Machine learning-driven multiscale modeling reveals lipid-dependent dynamics of RAS signaling proteins.

RAS is a signaling protein associated with the cell membrane that is mutated in up to 30% of human cancers. RAS signaling has been proposed to be regulated by dynamic heterogeneity of the cell membrane. Investigating such a mechanism requires near-atomistic detail at macroscopic temporal and spatial scales, which is not possible with conventional computational or experimental techniques. We demonstrate here a multiscale simulation infrastructure that uses machine learning to create a scale-bridging ensemble of over 100,000 simulations of active wild-type KRAS on a complex, asymmetric membrane. Initialized and validated with experimental data (including a new structure of active wild-type KRAS), these simulations represent a substantial advance in the ability to characterize RAS-membrane biology. We report distinctive patterns of local lipid composition that correlate with interfacially promiscuous RAS multimerization. These lipid fingerprints are coupled to RAS dynamics, predicted to influence effector binding, and therefore may be a mechanism for regulating cell signaling cascades.

Exploring CRD mobility during RAS/RAF engagement at the membrane.

During the activation of mitogen-activated protein kinase (MAPK) signaling, the RAS-binding domain (RBD) and cysteine-rich domain (CRD) of RAF bind to active RAS at the plasma membrane. The orientation of RAS at the membrane may be critical for formation of the RAS-RBDCRD complex and subsequent signaling. To explore how RAS membrane orientation relates to the protein dynamics within the RAS-RBDCRD complex, we perform multiscale coarse-grained and all-atom molecular dynamics (MD) simulations of KRAS4b bound to the RBD and CRD domains of RAF-1, both in solution and anchored to a model plasma membrane. Solution MD simulations describe dynamic KRAS4b-CRD conformations, suggesting that the CRD has sufficient flexibility in this environment to substantially change its binding interface with KRAS4b. In contrast, when the ternary complex is anchored to the membrane, the mobility of the CRD relative to KRAS4b is restricted, resulting in fewer distinct KRAS4b-CRD conformations. These simulations implicate membrane orientations of the ternary complex that are consistent with NMR measurements. While a crystal structure-like conformation is observed in both solution and membrane simulations, a particular intermolecular rearrangement of the ternary complex is observed only when it is anchored to the membrane. This configuration emerges when the CRD hydrophobic loops are inserted into the membrane and helices α3-5 of KRAS4b are solvent exposed. This membrane-specific configuration is stabilized by KRAS4b-CRD contacts that are not observed in the crystal structure. These results suggest modulatory interplay between the CRD and plasma membrane that correlate with RAS/RAF complex structure and dynamics, and potentially influence subsequent steps in the activation of MAPK signaling.

Anionic Lipids Impact RAS-Binding Site Accessibility and Membrane Binding Affinity of CRAF RBD-CRD.

CRAF activation requires binding to membrane-anchored and active GTP-bound RAS. Whereas its RAS-binding domain (RBD) contains the main binding interface to the RAS G domain, its cysteine-rich domain (CRD) is responsible for association to anionic lipid-rich membranes. Both RAF domains are connected by a short linker, and it remains unclear if the two domains act independently or if one domain can impact the function of the other. Here, we used a combination of coarse-grained and all-atom molecular dynamics simulations of a CRAF RBD-CRD construct to investigate the dynamics of the RBD when it is tethered to CRD that is anchored to a POPC:POPS model membrane. First, we show that the RBD positioning is very dynamic with a preferential localization near the membrane surface. Next, we show that membrane-localized RBD has its RAS-binding interface mostly inaccessible because of its proximity to the membrane. Several positively charged residues in this interface were identified from simulations as important for driving RBD association to the membrane. Surface plasmon resonance (SPR) measurements confirmed that mutations of these RBD residues reduced the liposome partitioning of RBD-CRD. Last, simulations indicated that the presence of RBD near the membrane led to a local enrichment of anionic lipids that could potentially enhance the membrane affinity of the entire RBD-CRD construct. This was supported by SPR measurements that showed stronger liposome partitioning of RBD-CRD relative to CRD alone. These findings thus suggest that the RBD and CRD have synergistic effects on their membrane dynamics, with CRD bringing RBD closer to the membrane that impacts its accessibility to RAS and with RBD causing local anionic lipid enrichment that enhances the overall affinity between the membrane and RBD-CRD. These mechanisms have potential implications on the order of events of the interactions between RAS and CRAF at the membrane.

Graph-Directed Approach for Downselecting Toxins for Experimental Structure Determination.

Conotoxins are short, cysteine-rich peptides of great interest as novel therapeutic leads and of great concern as lethal biological agents due to their high affinity and specificity for various receptors involved in neuromuscular transmission. Currently, of the approximately 6000 known conotoxin sequences, only about 3% have associated structural characterization, which leads to a bottleneck in rapid high-throughput screening (HTS) for identification of potential leads or threats. In this work, we combine a graph-based approach with homology modeling to expand the library of conotoxin structures and to identify those conotoxin sequences that are of the greatest value for experimental structural characterization. The latter would allow for the rapid expansion of the known structural space for generating high quality template-based models. Our approach generalizes to other evolutionarily-related, short, cysteine-rich venoms of interest. Overall, we present and validate an approach for venom structure modeling and experimental guidance and employ it to produce a 290%-larger library of approximate conotoxin structures for HTS. We also provide a set of ranked conotoxin sequences for experimental structure determination to further expand this library.

Snails In Silico: A Review of Computational Studies on the Conopeptides.

Marine cone snails are carnivorous gastropods that use peptide toxins called conopeptides both as a defense mechanism and as a means to immobilize and kill their prey. These peptide toxins exhibit a large chemical diversity that enables exquisite specificity and potency for target receptor proteins. This diversity arises in terms of variations both in amino acid sequence and length, and in posttranslational modifications, particularly the formation of multiple disulfide linkages. Most of the functionally characterized conopeptides target ion channels of animal nervous systems, which has led to research on their therapeutic applications. Many facets of the underlying molecular mechanisms responsible for the specificity and virulence of conopeptides, however, remain poorly understood. In this review, we will explore the chemical diversity of conopeptides from a computational perspective. First, we discuss current approaches used for classifying conopeptides. Next, we review different computational strategies that have been applied to understanding and predicting their structure and function, from machine learning techniques for predictive classification to docking studies and molecular dynamics simulations for molecular-level understanding. We then review recent novel computational approaches for rapid high-throughput screening and chemical design of conopeptides for particular applications. We close with an assessment of the state of the field, emphasizing important questions for future lines of inquiry.

Extensive Citrullination Promotes Immunogenicity of HSP90 through Protein Unfolding and Exposure of Cryptic Epitopes.

Post-translational protein modifications such as citrullination have been linked to the breach of immune tolerance and clinical autoimmunity. Previous studies from our laboratory support this concept, demonstrating that autoantibodies targeting citrullinated isoforms of heat shock protein 90 (HSP90) are associated with rheumatoid arthritis complicated by interstitial lung disease. To further explore the relationship between citrullination and structural determinants of HSP90 immunogenicity, we employed a combination of ELISA-based epitope profiling, computational modeling, and mass-spectrometric sequencing of peptidylarginine deiminase (PAD)-modified protein. Remarkably, ELISAs involving selected citrullinated HSP90ß/α peptides identified a key epitope corresponding to an internal Arg residue (R502 [HSP90ß]/R510 [HSP90α]) that is normally buried within the crystal structure of native/unmodified HSP90. In vitro time/dose-response experiments reveal an ordered pattern of PAD-mediated deimination events culminating in citrullination of R502/R510. Conventional as well as scaled molecular dynamics simulations further demonstrate that citrullination of selected Arg residues leads to progressive disruption of HSP90 tertiary structure, promoting exposure of R502/R510 to PAD modification and subsequent autoantibody binding. Consistent with this process, ELISAs incorporating variably deiminated HSP90 as substrate Ag indicate a direct relationship between the degree of citrullination and the level of ex vivo Ab recognition. Overall, these data support a novel structural paradigm whereby citrullination-induced shifts in protein structure generate cryptic epitopes capable of bypassing B cell tolerance in the appropriate genetic context.

Anti-citrullinated heat shock protein 90 antibodies identified in bronchoalveolar lavage fluid are a marker of lung-specific immune responses.

Previous work has demonstrated a correlation between serum anti-citrullinated HSP90 antibodies and rheumatoid arthritis-associated interstitial lung disease (RA-ILD). To further investigate this potential pathogenic relationship, we used ELISA-based techniques to assess anti-citrullinated HSP90 antibody profiles in bronchoalveolar lavage fluid (BALF) of patients with different stages of RA-ILD. 9/21 RA-derived BALF specimens demonstrated IgG and/or IgA antibodies targeting citrullinated HSP90 proteins/peptides, highlighting disease specific responses (with a predilection for RA-ILD) that did not occur in IPF patients (0/5) or healthy control subjects (0/5). Comparison of antibody profiles between BALF and matching serum specimens revealed various recognition patterns favoring predominant production of anti-citrullinated HSP90 antibodies within the lung microenvironment-further supporting the connection between this antibody specificity and parenchymal lung disease. Equally important, qualitative as well as quantitative differences in anti-citrullinated HSP90 profiles between BALF and serum indicate that the lung plays a direct role in shaping the immune repertoire of RA/RA-ILD.

Modeling the assembly of the multiple domains of α-actinin-4 and its role in actin cross-linking.

The assembly of proteins into multidomain complexes is critical for their function. In eukaryotic nonmuscle cells, regulation of the homodimeric actin cross-linking protein α-actinin-4 (ACTN4) during cell migration involves signaling receptors with intrinsic tyrosine kinase activity, yet the underlying molecular mechanisms are poorly understood. As a first step to address the latter, we validate here an atomic model for the ACTN4 end region, which corresponds to a ternary complex between the N-terminal actin-binding domain (ABD) and an adjacent helical neck region of one monomer, and the C-terminal calmodulin-like domain of the opposite antiparallel monomer. Mutagenesis experiments designed to disrupt this ternary complex confirm that its formation reduces binding to F-actin. Molecular dynamics simulations show that the phosphomimic mutation Y265E increases actin binding by breaking several interactions that tether the two calponin homology domains into a closed ABD conformation. Simulations also show a disorder-to-order transition in the double phosphomimic mutant Y4E/Y31E of the 45-residue ACTN4 N-terminal region, which can inhibit actin binding by latching both calponin homology domains more tightly. Collectively, these studies provide a starting point for understanding the role of external cues in regulating ACTN4, with different phenotypes resulting from changes in the multidomain assembly of the protein.

Combinatorial diversity of Syk recruitment driven by its multivalent engagement with FcεRIγ.

Syk/Zap70 family kinases are essential for signaling via multichain immune-recognition receptors such as tetrameric (αßγ2) FcεRI. Syk activation is generally attributed to cis binding of its tandem SH2 domains to dual phosphotyrosines within FcεRIγ-ITAMs (immunoreceptor tyrosine-based activation motifs). However, the mechanistic details of Syk docking on γ homodimers are unresolved. Here, we estimate that multivalent interactions for WT Syk improve cis-oriented binding by three orders of magnitude. We applied molecular dynamics (MD), hybrid MD/worm-like chain polymer modeling, and live cell imaging to evaluate relative binding and signaling output for all possible cis and trans Syk-FcεRIγ configurations. Syk binding is likely modulated during signaling by autophosphorylation on Y130 in interdomain A, since a Y130E phosphomimetic form of Syk is predicted to lead to reduced helicity of interdomain A and alter Syk's bias for cis binding. Experiments in reconstituted γ-KO cells, whose γ subunits are linked by disulfide bonds, as well as in cells expressing monomeric ITAM or hemITAM γ-chimeras, support model predictions that short distances between γ ITAM pairs are required for trans docking. We propose that the full range of docking configurations improves signaling efficiency by expanding the combinatorial possibilities for Syk recruitment, particularly under conditions of incomplete ITAM phosphorylation.

Sequence- and structure-based computational analyses of Gram-negative tripartite efflux pumps in the context of bacterial membranes.

Gram-negative multidrug resistance currently presents a serious threat to public health with infections effectively rendered untreatable. Multiple molecular mechanisms exist that cause antibiotic resistance and in addition, the last three decades have seen slowing rates of new drug development. In this review, we summarize the use of various computational techniques for investigating the mechanisms of multidrug resistance mediated by Gram-negative tripartite efflux pumps and membranes. Recent work in our lab combines data-driven sequence and structure analyses to study the interactions and dynamics of these bacterial components. Computational studies can complement experimental methodologies for gaining crucial insights into combatting multidrug resistance.

Molecular recognition of RAS/RAF complex at the membrane: Role of RAF cysteine-rich domain.

Activation of RAF kinase involves the association of its RAS-binding domain (RBD) and cysteine-rich domain (CRD) with membrane-anchored RAS. However, the overall architecture of the RAS/RBD/CRD ternary complex and the orientations of its constituent domains at the membrane remain unclear. Here, we have combined all-atom and coarse-grained molecular dynamics (MD) simulations with experimental data to construct and validate a model of membrane-anchored CRD, and used this as a basis to explore models of membrane-anchored RAS/RBD/CRD complex. First, simulations of the CRD revealed that it anchors to the membrane via insertion of its two hydrophobic loops, which is consistent with our NMR measurements of CRD bound to nanodiscs. Simulations of the CRD in the context of membrane-anchored RAS/RBD then show how CRD association with either RAS or RBD could play an unexpected role in guiding the membrane orientations of RAS/RBD. This finding has implications for the formation of RAS-RAS dimers, as different membrane orientations of RAS expose distinct putative dimerization interfaces.

Dynamics of Intact MexAB-OprM Efflux Pump: Focusing on the MexA-OprM Interface.

Antibiotic efflux is one of the most critical mechanisms leading to bacterial multidrug resistance. Antibiotics are effluxed out of the bacterial cell by a tripartite efflux pump, a complex machinery comprised of outer membrane, periplasmic adaptor, and inner membrane protein components. Understanding the mechanism of efflux pump assembly and its dynamics could facilitate discovery of novel approaches to counteract antibiotic resistance in bacteria. We built here an intact atomistic model of the Pseudomonas aeruginosa MexAB-OprM pump in a Gram-negative membrane model that contained both inner and outer membranes separated by a periplasmic space. All-atom molecular dynamics (MD) simulations confirm that the fully assembled pump is stable in the microsecond timescale. Using a combination of all-atom and coarse-grained MD simulations and sequence covariation analysis, we characterized the interface between MexA and OprM in the context of the entire efflux pump. These analyses suggest a plausible mechanism by which OprM is activated via opening of its periplasmic aperture through a concerted interaction with MexA.

Switch Loop Flexibility Affects Substrate Transport of the AcrB Efflux Pump.

The functionally important switch loop of the trimeric multidrug transporter AcrB separates the access and deep drug binding pockets in every protomer. This loop, comprising 11-amino-acid residues, has been shown to be crucial for substrate transport, as drugs have to travel past the loop to reach the deep binding pocket and from there are transported outside the cell via the connected AcrA and TolC channels. It contains four symmetrically arranged glycine residues suggesting that flexibility is a key feature for pump activity. Upon combinatorial substitution of these glycine residues to proline, functional and structural asymmetry was observed. Proline substitutions on the PC1-proximal side completely abolished transport and reduced backbone flexibility of the switch loop, which adopted a conformation restricting the pathway toward the deep binding pocket. Two phenylalanine residues located adjacent to the substitution sensitive glycine residues play a role in blocking the pathway upon rigidification of the loop, since the removal of the phenyl rings from the rigid loop restores drug transport activity.

Tandem phosphorylation within an intrinsically disordered region regulates ACTN4 function.

Phosphorylated residues occur preferentially in the intrinsically disordered regions of eukaryotic proteins. In the disordered amino-terminal region of human α-actinin-4 (ACTN4), Tyr(4) and Tyr(31) are phosphorylated in cells stimulated with epidermal growth factor (EGF), and a mutant with phosphorylation-mimicking mutations of both tyrosines exhibits reduced interaction with actin in vitro. Cleavage of ACTN4 by m-calpain, a protease that in motile cells is predominantly activated at the rear, removes the Tyr(4) site. We found that introducing a phosphomimetic mutation at only Tyr(31) was sufficient to inhibit the interaction with actin in vitro. However, molecular dynamics simulations predicted that Tyr(31) is mostly buried and that phosphorylation of Tyr(4) would increase the solvent exposure and thus kinase accessibility of Tyr(31). In fibroblast cells, EGF stimulation increased tyrosine phosphorylation of a mutant form of ACTN4 with a phosphorylation-mimicking residue at Tyr(4), whereas a truncated mutant representing the product of m-calpain cleavage exhibited EGF-stimulated tyrosine phosphorylation at a background amount similar to that observed for a double phosphomimetic mutant of Tyr(4) and Tyr(31). We also found that inhibition of the receptor tyrosine kinases of the TAM family, such as AXL, blocked EGF-stimulated tyrosine phosphorylation of ACTN4. Mathematical modeling predicted that the kinetics of phosphorylation at Tyr(31) can be dictated by the kinase affinity for Tyr(4). This study suggests that tandem-site phosphorylation within intrinsically disordered regions provides a mechanism for a site to function as a switch to reveal a nearby function-regulating site.

FixingTIM: interactive exploration of sequence and structural data to identify functional mutations in protein families.

BACKGROUND: Knowledge of the 3D structure and functionality of proteins can lead to insight into the associated cellular processes, speed up the creation of pharmaceutical products, and develop drugs that are more effective in combating disease. METHODS: We present the design and implementation of a visual mining and analysis tool to help identify protein mutations across a family of structural models and to help discover the effect of these mutations on protein function. We integrate 3D structure and sequence information in a common visual interface; multiple linked views and a computational backbone allow comparison at the molecular and atomic levels, while a novel trend-image visual abstraction allows for the sorting and mining of large collections of sequences and of their residues. RESULTS: We evaluate our approach on the triosephosphate isomerase (TIM) family structural models and sequence data and show that our tool provides an effective, scalable way to navigate a family of proteins, as well as a means to inspect the structure and sequence of individual proteins. CONCLUSIONS: The TIM application shows that our tool can assist in the navigation of families of proteins, as well as in the exploration of individual protein structures. In conjunction with domain expert knowledge, this interactive tool can help provide biophysical insight into why specific mutations affect function and potentially suggest additional modifications to the protein that could be used to rescue functionality.

The carboxyl tail of alpha-actinin-4 regulates its susceptibility to m-calpain and thus functions in cell migration and spreading.

Alpha-actinin-4 links the cytoskeleton to sites of adhesion and has been shown to be modulated to enable cell migration. Such focal adhesions must be labile to accomplish migration, with this detachment occurring at least in part via m-calpain activation (Glading et al., 2001, 2002; Xie et al., 1998). In this study, we report that alpha-actinin-4 is initially cleaved by m-calpain between tyrosine 13 and glycine. Removal of the first 13 amino acids does not affect alpha-actinin-4 binding to actin filaments and its localization within fibroblasts but drives cell migration with less persistence. Binding of phosphoinositides PI(4,5)P2, PI(3,4,5)P3 and PI(3,4)P2 to alpha-actinin-4, as well as binding of alpha-actinin-4 to actin filaments all inhibit m-calpain cleavage of ACTN4 between tyrosine 13 and glycine 14. Interestingly, the carboxyl terminus of alpha-actinin-4 including its calcium binding motifs, is inhibitory for a secondary cleavage of alpha-actinin-4 between lysine 283 and valine 284. The minimal length of inhibitory domain is mapped to the last 11 amino acids of alpha-actinin-4. The C-terminal tail of alpha-actinin-4 is essential for maintaining its normal actin binding activity and localization within cytoplasm and also its colocalization with actin in the lamellipodia of locomoting fibroblasts. Live cell imaging reveals that the 1-890 fragment fails to rescue neither the basal or growth factor-stimulated migration nor the revert the spread area of fibroblasts to the level of NR6WT. These findings suggest that the C-terminal tail of alpha-actinin-4 is essential for its function in cell migration and adhesion to substratum.