Structural and functional interactions between the EF hand domain and S2-S3 loop in the type-1 ryanodine receptor ion channel.

Previous cryo-electron micrographs suggested that the skeletal muscle Ca2+ release channel, ryanodine receptor (RyR)1, is regulated by intricate interactions between the EF hand Ca2+ binding domain and the cytosolic loop (S2-S3 loop). However, the precise molecular details of these interactions and functional consequences of the interactions remain elusive. Here, we used molecular dynamics simulations to explore the specific amino acid pairs involved in hydrogen bond interactions within the EF hand-S2-S3 loop interface. Our simulations unveiled two key interactions: (1) K4101 (EF hand) with D4730 (S2-S3 loop) and (2) E4075, Q4078, and D4079 (EF hand) with R4736 (S2-S3 loop). To probe the functional significance of these interactions, we constructed mutant RyR1 complementary DNAs and expressed them in HEK293 cells for [3H]ryanodine binding assays. Our results demonstrated that mutations in the EF hand, specifically K4101E and K4101M, resulted in reduced affinities for Ca2+/Mg2+-dependent inhibitions. Interestingly, the K4101E mutation increased the affinity for Ca2+-dependent activation. Conversely, mutations in the S2-S3 loop, D4730K and D4730N, did not significantly change the affinities for Ca2+/Mg2+-dependent inhibitions. Our previous finding that skeletal disease-associated RyR1 mutations, R4736Q and R4736W, impaired Ca2+-dependent inhibition, is consistent with the current results. In silico mutagenesis analysis aligned with our functional data, indicating altered hydrogen bonding patterns upon mutations. Taken together, our findings emphasize the critical role of the EF hand-S2-S3 loop interaction in Ca2+/Mg2+-dependent inhibition of RyR1 and provide insights into potential therapeutic strategies targeting this domain interaction for the treatment of skeletal myopathies.

Oncogenic KRAS G12C: Kinetic and redox characterization of covalent inhibition.

The recent development of mutant-selective inhibitors for the oncogenic KRASG12C allele has generated considerable excitement. These inhibitors covalently engage the mutant C12 thiol located within the phosphoryl binding loop of RAS, locking the KRASG12C protein in an inactive state. While clinical trials of these inhibitors have been promising, mechanistic questions regarding the reactivity of this thiol remain. Here, we show by NMR and an independent biochemical assay that the pKa of the C12 thiol is depressed (pKa ∼7.6), consistent with susceptibility to chemical ligation. Using a validated fluorescent KRASY137W variant amenable to stopped-flow spectroscopy, we characterized the kinetics of KRASG12C fluorescence changes upon addition of ARS-853 or AMG 510, noting that at low temperatures, ARS-853 addition elicited both a rapid first phase of fluorescence change (attributed to binding, Kd = 36.0 ± 0.7 µM) and a second, slower pH-dependent phase, taken to represent covalent ligation. Consistent with the lower pKa of the C12 thiol, we found that reversible and irreversible oxidation of KRASG12C occurred readily both in vitro and in the cellular environment, preventing the covalent binding of ARS-853. Moreover, we found that oxidation of the KRASG12C Cys12 to a sulfinate altered RAS conformation and dynamics to be more similar to KRASG12D in comparison to the unmodified protein, as assessed by molecular dynamics simulations. Taken together, these findings provide insight for future KRASG12C drug discovery efforts, and identify the occurrence of G12C oxidation with currently unknown biological ramifications.

Structural and functional interactions between the Ca2+-, ATP-, and caffeine-binding sites of skeletal muscle ryanodine receptor (RyR1).

Ryanodine receptor type 1 (RyR1) releases Ca2+ ions from the sarcoplasmic reticulum of skeletal muscle cells to initiate muscle contraction. Multiple endogenous and exogenous effectors regulate RyR1, such as ATP, Ca2+, caffeine (Caf), and ryanodine. Cryo-EM identified binding sites for the three coactivators Ca2+, ATP, and Caf. However, the mechanism of coregulation and synergy between these activators remains to be determined. Here, we used [3H]ryanodine ligand-binding assays and molecular dynamics simulations to test the hypothesis that both the ATP- and Caf-binding sites communicate with the Ca2+-binding site to sensitize RyR1 to Ca2+. We report that either phosphomethylphosphonic acid adenylate ester (AMPPCP), a nonhydrolyzable ATP analog, or Caf can activate RyR1 in the absence or the presence of Ca2+. However, enhanced RyR1 activation occurred in the presence of Ca2+, AMPPCP, and Caf. In the absence of Ca2+, Na+ inhibited [3H]ryanodine binding without impairing RyR1 activation by AMPPCP and Caf. Computational analysis suggested that Ca2+-, ATP-, and Caf-binding sites modulate RyR1 protein stability through interactions with the carboxyterminal domain and other domains in the activation core. In the presence of ATP and Caf but the absence of Ca2+, Na+ is predicted to inhibit RyR1 by interacting with the Ca2+-binding site. Our data suggested that ATP and Caf binding affected the conformation of the Ca2+-binding site, and conversely, Ca2+ binding affected the conformation of the ATP- and Caf-binding sites. We conclude that Ca2+, ATP, and Caf regulate RyR1 through a network of allosteric interactions involving the Ca2+-, ATP-, and Caf-binding sites.

Mapping co-regulatory interactions among ligand-binding sites in ryanodine receptor 1.

Ryanodine receptor 1 (RyR1) is an intracellular calcium ion (Ca2+ ) release channel required for skeletal muscle contraction. Although cryo-electron microscopy identified binding sites of three coactivators Ca2+ , ATP, and caffeine (CFF), the mechanism of co-regulation and synergy of these activators is unknown. Here, we report allosteric connections among the three ligand-binding sites and pore region in (i) Ca2+ bound-closed, (ii) ATP/CFF bound-closed, (iii) Ca2+ /ATP/CFF bound-closed, and (iv) Ca2+ /ATP/CFF bound-open RyR1 states. We identified two dominant networks of interactions that mediate communication between the Ca2+ -binding site and pore region in Ca2+ bound-closed state, which partially overlapped with the pore communications in ATP/CFF bound-closed RyR1 state. In Ca2+ /ATP/CFF bound-closed and -open RyR1 states, co-regulatory interactions were analogous to communications in the Ca2+ bound-closed and ATP/CFF bound-closed states. Both ATP- and CFF-binding sites mediate communication between the Ca2+ -binding site and the pore region in Ca2+ /ATP/CFF bound-open RyR1 structure. We conclude that Ca2+ , ATP, and CFF propagate their effects to the pore region through a network of overlapping interactions that mediate allosteric control and molecular synergy in channel regulation.

Identifying crucial E-protein residues responsible for unusual stability of Zika virus envelope.

An outbreak of Zika virus (ZIKV) infections in 2015-16 that caused microcephaly and other congenital abnormalities in newborns prompted intense research across the globe. These studies have suggested that ZIKV can survive high temperatures and harsh physiological conditions, unlike the other flaviviruses such as dengue virus (DENV). In contrast, recent cryo-electron microscopy studies have shown very similar architecture of the ZIKV and DENV envelopes that constitute the primary level of viral protection. Encouraged by these findings, here we attempt to identify the crucial protein residues that make the ZIKV envelope so robust by employing coarse-grained and all-atomic molecular dynamics simulations and computational mutagenesis studies. In accordance with more recent cryo-electron microscopy findings, our simulation results exhibited stable ZIKV envelope protein shell both at 29oC and 40°C, whereas the DENV2 shell loosened up significantly at 40°C. Subsequently, we simulated a series of ZIKV variants to identify the specific domain and residues involved in maintaining the structural integrity of the viral protein shell at high temperatures. Our results suggest that the DIII domain-more specifically, the CD- and FG-loop residues of the ZIKV protein shell-play a crucial role in making the virus envelope thermostable by inducing strong raft-raft interactions. These findings can accelerate the rational design of ZIKV therapeutics.

A central core disease mutation in the Ca2+-binding site of skeletal muscle ryanodine receptor impairs single-channel regulation.

Cryoelectron microscopy and mutational analyses have shown that type 1 ryanodine receptor (RyR1) amino acid residues RyR1-E3893, -E3967, and -T5001 are critical for Ca2+-mediated activation of skeletal muscle Ca2+ release channel. De novo missense mutation RyR1-Q3970K in the secondary binding sphere of Ca2+ was reported in association with central core disease (CCD) in a 2-yr-old boy. Here, we characterized recombinant RyR1-Q3970K mutant by cellular Ca2+ release measurements, single-channel recordings, and computational methods. Caffeine-induced Ca2+ release studies indicated that RyR1-Q3970K formed caffeine-sensitive, Ca2+-conducting channel in HEK293 cells. However, in single-channel recordings, RyR1-Q3970K displayed low Ca2+-dependent channel activity and greatly reduced activation by caffeine or ATP. A RyR1-Q3970E mutant corresponds to missense mutation RyR2-Q3925E associated with arrhythmogenic syndrome in cardiac muscle. RyR1-Q3970E also formed caffeine-induced Ca2+ release in HEK293 cells and exhibited low activity in the presence of the activating ligand Ca2+ but, in contrast to RyR1-Q3970K, was activated by ATP and caffeine in single-channel recordings. Computational analyses suggested distinct structural rearrangements in the secondary binding sphere of Ca2+ of the two mutants, whereas the interaction of Ca2+ with directly interacting RyR1 amino acid residues Glu3893, Glu3967, and Thr5001 was only minimally affected. We conclude that RyR1-Q3970 has a critical role in Ca2+-dependent activation of RyR1 and that a missense RyR1-Q3970K mutant may give rise to myopathy in skeletal muscle.

Ca2+-mediated activation of the skeletal-muscle ryanodine receptor ion channel.

Cryo-electron micrograph studies recently have identified a Ca2+-binding site in the 2,200-kDa ryanodine receptor ion channel (RyR1) in skeletal muscle. To clarify the role of this site in regulating RyR1 activity, here we applied mutational, electrophysiological, and computational methods. Three amino acid residues that interact directly with Ca2+ were replaced, and these RyR1 variants were expressed in HEK293 cells. Single-site RyR1-E3893Q, -E3893V, -E3967Q, -E3967V, and -T5001A variants and double-site RyR1-E3893Q/E3967Q and -E3893V/E3967V variants displayed cellular Ca2+ release in response to caffeine, which indicated that they retained functionality as caffeine-sensitive, Ca2+-conducting channels in the HEK293 cell system. Using [3H]ryanodine binding and single-channel measurements of membrane isolates, we found that single- and double-site RyR1-E3893 and -E3967 variants are not activated by Ca2+ We also noted that RyR1-E3893Q/E3967Q and -E3893V/E3967V variants maintain caffeine- and ATP-induced activation and that RyR1-E3893Q/E3967Q is inhibited by Mg2+ and elevated Ca2+ RyR1-T5001A exhibited decreased Ca2+ sensitivity compared with WT-RyR1 in single-channel measurements. Computational methods suggested that electrostatic interactions between Ca2+ and negatively charged glutamate residues have a critical role in transducing the functional effects of Ca2+ on RyR1. We conclude that the removal of negative charges in the recently identified RyR1 Ca2+-binding site impairs RyR1 activation by physiological Ca2+ concentrations and results in loss of binding to Ca2+ or reduced Ca2+ affinity of the binding site.

G4941K substitution in the pore-lining S6 helix of the skeletal muscle ryanodine receptor increases RyR1 sensitivity to cytosolic and luminal Ca2.

The ryanodine receptor ion channel RyR1 is present in skeletal muscle and has a large cytoplasmic N-terminal domain and smaller C-terminal pore-forming domain comprising six transmembrane helices, a pore helix, and a selectivity filter. The RyR1 S6 pore-lining helix has two conserved glycines, Gly-4934 and Gly-4941, that facilitate RyR1 channel gating by providing S6 flexibility and minimizing amino acid clashes. Here, we report that substitution of Gly-4941 with Asp or Lys results in functional channels as indicated by caffeine-induced Ca2+ release response in HEK293 cells, whereas a low response of the corresponding Gly-4934 variants suggested loss of function. Following purification, the RyR1 mutants G4934D, G4934K, and G4941D did not noticeably conduct Ca2+ in single-channel measurements. Gly-4941 replacement with Lys resulted in channels having reduced K+ conductance and reduced selectivity for Ca2+ compared with wildtype. RyR1-G4941K did not fully close at nanomolar cytosolic Ca2+ concentrations and nearly fully opened at 2 µm cytosolic or sarcoplasmic reticulum luminal Ca2+, and Ca2+- and voltage-dependent regulation of RyR1-G4941K mutant channels was demonstrated. Computational methods and single-channel recordings indicated that the open G4941K variant results in the formation of a salt bridge to Asp-4938. In contrast, wildtype RyR1 was closed and not activated by luminal Ca2+ at low cytosolic Ca2+ levels. A model suggested that luminal Ca2+ activates RyR1 by accessing a recently identified cytosolic Ca2+-binding site in the open channel as the Ca2+ ions pass through the pore.

Structural Plasticity of Cholesteryl Ester Transfer Protein Assists the Lipid Transfer Activity.

Cholesteryl ester transfer protein (CETP) mediates the transfer of cholesteryl esters (CEs) and triglycerides between different lipoproteins. Recent studies have shown that blocking the function of CETP can increase the level of HDL cholesterol in blood plasma and suppress the risk of cardiovascular disease. Hence, understanding the structure, dynamics, and mechanism by which CETP transfers the neutral lipids has received tremendous attention in last decade. Although the recent crystal structure has provided direct evidence of the existence of strongly bound CEs in the CETP core, very little is known about the mechanism of CE/triglyceride transfer by CETP. In this study, we explore the large scale dynamics of CETP by means of multimicrosecond molecular dynamics simulations and normal mode analysis, which provided a wealth of detailed information about the lipid transfer mechanism of CETP. Results show that the bound CEs intraconvert between bent and linear conformations in the CETP core tunnel as a consequence of the high degree of conformational flexibility of the protein. During the conformational switching, there occurred a significant reduction in hydrophobic contacts between the CEs and CETP, and a continuous tunnel traversing across the CETP long axis appeared spontaneously. Thus, our results support the recently proposed "tunnel mechanism" of CETP from cryo-EM studies for the transfer of neutral lipids between different lipoproteins. The detailed understanding obtained here could help in devising methods to prevent CETP function as a cardiovascular disease therapeutic.

Modulation of the mitochondrial voltage dependent anion channel (VDAC) by curcumin.

Voltage dependent anion channel (VDAC) of mitochondria plays a crucial role in apoptosis. Human VDAC-1, reconstituted in planar lipid bilayer showed reduced conductance when treated with curcumin. Curcumin interacts with residues in the α helical N-terminus of VDAC and in the channel wall, as revealed by molecular docking, followed by mutational analysis. N-terminus mimicking peptide showed conformational changes in circular dichroism, upon curcumin treatment. We propose that the interaction of curcumin with amino acids in N-terminus and in channel wall fixes the α helix in closed conformation. This restricts its movement which is required for the opening of the channel.

Regulation of intestinal SGLT1 by catestatin in hyperleptinemic type 2 diabetic mice.

The small intestine is the major site for nutrient absorption that is critical in maintenance of euglycemia. Leptin, a key hormone involved in energy homeostasis, directly affects nutrient transport across the intestinal epithelium. Catestatin (CST), a 21-amino acid peptide derived from proprotein chromogranin A, has been shown to modulate leptin signaling. Therefore, we reasoned that leptin and CST could modulate intestinal Na(+)-glucose transporter 1 (SGLT1) expression in the context of obesity and diabetes. We found that hyperleptinemic db/db mice exhibit increased mucosal mass, associated with an enhanced proliferative response and decreased apoptosis in intestinal crypts, a finding absent in leptin-deficient ob/ob mice. Intestinal SGLT1 abundance was significantly decreased in hyperleptinemic but not leptin-deficient mice, indicating leptin regulation of SGLT1 expression. Phlorizin, a SGLT1/2 inhibitor, was without effect in an oral glucose tolerance test in db/db mice. The alterations in architecture and SGLT1 abundance were not accompanied by changes in the localization of intestinal alkaline phosphatase, indicating intact differentiation. Treatment of db/db mice with CST restored intestinal SGLT1 abundance and intestinal turnover, suggesting a cross-talk between leptin and CST, without affecting plasma leptin levels. Consistent with this hypothesis, we identified structural homology between CST and the AB-loop of leptin and protein-protein docking revealed binding of CST and leptin with the Ig-like binding site-III of the leptin receptor. In summary, downregulation of SGLT1 in an obese type 2 diabetic mouse model with hyperleptinemia is presumably mediated via the short form of the leptin receptor and reduces overt hyperglycemia.

Naturally occurring variants of the dysglycemic peptide pancreastatin: differential potencies for multiple cellular functions and structure-function correlation.

Pancreastatin (PST), a chromogranin A-derived peptide, is a potent physiological inhibitor of glucose-induced insulin secretion. PST also triggers glycogenolysis in liver and reduces glucose uptake in adipocytes and hepatocytes. Here, we probed for genetic variations in PST sequence and identified two variants within its functionally important carboxyl terminus domain: E287K and G297S. To understand functional implications of these amino acid substitutions, we tested the effects of wild-type (PST-WT), PST-287K, and PST-297S peptides on various cellular processes/events. The rank order of efficacy to inhibit insulin-stimulated glucose uptake was: PST-297S > PST-287K > PST-WT. The PST peptides also displayed the same order of efficacy for enhancing intracellular nitric oxide and Ca(2+) levels in various cell types. In addition, PST peptides activated gluconeogenic genes in the following order: PST-297S ≈ PST-287K > PST-WT. Consistent with these in vitro results, the common PST variant allele Ser-297 was associated with significantly higher (by ∼17 mg/dl, as compared with the wild-type Gly-297 allele) plasma glucose level in our study population (n = 410). Molecular modeling and molecular dynamics simulations predicted the following rank order of α-helical content: PST-297S > PST-287K > PST-WT. Corroboratively, circular dichroism analysis of PST peptides revealed significant differences in global structures (e.g. the order of propensity to form α-helix was: PST-297S ≈ PST-287K > PST-WT). This study provides a molecular basis for enhanced potencies/efficacies of human PST variants (likely to occur in ∼300 million people worldwide) and has quantitative implications for inter-individual variations in glucose/insulin homeostasis.

Elucidation of the Molecular Basis and Cellular Functions of Vinculin-Actin Directional Catch Bonding.

The ability of cells and tissues to differentially resist or adapt to mechanical forces applied in distinct directions is mediated by the ability of load-bearing proteins to preferentially maintain physical linkages in certain directions. However, the molecular basis and biological consequences of directional force-sensitive binding are unclear. Vinculin (Vcn) is a load-bearing linker protein that exhibits directional catch bonding due to interactions between the Vcn tail domain (Vt) and filamentous (F)-actin. We developed a computational approach to predict Vcn residues involved in directional catch bonding and produced a set of associated Vcn variants with unaltered Vt structure, actin binding, or phospholipid interactions. Incorporation of these variants into Vcn biosensors did not perturb Vcn conformation, but reduced Vcn loading consistent with loss of directional catch bonding. Expression of Vcn variants perturbed the coalignment of FAs and F-actin and directed cell migration, establishing key cellular functions for Vcn directional catch bonding.

Molecular basis and cellular functions of vinculin-actin directional catch bonding.

The ability of cells and tissues to respond differentially to mechanical forces applied in distinct directions is mediated by the ability of load-bearing proteins to preferentially maintain physical linkages in certain directions. However, the molecular basis and biological consequences of directional force-sensitive binding remain unclear. Vinculin (Vcn) is a load-bearing linker protein that exhibits directional catch bonding due to interactions between the Vcn tail domain (Vt) and filamentous (F)-actin. We developed a computational approach to predict Vcn residues involved in directional catch bonding and produced a set of associated Vcn variants with unaltered Vt structure, actin binding, or phospholipid interactions. Incorporation of the variants did not affect Vcn activation but reduced Vcn loading and altered exchange dynamics, consistent with the loss of directional catch bonding. Expression of Vcn variants perturbed the coordination of subcellular structures and cell migration, establishing key cellular functions for Vcn directional catch bonding.

Urolithin A analog inhibits castration-resistant prostate cancer by targeting the androgen receptor and its variant, androgen receptor-variant 7.

We investigated the efficacy of a small molecule ASR-600, an analog of Urolithin A (Uro A), on blocking androgen receptor (AR) and its splice variant AR-variant 7 (AR-V7) signaling in castration-resistant prostate cancer (CRPC). ASR-600 effectively suppressed the growth of AR+ CRPC cells by inhibiting AR and AR-V7 expressions; no effect was seen in AR- CRPC and normal prostate epithelial cells. Biomolecular interaction assays revealed ASR-600 binds to the N-terminal domain of AR, which was further confirmed by immunoblot and subcellular localization studies. Molecular studies suggested that ASR-600 promotes the ubiquitination of AR and AR-V7 resulting in the inhibition of AR signaling. Microsomal and plasma stability studies suggest that ASR-600 is stable, and its oral administration inhibits tumor growth in CRPC xenografted castrated and non-castrated mice. In conclusion, our data suggest that ASR-600 enhances AR ubiquitination in both AR+ and AR-V7 CRPC cells and inhibits their growth in vitro and in vivo models.

Whole proteome mapping of compound-protein interactions.

Off-target binding is one of the primary causes of toxic side effects of drugs in clinical development, resulting in failures of clinical trials. While off-target drug binding is a known phenomenon, experimental identification of the undesired protein binders can be prohibitively expensive due to the large pool of possible biological targets. Here, we propose a new strategy combining chemical similarity principle and deep learning to enable proteome-wide mapping of compound-protein interactions. We have developed a pipeline to identify the targets of bioactive molecules by matching them with chemically similar annotated "bait" compounds and ranking them with deep learning. We have constructed a user-friendly web server for drug-target identification based on chemical similarity (DRIFT) to perform searches across annotated bioactive compound datasets, thus enabling high-throughput, multi-ligand target identification, as well as chemical fragmentation of target-binding moieties.

Enhanced BRAF engagement by NRAS mutants capable of promoting melanoma initiation.

A distinct profile of NRAS mutants is observed in each tumor type. It is unclear whether these profiles are determined by mutagenic events or functional differences between NRAS oncoproteins. Here, we establish functional hallmarks of NRAS mutants enriched in human melanoma. We generate eight conditional, knock-in mouse models and show that rare melanoma mutants (NRAS G12D, G13D, G13R, Q61H, and Q61P) are poor drivers of spontaneous melanoma formation, whereas common melanoma mutants (NRAS Q61R, Q61K, or Q61L) induce rapid tumor onset with high penetrance. Molecular dynamics simulations, combined with cell-based protein-protein interaction studies, reveal that melanomagenic NRAS mutants form intramolecular contacts that enhance BRAF binding affinity, BRAF-CRAF heterodimer formation, and MAPK > ERK signaling. Along with the allelic series of conditional mouse models we describe, these results establish a mechanistic basis for the enrichment of specific NRAS mutants in human melanoma.

Functional Gly297Ser Variant of the Physiological Dysglycemic Peptide Pancreastatin Is a Novel Risk Factor for Cardiometabolic Disorders.

Pancreastatin (PST), a chromogranin A-derived potent physiological dysglycemic peptide, regulates glucose/insulin homeostasis. We have identified a nonsynonymous functional PST variant (p.Gly297Ser; rs9658664) that occurs in a large section of human populations. Association analysis of this single nucleotide polymorphism with cardiovascular/metabolic disease states in Indian populations (n = 4,300 subjects) displays elevated plasma glucose, glycosylated hemoglobin, diastolic blood pressure, and catecholamines in Gly/Ser subjects as compared with wild-type individuals (Gly/Gly). Consistently, the 297Ser allele confers an increased risk (∼1.3-1.6-fold) for type 2 diabetes/hypertension/coronary artery disease/metabolic syndrome. In corroboration, the variant peptide (PST-297S) displays gain-of-potency in several cellular events relevant for cardiometabolic disorders (e.g., increased expression of gluconeogenic genes, increased catecholamine secretion, and greater inhibition of insulin-stimulated glucose uptake) than the wild-type peptide. Computational docking analysis and molecular dynamics simulations show higher affinity binding of PST-297S peptide with glucose-regulated protein 78 (GRP78) and insulin receptor than the wild-type peptide, providing a mechanistic basis for the enhanced activity of the variant peptide. In vitro binding assays validate these in silico predictions of PST peptides binding to GRP78 and insulin receptor. In conclusion, the PST 297Ser allele influences cardiovascular/metabolic phenotypes and emerges as a novel risk factor for type 2 diabetes/hypertension/coronary artery disease in human populations.

Two-input protein logic gate for computation in living cells.

Advances in protein design have brought us within reach of developing a nanoscale programming language, in which molecules serve as operands and their conformational states function as logic gates with precise input and output behaviors. Combining these nanoscale computing agents into larger molecules and molecular complexes will allow us to write and execute "code". Here, in an important step toward this goal, we report an engineered, single protein design that is allosterically regulated to function as a 'two-input logic OR gate'. Our system is based on chemo- and optogenetic regulation of focal adhesion kinase. In the engineered FAK, all of FAK domain architecture is retained and key intramolecular interactions between the kinase and the FERM domains are externally controlled through a rapamycin-inducible uniRapR module in the kinase domain and a light-inducible LOV2 module in the FERM domain. Orthogonal regulation of protein function was possible using the chemo- and optogenetic switches. We demonstrate that dynamic FAK activation profoundly increased cell multiaxial complexity in the fibrous extracellular matrix microenvironment and decreased cell motility. This work provides proof-of-principle for fine multimodal control of protein function and paves the way for construction of complex nanoscale computing agents.

Divergent Mechanisms Activating RAS and Small GTPases Through Post-translational Modification.

RAS is a founding member of the RAS superfamily of GTPases. These small 21 kDa proteins function as molecular switches to initialize signaling cascades involved in various cellular processes, including gene expression, cell growth, and differentiation. RAS is activated by GTP loading and deactivated upon GTP hydrolysis to GDP. Guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs) accelerate GTP loading and hydrolysis, respectively. These accessory proteins play a fundamental role in regulating activities of RAS superfamily small GTPase via a conserved guanine binding (G)-domain, which consists of five G motifs. The Switch regions lie within or proximal to the G2 and G3 motifs, and undergo dynamic conformational changes between the GDP-bound "OFF" state and GTP-bound "ON" state. They play an important role in the recognition of regulatory factors (GEFs and GAPs) and effectors. The G4 and G5 motifs are the focus of the present work and lie outside Switch regions. These motifs are responsible for the recognition of the guanine moiety in GTP and GDP, and contain residues that undergo post-translational modifications that underlie new mechanisms of RAS regulation. Post-translational modification within the G4 and G5 motifs activates RAS by populating the GTP-bound "ON" state, either through enhancement of intrinsic guanine nucleotide exchange or impairing GAP-mediated down-regulation. Here, we provide a comprehensive review of post-translational modifications in the RAS G4 and G5 motifs, and describe the role of these modifications in RAS activation as well as potential applications for cancer therapy.