes caused by de novo KCNC2 variants.

Whole-exome sequencing (WES) in the clinic has identified several rare monogenic developmental and epileptic encephalopathies (DEE) caused by ion channel variants. However, WES often fails to provide actionable insight for rare diseases, such as DEEs, due to the challenges of interpreting variants of unknown significance (VUS). Here, we describe a "personalized structural biology" (PSB) approach that leverages recent innovations in the analysis of protein 3D structures to address this challenge. We illustrate this approach in an Undiagnosed Diseases Network (UDN) individual with DEE symptoms and a de novo VUS in KCNC2 (p.V469L), the Kv3.2 voltage-gated potassium channel. A nearby KCNC2 variant (p.V471L) was recently suggested to cause DEE-like phenotypes. Computational structural modeling suggests that both affect protein function. However, despite their proximity, the p.V469L variant is likely to sterically block the channel pore, while the p.V471L variant is likely to stabilize the open state. Biochemical and electrophysiological analyses demonstrate heterogeneous loss-of-function and gain-of-function effects, as well as differential response to 4-aminopyridine treatment. Molecular dynamics simulations illustrate that the pore of the p.V469L variant is more constricted, increasing the energetic barrier for K+ permeation, whereas the p.V471L variant stabilizes the open conformation. Our results implicate variants in KCNC2 as causative for DEE and guide the interpretation of a UDN individual. They further delineate the molecular basis for the heterogeneous clinical phenotypes resulting from two proximal pathogenic variants. This demonstrates how the PSB approach can provide an analytical framework for individualized hypothesis-driven interpretation of protein-coding VUS.

Glycosylation of {beta}2 subunits regulates GABAA receptor biogenesis and channel gating.

γ-aminobutyric acid type A (GABA(A)) receptors are heteropentameric glycoproteins. Based on consensus sequences, the GABA(A) receptor ß2 subunit contains three potential N-linked glycosylation sites, Asn-32, Asn-104, and Asn-173. Homology modeling indicates that Asn-32 and Asn-104 are located before the α1 helix and in loop L3, respectively, near the top of the subunit-subunit interface on the minus side, and that Asn-173 is located in the Cys-loop near the bottom of the subunit N-terminal domain. Using site-directed mutagenesis, we demonstrated that all predicted ß2 subunit glycosylation sites were glycosylated in transfected HEK293T cells. Glycosylation of each site, however, produced specific changes in α1ß2 receptor surface expression and function. Although glycosylation of Asn-173 in the Cys-loop was important for stability of ß2 subunits when expressed alone, results obtained with flow cytometry, brefeldin A treatment, and endo-ß-N-acetylglucosaminidase H digestion suggested that glycosylation of Asn-104 was required for efficient α1ß2 receptor assembly and/or stability in the endoplasmic reticulum. Patch clamp recording revealed that mutation of each site to prevent glycosylation decreased peak α1ß2 receptor current amplitudes and altered the gating properties of α1ß2 receptor channels by reducing mean open time due to a reduction in the proportion of long open states. In addition to functional heterogeneity, endo-ß-N-acetylglucosaminidase H digestion and glycomic profiling revealed that surface ß2 subunit N-glycans at Asn-173 were high mannose forms that were different from those of Asn-32 and N104. Using a homology model of the pentameric extracellular domain of α1ß2 channel, we propose mechanisms for regulation of GABA(A) receptors by glycosylation.

Measurement of aptamer-protein interactions with back-scattering interferometry.

We report the quantitative measurement of aptamer-protein interactions using backscattering interferometry (BSI) and show that BSI can determine when distinct binding regions are accessed. As a model system, we utilized two DNA aptamers (Tasset and Bock) that bind to distinct sites of a target protein (human α-thrombin). This is the first time BSI has been used to study a multivalent system in free solution wherein more than one ligand binds to a single target. We measured aptamer equilibrum dissociation constants (K(d)) of 3.84 nM (Tasset-thrombin) and 5.96 nM (Bock-thrombin), in close agreement with the literature. Unexpectedly, we observed allosteric effects such that the binding of the first aptamer resulted in a significant change in the binding affinity of the second aptamer. For example, the K(d) of Bock aptamer binding to preformed Tasset-thrombin complexes was 7-fold lower (indicating higher affinity) compared to binding to thrombin alone. Preliminary modeling efforts suggest evidence for allosteric linkage between the two exosites.

Familial Autonomic Ganglionopathy Caused by Rare CHRNA3 Genetic Variants.

OBJECTIVE: To determine the molecular basis of a new monogenetic recessive disorder that results in familial autonomic ganglionopathy with diffuse autonomic failure. METHODS: Two adult siblings from one family (I-4 and I-5) and another participant from a second family (II-3) presented with severe neurogenic orthostatic hypotension (nOH), small nonreactive pupils, and constipation. All 3 affected members had low norepinephrine levels and diffuse panautonomic failure. RESULTS: Whole exome sequencing of DNA from I-4 and I-5 showed compound heterozygosity for c.907_908delCT (p.L303Dfs*115)/c.688 G>A (p.D230N) pathologic variants in the acetylcholine receptor, neuronal nicotinic, α3 subunit gene (CHRNA3). II-3 from the second family was homozygous for the same frameshift (fs) variant (p.L303Dfs*115//p.L303Dfs*115). CHRNA3 encodes a critical subunit of the nicotinic acetylcholine receptors (nAChRs) responsible for fast synaptic transmission in the autonomic ganglia. The fs variant is clearly pathogenic and the p.D230N variant is predicted to be damaging (SIFT)/probably damaging (PolyPhen2). The p.D230N variant lies on the interface between CHRNA3 and other nAChR subunits based on structural modeling and is predicted to destabilize the nAChR pentameric complex. CONCLUSIONS: We report a novel genetic disease that affected 3 individuals from 2 unrelated families who presented with severe nOH, miosis, and constipation. These patients had rare pathologic variants in the CHRNA3 gene that cosegregate with and are predicted to be the likely cause of their diffuse panautonomic failure.

Co-occurring gain-of-function mutations in HER2 and HER3 modulate HER2/HER3 activation, oncogenesis, and HER2 inhibitor sensitivity.

Activating mutations in HER2 (ERBB2) drive the growth of a subset of breast and other cancers and tend to co-occur with HER3 (ERBB3) missense mutations. The HER2 tyrosine kinase inhibitor neratinib has shown clinical activity against HER2-mutant tumors. To characterize the role of HER3 mutations in HER2-mutant tumors, we integrate computational structural modeling with biochemical and cell biological analyses. Computational modeling predicts that the frequent HER3E928G kinase domain mutation enhances the affinity of HER2/HER3 and reduces binding of HER2 to its inhibitor neratinib. Co-expression of mutant HER2/HER3 enhances HER2/HER3 co-immunoprecipitation and ligand-independent activation of HER2/HER3 and PI3K/AKT, resulting in enhanced growth, invasiveness, and resistance to HER2-targeted therapies, which can be reversed by combined treatment with PI3Kα inhibitors. Our results provide a mechanistic rationale for the evolutionary selection of co-occurring HER2/HER3 mutations and the recent clinical observations that HER3 mutations are associated with a poor response to neratinib in HER2-mutant cancers.

Practically useful: what the Rosetta protein modeling suite can do for you.

The objective of this review is to enable researchers to use the software package Rosetta for biochemical and biomedicinal studies. We provide a brief review of the six most frequent research problems tackled with Rosetta. For each of these six tasks, we provide a tutorial that illustrates a basic Rosetta protocol. The Rosetta method was originally developed for de novo protein structure prediction and is regularly one of the best performers in the community-wide biennial Critical Assessment of Structure Prediction. Predictions for protein domains with fewer than 125 amino acids regularly have a backbone root-mean-square deviation of better than 5.0 A. More impressively, there are several cases in which Rosetta has been used to predict structures with atomic level accuracy better than 2.5 A. In addition to de novo structure prediction, Rosetta also has methods for molecular docking, homology modeling, determining protein structures from sparse experimental NMR or EPR data, and protein design. Rosetta has been used to accurately design a novel protein structure, predict the structure of protein-protein complexes, design altered specificity protein-protein and protein-DNA interactions, and stabilize proteins and protein complexes. Most recently, Rosetta has been used to solve the X-ray crystallographic phase problem.

Phenotypic Profiling in Subjects Heterozygous for 1 of 2 Rare Variants in the Hypophosphatasia Gene (ALPL).

CONTEXT: Hypophosphatasia (HPP) is a syndrome marked by low serum alkaline phosphatase (AlkP) activity as well as musculoskeletal and/or dental disease. While the majority of subjects with HPP carry a pathogenic variant in the ALPL gene or its regulatory regions, individual pathogenic variants are often not tightly correlated with clinical symptomatology. We sought to better understand the genotype/phenotype correlation in HPP by examining the clinical and biochemical data of 37 subjects with 2 rare variants in ALPL. METHODS: Through BioVU, a DNA biobank that pairs individuals' genetic information with their de-identified medical records, we identified subjects with 2 rare variants with distinct reported clinical phenotypes (p.D294A and p.T273M). We then performed a manual review of these subjects' de-identified medical records along with computational modeling of protein structure to construct a genetic, biochemical and clinical phenotype for each subject and variant. RESULTS: Twenty subjects with the p.D294A variant and 17 with the p.T273M variant had sufficient data for analysis. Among subjects in our cohort with the p.D294A variant, 6 (30.0%) had both clinical bone disease and serum AlkP activity below 40 IU/L while 4 subjects (23.5%) with the p.T273M variant met the same criteria despite the distinct clinical phenotypes of these variants. CONCLUSIONS: Given the loose genotype/phenotype correlation in HPP seen in our cohort, clinical context is crucial for the interpretation of genetic test results to guide clinical care in this population. Otherwise, over- or under-diagnosis may occur, resulting in misidentification of those who may benefit from additional screening and perhaps pharmacologic intervention.

Integrating structural and evolutionary data to interpret variation and pathogenicity in adapter protein complex 4.

Genetic variation in the membrane trafficking adapter protein complex 4 (AP-4) can result in pathogenic neurological phenotypes including microencephaly, spastic paraplegias, epilepsy, and other developmental defects. We lack molecular mechanisms responsible for impaired AP-4 function arising from genetic variation, because AP-4 remains poorly understood structurally. Here, we analyze patterns of AP-4 genetic evolution and conservation to identify regions that are likely important for function and thus more susceptible to pathogenic variation. We map known variants onto an AP-4 homology model and predict the likelihood of pathogenic variation at a given location on the structure of AP-4. We find significant clustering of likely pathogenic variants located at the interface between the ß4 and N-µ4 subunits, as well as throughout the C-µ4 subunit. Our work offers an integrated perspective on how genetic and evolutionary forces affect AP-4 structure and function. As more individuals with uncharacterized AP-4 variants are identified, our work provides a foundation upon which their functional effects and disease relevance can be interpreted.