Inactive Conformational States Research Articles

Side-chain dynamics of the α1B -adrenergic receptor determined by NMR via methyl relaxation.

G protein-coupled receptors (GPCRs) are medically important membrane proteins that sample inactive, intermediate, and active conformational states characterized by relatively slow interconversions (~μs-ms). On a faster timescale (~ps-ns), the conformational landscape of GPCRs is governed by the rapid dynamics of amino acid side chains. Such dynamics are essential for protein functions such as ligand recognition and allostery. Unfortunately, technical challenges have almost entirely precluded the study of side-chain dynamics for GPCRs. Here, we investigate the rapid side-chain dynamics of a thermostabilized α1B -adrenergic receptor (α1B -AR) as probed by methyl relaxation. We determined order parameters for Ile, Leu, and Val methyl groups in the presence of inverse agonists that bind orthosterically (prazosin, tamsulosin) or allosterically (conopeptide ρ-TIA). Despite the differences in the ligands, the receptor's overall side-chain dynamics are very similar, including those of the apo form. However, ρ-TIA increases the flexibility of Ile1764×56 and possibly of Ile2145×49 , adjacent to Pro2155×50 of the highly conserved P5×50 I3×40 F6×44 motif crucial for receptor activation, suggesting differences in the mechanisms for orthosteric and allosteric receptor inactivation. Overall, increased Ile side-chain rigidity was found for residues closer to the center of the membrane bilayer, correlating with denser packing and lower protein surface exposure. In contrast to two microbial membrane proteins, in α1B -AR Leu exhibited higher flexibility than Ile side chains on average, correlating with the presence of Leu in less densely packed areas and with higher protein-surface exposure than Ile. Our findings demonstrate the feasibility of studying receptor-wide side-chain dynamics in GPCRs to gain functional insights.

Coupling of conformational dynamics and inhibitor binding in the phosphodiesterase-5 family.

Phosphodiesterase-5 (PDE5) is responsible for regulating the concentration of the second messenger molecule cGMP by hydrolyzing it into 5'-GMP. PDE5 is implicated in erectile dysfunction and cardiovascular diseases. The substrate binding site in the catalytic domain of PDE5 is surrounded by several dynamic structural motifs (including the α14 helix, M-loop, and H-loop) that are known to switch between inactive and active conformational states via currently unresolved structural intermediates. We evaluated the conformational dynamics of these structural motifs in the apo state and upon binding of an allosteric inhibitor (evodiamine) or avanafil, a competitive inhibitor. We employed enhanced sampling-based replica exchange solute scaling (REST2) method, principal component analysis (PCA), time-lagged independent component analysis (tICA), molecular dynamics (MD) simulations, and well-tempered metadynamics simulations to probe the conformational changes in these structural motifs. Our results support a regulatory mechanism for PDE5, where the α14 helix alternates between an inward (lower activity) conformation and an outward (higher activity) conformation that is accompanied by the folding/unfolding of the α8' and α8″ helices of the H-loop. When the allosteric inhibitor evodiamine is bound to PDE5, the inward (inactive) state of the α14 helix is preferred, thus preventing substrate access to the catalytic site. In contrast, competitive inhibitors of PDE5 block catalysis by occupying the active site accompanied by stabilization of the outward conformation of the α14 helix. Defining the conformational dynamics underlying regulation of PDE5 activation will be helpful in rational design of next-generation small molecules modulators of PDE5 activity.

Classification of GTP-dependent K-Ras4B active and inactive conformational states.

Classifying reliably active and inactive molecular conformations of wildtype (WT) and mutated oncogenic proteins is a key, ongoing challenge in molecular cancer studies. Here, we probe the GTP-bound K-Ras4B conformational dynamics using long-time atomistic molecular dynamics (MD) simulations. We extract and analyze the detailed underlying free energy landscape of WT K-Ras4B. We use two key reaction coordinates, labeled d1 and d2 (i.e., distances coordinating the Pβ atom of the GTP ligand with two key residues, T35 and G60), shown to correlate closely with activities of WT and mutated K-Ras4B. However, our new K-Ras4B conformational kinetics study reveals a more complex network of equilibrium Markovian states. We show that a new reaction coordinate is required to account for the orientation of acidic K-Ras4B sidechains such as D38 with respect to the interface with binding effector RAF1 and rationalize the activation/inactivation propensities and the corresponding molecular binding mechanisms. We use this understanding to unveil how a relatively conservative mutation (i.e., D33E, in the switch I region) can lead to significantly different activation propensities compared with WT K-Ras4B. Our study sheds new light on the ability of residues near the K-Ras4B-RAF1 interface to modulate the network of salt bridges at the binding interface with the RAF1 downstream effector and, thus, to influence the underlying GTP-dependent activation/inactivation mechanism. Altogether, our hybrid MD-docking modeling approach enables the development of new in silico methods for quantitative assessment of activation propensity changes (e.g., due to mutations or local binding environment). It also unveils the underlying molecular mechanisms and facilitates the rational design of new cancer drugs.

Abstract PR013: Novel mechanisms of ezrin function in osteosarcoma metastasis

Abstract Ezrin, a member of the ezrin/radixin/moesin protein family, functions as a cytoskeletal linker to mediate cell adhesion, motility, proliferation, and programmed death in different tissues. Ezrin plays a critical role in osteosarcoma (OS) metastasis to the lungs. Even though ezrin has no function in proliferation of primary tumor cells, OS cells lacking ezrin do not metastasize to the lungs. Even the OS cells that extravasate in lung capillaries fail to grow metastatic foci in the absence of ezrin. The protein is believed to be in an inactive (closed) conformational state in the cytoplasm. Phosphorylated ezrin acquired an active (open) conformation and relocates to the plasma membrane. The exact molecular mechanism of how ezrin in the membrane contributes to OS metastasis remains elusive. In this study, we propose an alternative mechanism for ezrin in the cytoplasm through direct interaction with mRNA and new protein partners that are involved in stress granules. We discovered the first in class ezrin inhibitor, NSC305787, though a surface plasmon resonance screening experiment and validated its biological activities. We demonstrated that NSC305787 directly binds to ezrin protein and inhibits ezrin mediated OS metastasis in three different mouse models including a transgenic mouse with spontaneous lung metastasis and primary xenografts tumors that were removed by amputation for metastasis development. We then used the small molecule inhibitor ezrin as molecular probe to discover novel interaction partners of ezrin in the cytoplasm. Affinity column capture of ezrin and NSC305787 competition followed by mass spec analysis identified a number of novel ezrin binding proteins whose interaction was disrupted by NSC305787 in OS cell lysates. At least seven of these novel interactors were RNA binding proteins (DDX3, Caprin-1, FAK, FXR1, PABP1, EWS, FUS) that were implicated in translation regulation and found in stress granules, which are membrane-less cytoplasmic granules where particular mRNA species are stored during stress. Supporting ezrin’s role in stress granule formation, we demonstrated that ezrin knockdown or inhibition by NSC305787 affects the expression levels of a set of stress response genes. In addition to interacting with RNA binding proteins, we also identified several mRNAs preferentially associated with ezrin in OS cells by RIP-Seq (Ribonuclear protein Immuno-precipitation). Expression of ezrin binding target mRNAs correlated with presence of ezrin and can be modulated by the ezrin inhibitor. Therefore, we propose that ezrin may regulate target gene expression by directly binding to mRNA and interacting with a number of stress granule proteins. Our data challenges the conventional molecular mechanism in the field that required ezrin phosphorylation and membrane localization and proposes a novel mechanism of action in the cytoplasm. Citation Format: Aykut Uren, Emre Deniz, Taryn Shaw, Jeffrey Toretsky, Luiz O. Penalva. Novel mechanisms of ezrin function in osteosarcoma metastasis [abstract]. In: Proceedings of the AACR Special Conference: Sarcomas; 2022 May 9-12; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(18_Suppl):Abstract nr PR013.

Structural basis of retinal membrane guanylate cyclase regulation by GCAP1 and RD3.

Retinal membrane guanylate cyclases (RetGC1 and RetGC2) are expressed in photoreceptor rod and cone cells, where they promote the onset of visual recovery during phototransduction. The catalytic activity of RetGCs is regulated by their binding to regulatory proteins, guanylate cyclase activating proteins (GCAP1-5) and the retinal degeneration 3 protein (RD3). RetGC1 is activated by its binding to Ca2+-free/Mg2+-bound GCAP1 at low cytosolic Ca2+ levels in light-activated photoreceptors. By contrast, RetGC1 is inactivated by its binding to Ca2+-bound GCAP1 and/or RD3 at elevated Ca2+ levels in dark-adapted photoreceptors. The Ca2+ sensitive cyclase activation helps to replenish the cytosolic cGMP levels in photoreceptors during visual recovery. Mutations in RetGC1, GCAP1 or RD3 that disable the Ca2+-dependent regulation of cyclase activity are genetically linked to rod/cone dystrophies and other inherited forms of blindness. Here I review the structural interaction of RetGC1 with GCAP1 and RD3. I propose a two-state concerted model in which the dimeric RetGC1 allosterically switches between active and inactive conformational states with distinct quaternary structures that are oppositely stabilized by the binding of GCAP1 and RD3. The binding of Ca2+-free/Mg2+-bound GCAP1 is proposed to activate the cyclase by stabilizing RetGC1 in an active conformation (R-state), whereas Ca2+-bound GCAP1 and/or RD3 inhibit the cyclase by locking RetGC1 in an inactive conformation (T-state). Exposed hydrophobic residues in GCAP1 (residues H19, Y22, M26, F73, V77, W94) are essential for cyclase activation and could be targeted by rational drug design for the possible treatment of rod/cone dystrophies.

Functional Loop Dynamics and Characterization of the Inactive State of the NS2B-NS3 Dengue Protease due to Allosteric Inhibitor Binding.

Dengue virus, a flavivirus that causes dengue shock syndrome and dengue hemorrhagic fever, is currently prevalent worldwide. A two-component protease (NS2B-NS3) is essential for maturation, representing an important target for designing anti-flavivirus drugs. Previously, consideration has been centered on developing active-site inhibitors of NS2B-NS3pro. However, the flat and charged nature of its active site renders difficulties in developing inhibitors, suggesting an alternative strategy for identifying allosteric inhibitors. The allosterically sensitive site of the dengue protease is located near Ala125, between the 120s loop and 150s loop. Using atomistic molecular dynamics simulations, we have explored the protease's conformational dynamics upon binding of an allosteric inhibitor. Furthermore, characterization of the inherent flexible loops (71-75s loop, 120s loop, and 150s loop) is carried out for allosteric-inhibitor-bound wild-type and mutant A125C variants and a comparison is performed with its unbound state to extract the structural changes describing the inactive state of the protease. Our study reveals that compared to the unliganded system, the inhibitor-bound system shows large structural changes in the 120s loop and 150s loop in contrast to the rigid 71-75s loop. The unliganded system shows a closed-state pocket in contrast to the open state for the wild-type complex that locks the protease into the open and inactive-state conformations. However, the mutant complex fluctuates between open and closed states. Also, we tried to see how mutation and binding of an allosteric inhibitor perturb the connectivity in a protein structure network (PSN) at contact levels. Altogether, our study reveals the mechanism of conformational rearrangements of loops at the molecular level, locking the protein in an inactive conformation, which may be useful for developing allosteric inhibitors.

Extent of Internal Hydration influence the Activation of GPCR Rhodopsin

Rhodopsin is an archetypical GPCR responsible for dim‐light vision, which adopts a dynamic equilibrium between two metastable intermediates, metarhodopsin‐I (MI) and metarhodopsin‐II (MII), upon absorption of light. MI is an inactive conformational state of this GPCR, while MII is a fully active conformational state. We hypothesized that the MI to MII transition following isomerization of the retinal ligand involves a flood of bulk water into the protein core. To observe this change in the hydration level of the receptor during activation that is typically invisible to structural techniques such as X‐ray crystallography or cryo‐EM, we carried out a set of experiments using an osmotic stress approach. In this approach, the hydration level of rhodopsin was changed by placing the protein in native disk membranes in solutions with varying levels of osmotic pressure. Inert hydrophilic polyethylene glycol (PEG) solutes with different molecular weights and concentrations were used as the osmolytes. The effect of osmotic pressure on conformational dynamics was then quantified by measuring the reversible shifting of the metarhodopsin equilibrium using UV‐Visible spectroscopy. Results from these experiments show an influx of 80–100 water molecules into the rhodopsin interior, forming a partially disordered, solvent‐swollen active MII conformational state during photoactivation, a result supported by atomistic molecular dynamics simulations work [1, 2]. Additionally, we observed that the size of the polymer osmolyte played an important role in the modulation of rhodopsin’s activation: large osmolytes (PEG > 600) back shift the equilibrium to inactive MI state, while small osmolytes (PEG < 600) forward‐shift the equilibrium to MII state. We attribute this size dependence to the degree of osmolyte penetration into the rhodopsin core. Large polymers behave similarly to ideal osmolytes and dehydrate rhodopsin, while smaller polymers wriggle into the rhodopsin interior and stabilize the open MII conformation. These results necessitate a new understanding of GPCR activation in which the hydration level of protein is paramount in governing conformational energy landscapes. [1] U. Chawla et al. (2021) Angew. Chem. Int. Ed. 60, 2288–2295. [2] N. Leioatts et al. (2014) Biochemistry 53, 376−385.

Ligand-Dependent Modulation of the Dynamics of Intracellular Loops Dictates Functional Selectivity of 5-HT2AR

The serotonin 2A receptor (5-HT2AR) subtype of the G protein-coupled receptor (GPCR) family is involved in a plethora of neuromodulatory functions (e.g., neurogenesis, sleep, and cognitive processes). 5-HT2AR is the target of pharmacologically distinct classes of ligands, binding of which either activate or inactivate the receptor. Although high-resolution structures of 5-HT2AR as well as several other 5-HT GPCRs provided snapshots of both active and inactive conformational states, these structures, representing a truncated form of the receptor, cannot fully explain the mechanism of conformational transitions during their function. Importantly, biochemical studies have suggested the importance of intracellular loops in receptor functions. In our previous study, a model of the ligand-free form of 5-HT2AR with the third intracellular loop (ICL3) has been meticulously built. Here, we have investigated the functional regulation of 5-HT2AR with intact intracellular loops in ligand-free and five distinct ligand-bound configurations using unbiased atomistic molecular dynamics (MD) simulations. The selected ligands belong to either of the full, partial, or inverse agonist classes, which exert distinct pharmacological responses. We have observed significant structural, dynamic, and thermodynamic differences within ligand-bound complexes. Our results revealed, for the first time, that either activation or inactivation of the receptor upon specific ligand binding is primarily achieved through conformational transitions of its second and third intracellular loops (ICL2 and ICL3). A remarkable allosteric cross-talk between the ligand-binding site and the distal intracellular parts of the receptor, where binding of a specific ligand thermodynamically controls (either stabilizes or destabilizes) the intracellular region, consisting of crucial dynamic elements ICL2 and ICL3, and differential conformational transitions of these loops determine ligand-dependent functional selectivity.

Role of Cholesterol in Transmembrane Dimerization of the ErbB2 Growth Factor Receptor.

The association of ErbB2 growth factor receptors is critical for cell growth and potentiates tumor proliferation in several cancer types. An important aspect in ErbB2 association is the role of lipids such as cholesterol, especially since their metabolism is often reprogrammed in cancer cells. Here, we have coupled metadynamics with coarse-grain simulations to identify cholesterol effects in the transmembrane dimerization of ErbB2 receptors. Overall, cholesterol interactions are observed with the receptor that directly tunes the association energetics. Several dimer conformations are identified both in the presence and absence of cholesterol, although the dimer regime appears to be more favorable in the presence of cholesterol. We observe an overall modulation of the underlying energy profile and the symmetric active and inactive conformational states are not distinguished in the presence of cholesterol. We show that cholesterol binds to the receptor transmembrane domain at a site (CRAC motif) that overlaps with the dimer interface (SmXXXSm motif). The competition between the transmembrane interactions and cholesterol interactions decides the final conformational landscape. Our work is an important step toward characterizing cholesterol effects in ErbB2 membrane receptor function.

De novo STXBP1 mutation in a child with developmental delay and spasticity reveals a major structural alteration in the interface with syntaxin 1A

STXBP1, also known as Munc-18, is a master regulator of neurotransmitter release and synaptic function in the human brain through its direct interaction with syntaxin 1A. STXBP1 binds syntaxin 1A is an inactive conformational state. STXBP1 decreases its binding affinity to syntaxin upon phosphorylation, enabling syntaxin 1A to engage in the SNARE complex, leading to neurotransmitter release. STXBP1-related disorders are well characterized by encephalopathy with epilepsy, and a diverse range of neurological and neurodevelopmental conditions. Through exome sequencing of a child with developmental delay, hypotonia, and spasticity, we found a novel de novo insertion mutation of three nucleotides in the STXBP1 coding region, resulting in an additional arginine after position 39 (R39dup). Inconclusive results from state-of-the-art variant prediction tools mandated a structure-based approach using molecular dynamics (MD) simulations of the STXBP1-syntaxin 1A complex. Comparison of the interaction interfaces of the wild-type and the R39dup complexes revealed a reduced interaction surface area in the mutant, leading to destabilization of the protein complex. Moreover, the decrease in affinity toward syntaxin 1A is similar for the phosphorylated STXBP1 and the R39dup. We applied the same MD methodology to seven additional previously reported STXBP1 mutations and reveal that the stability of the STXBP1-syntaxin 1A interface correlates with the reported clinical phenotypes. This study provides a direct link between the outcome of a novel variant in STXBP1 and protein structure and dynamics. The structural change upon mutation drives an alteration in synaptic function.

A shift of dynamic equilibrium between the KIT active and inactive states causes drug resistance.

Tyrosine phosphorylation, a highly regulated post-translational modification, is carried out by the enzyme tyrosine kinase (TK). TKs are important mediators in signaling cascades, facilitating diverse biological processes in response to stimuli. TKs may acquire mutations leading to malignancy and are viable targets for anti-cancer drugs. Mast/stem cell growth factor receptor KIT is a TK involved in cell differentiation, whose dysregulation leads to various types of cancer, including gastrointestinal stromal tumors, leukemia, and melanoma. KIT can be targeted by a range of inhibitors that predominantly bind to the inactive state of the enzyme. A mutation Y823D in the activation loop of KIT is known to be responsible for the loss of sensitivity to some drugs in metastatic tumors. We used all-atom molecular dynamics simulations to study the impact of Y823D on the KIT conformation and dynamics and compared it to the effect of phosphorylation of Y823. We simulated in total 6.4 μs of wild-type, mutant and phosphorylated KIT in the active- and inactive-state conformations. We found that Y823D affects the protein dynamics differently: in the active state, the mutation increases the protein stability, whereas in the inactive state it induces local destabilization, thus shifting the dynamic equilibrium towards the active state, altering the communication between distant regulatory regions. The observed dynamics of the Y823D mutant is similar to the dynamics of KIT phosphorylated at position Y823, thus we hypothesize that this mutation mimics a constitutively active kinase, which is not responsive to inhibitors that bind its inactive conformation.

Interdependence of hypoxia and β-adrenergic receptor signaling in pulmonary arterial hypertension.

The β-adrenergic receptor (βAR) exists in an equilibrium of inactive and active conformational states, which shifts in response to different ligands and results in downstream signaling. In addition to cAMP, βAR signals to hypoxia-inducible factor 1 (HIF-1). We hypothesized that a βAR-active conformation (R**) that leads to HIF-1 is separable from the cAMP-activating conformation (R*) and that pulmonary arterial hypertension (PAH) patients with HIF-biased conformations would not respond to a cAMP agonist. We compared two cAMP agonists, isoproterenol and salbutamol, in vitro. Isoproterenol increased cAMP and HIF-1 activity, while salbutamol increased cAMP and reduced HIF-1. Hypoxia blunted agonist-stimulated cAMP, consistent with receptor equilibrium shifting toward HIF-activating conformations. Similarly, isoproterenol increased HIF-1 and erythropoiesis in mice, while salbutamol decreased erythropoiesis. βAR overexpression in cells increased glycolysis, which was blunted by HIF-1 inhibitors, suggesting increased βAR leads to increased hypoxia-metabolic effects. Because PAH is also characterized by HIF-related glycolytic shift, we dichotomized PAH patients in the Pulmonary Arterial Hypertension Treatment with Carvedilol for Heart Failure trial (NCT01586156) based on right ventricular (RV) glucose uptake to evaluate βAR ligands. Patients with high glucose uptake had more severe disease than those with low uptake. cAMP increased in response to isoproterenol in mononuclear cells from low-uptake patients but not in high-uptake patients' cells. When patients were treated with carvedilol for 1 wk, the low-uptake group decreased RV systolic pressures and pulmonary vascular resistance, but high-uptake patients had no physiologic responses. The findings expand the paradigm of βAR activation and uncover a novel PAH subtype that might benefit from β-blockers.

Rigidification Dramatically Improves Inhibitor Selectivity for RAF Kinases.

One effective means to achieve inhibitor specificity for RAF kinases, an important family of cancer drug targets, has been to target the monomeric inactive state conformation of the kinase domain, which, unlike most other kinases, can accommodate sulfonamide-containing drugs such as vemurafenib and dabrafenib because of the presence of a unique pocket specific to inactive RAF kinases. We previously reported an alternate strategy whereby rigidification of a nonselective pyrazolo[3,4-d]pyrimidine-based inhibitor through ring closure afforded moderate but appreciable increases in selectivity for RAF kinases. Here, we show that a further application of the rigidification strategy to a different pyrazolopyrimidine-based scaffold dramatically improved selectivity for RAF kinases. Crystal structure analysis confirmed our inhibitor design hypothesis revealing that 2l engages an active-like state conformation of BRAF normally associated with poorly discriminating inhibitors. When screened against a panel of distinct cancer cell lines, the optimized inhibitor 2l primarily inhibited the proliferation of the expected BRAFV600E-harboring cell lines consistent with its kinome selectivity profile. These results suggest that rigidification could be a general and powerful strategy for enhancing inhibitor selectivity against protein kinases, which may open up therapeutic opportunities not afforded by other approaches.

Mutations in the NPxxY motif stabilize pharmacologically distinct conformational states of the α1B- and β2-adrenoceptors.

G protein-coupled receptors (GPCRs) convert extracellular stimuli to intracellular responses that regulate numerous physiological processes. Crystallographic and biophysical advances in GPCR structural analysis have aided investigations of structure-function relationships that clarify our understanding of these dynamic receptors, but the molecular mechanisms associated with activation and signaling for individual GPCRs may be more complex than was previously appreciated. Here, we investigated the proposed water-mediated, hydrogen-bonded activation switch between the conserved NPxxY motif on transmembrane helix 7 (TMH7) and a conserved tyrosine in TMH5, which contributes to α1B-adrenoceptor (α1B-AR) and β2-AR activation. Disrupting this bond by mutagenesis stabilized the α1B-AR and the β2-AR in inactive-state conformations, which displayed decreased agonist potency for stimulating downstream IP1 and cAMP signaling, respectively. Compared to that for wild-type receptors, agonist-mediated β-arrestin recruitment was substantially reduced or abolished for all α1B-AR and β2-AR inactive-state mutants. However, the inactive-state β2-ARs exhibited decreased agonist affinity, whereas the inactive-state α1B-ARs had enhanced agonist affinity. Conversely, antagonist affinity was unchanged for inactive-state conformations of both α1B-AR and β2-AR. Removing the influence of agonist affinity on agonist potency gave a measure of signaling efficacy, which was markedly decreased for the α1B-AR mutants but little altered for the β2-AR mutants. These findings highlight the pharmacological heterogeneity of inactive-state GPCR conformations, which may facilitate the rational design of drugs that target distinct conformational states of GPCRs.

Distinct but overlapping binding sites of agonist and antagonist at the relaxin family peptide 3 (RXFP3) receptor

The relaxin-3 neuropeptide activates the relaxin family peptide 3 (RXFP3) receptor to modulate stress, appetite, and cognition. RXFP3 shows promise as a target for treating neurological disorders, but realization of its clinical potential requires development of smaller RXFP3-specific drugs that can penetrate the blood-brain barrier. Designing such drugs is challenging and requires structural knowledge of agonist- and antagonist-binding modes. Here, we used structure-activity data for relaxin-3 and a peptide RXFP3 antagonist termed R3 B1-22R to guide receptor mutagenesis and develop models of their binding modes. RXFP3 residues were alanine-substituted individually and in combination and tested in cell-based binding and functional assays to refine models of agonist and antagonist binding to active- and inactive-state homology models of RXFP3, respectively. These models suggested that both agonists and antagonists interact with RXFP3 via similar residues in their B-chain central helix. The models further suggested that the B-chain Trp27 inserts into the binding pocket of RXFP3 and interacts with Trp138 and Lys271, the latter through a salt bridge with the C-terminal carboxyl group of Trp27 in relaxin-3. R3 B1-22R, which does not contain Trp27, used a non-native Arg23 residue to form cation-π and salt-bridge interactions with Trp138 and Glu141 in RXFP3, explaining a key contribution of Arg23 to affinity. Overall, relaxin-3 and R3 B1-22R appear to share similar binding residues but may differ in binding modes, leading to active and inactive RXFP3 conformational states, respectively. These mechanistic insights may assist structure-based drug design of smaller relaxin-3 mimetics to manage neurological disorders.

Solution structure and dynamics of Xanthomonas albilineans Cas2 provide mechanistic insight on nuclease activity.

Cas2 protein in the CRISPR-Cas system functions as a scaffold for the acquisition of foreign DNA fragments, and as a nuclease against DNA and RNA substrates. Crystal structures of Cas2 have shown catalytically inactive conformational states that do not explain the mechanism of Cas2 nuclease activity. Here, we report that Xanthomonas albilineans Cas2 (XaCas2) assumes an inactive conformation in solution. Residual dipolar couplings and NMR relaxation, however, provide direct evidence on conformational dynamics at the predicted hinge region. Furthermore, XaCas2 transiently associates with metal ions for nuclease activity via highly mobile Asp8. Taken together, the dual function of Cas2 can be explained by a dynamic equilibrium of conformational states that serve as a scaffold or as a nuclease on demand.

Structural and dynamic insights into the role of conformational switching in the nuclease activity of the Xanthomonas albilineans Cas2 in CRISPR-mediated adaptive immunity.

Clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (Cas) proteins constitute a microbial, adaptive immune system countering invading nucleic acids. Cas2 is a universal Cas protein found in all types of CRISPR-Cas systems, and its role is implicated in new spacer acquisition into CRISPR loci. In subtype I-C CRISPR-Cas systems, Cas2 proteins are metal-dependent double-stranded DNA (dsDNA) nucleases, and a pH-dependent conformational transition has been proposed as a prerequisite for catalytic action. Here, we report the crystal structure of Xanthomonas albilineans Cas2 (XaCas2) and provide experimental evidence of a pH-dependent conformational change during functional activation. XaCas2 crystallized at an acidic pH represented a catalytically inactive conformational state in which two Asp8 residues were too far apart to coordinate a single catalytic metal ion. Consistently, XaCas2 exhibited dsDNA nuclease activity only under neutral and basic conditions. Despite the overall structural similarity of the two protomers, significant conformational heterogeneity was evident in the putative hinge regions, suggesting that XaCas2 engages in hinge-bending conformational switching. The presence of a Trp residue in the hinge region enabled the investigation of hinge dynamics by fluorescence spectroscopy. The pH dependence of the fluorescence intensity overlapped precisely with that of nuclease activity. Mutational analyses further suggested that conformational activation proceeded via a rigid-body hinge-bending motion as both D8E and hinge mutations significantly reduced nuclease activity. Together, our results reveal strong correlations between the conformational states, catalytic activity, and hinge dynamics of XaCas2, and provide structural and dynamic insights into the conformational activation of the nuclease function of Cas2.

A thermodynamically consistent model of the post-translational Kai circadian clock.

The principal pacemaker of the circadian clock of the cyanobacterium S. elongatus is a protein phosphorylation cycle consisting of three proteins, KaiA, KaiB and KaiC. KaiC forms a homohexamer, with each monomer consisting of two domains, CI and CII. Both domains can bind and hydrolyze ATP, but only the CII domain can be phosphorylated, at two residues, in a well-defined sequence. While this system has been studied extensively, how the clock is driven thermodynamically has remained elusive. Inspired by recent experimental observations and building on ideas from previous mathematical models, we present a new, thermodynamically consistent, statistical-mechanical model of the clock. At its heart are two main ideas: i) ATP hydrolysis in the CI domain provides the thermodynamic driving force for the clock, switching KaiC between an active conformational state in which its phosphorylation level tends to rise and an inactive one in which it tends to fall; ii) phosphorylation of the CII domain provides the timer for the hydrolysis in the CI domain. The model also naturally explains how KaiA, by acting as a nucleotide exchange factor, can stimulate phosphorylation of KaiC, and how the differential affinity of KaiA for the different KaiC phosphoforms generates the characteristic temporal order of KaiC phosphorylation. As the phosphorylation level in the CII domain rises, the release of ADP from CI slows down, making the inactive conformational state of KaiC more stable. In the inactive state, KaiC binds KaiB, which not only stabilizes this state further, but also leads to the sequestration of KaiA, and hence to KaiC dephosphorylation. Using a dedicated kinetic Monte Carlo algorithm, which makes it possible to efficiently simulate this system consisting of more than a billion reactions, we show that the model can describe a wealth of experimental data.

Allosteric Modulation of a Serine Protease by Conformationally Selective Nanobodies

Trypsin-like serine proteases regulate many important physiological processes including digestion, blood coagulation, tissue remodelling, complement activation and fibrinolysis. It is well known that ligand binding to allosteric sites modulates the function of the active site region through conformational and/or dynamic changes leading to a change in protease activity and altered substrate specificity. Furthermore, increasing biophysical evidence supports the existence of multiple active and inactive conformational states. However, molecular details about sparsely populated protease conformations, and how ligands modulate the activity of a given protease remains largely elusive. Here we report the X-ray crystal structure of an unappreciated inactive conformational state of the serine protease urokinase-type plasminogen activator (uPA). Next, two distinct uPA conformations were stabilized by different single domain camelid antibodies – an active conformation captured by an active site binding (orthosteric) nanobody (Nb22) and an inactive conformation captured by an allosteric nanobody (Nb7). By combining X-ray crystallography and hydrogen deuterium-exchange mass spectrometry, we identify the mechanism of allostery, which explains at the molecular level how ligand binding regulates uPA activity by changing the dynamics of several surface-exposed loops surrounding the active site region. Collectively, our data supports a model which assumes equilibrium amongst at least three conformational states and shows that ligands modulate the biological activity of the protease by differentially stabilizing a specific protease conformation.

Identifying multiple active conformations in the G protein-coupled receptor activation landscape using computational methods.

G protein-coupled receptors (GPCRs) are membrane proteins critical in cellular signaling, making them important targets for therapeutics. The activation of GPCRs is central to their function, requiring multiple conformations of the GPCRs in their activation landscape. To enable rational design of GPCR-targeting drugs, it is essential to obtain the ensemble of atomistic structures of GPCRs along their activation pathways. This is most challenging for structure determination experiments, making it valuable to develop reliable computational structure prediction methods. In particular, since the active-state conformations are higher in energy (less stable) than inactive-state conformations, they are difficult to stabilize. In addition, the computational methods are generally biased toward lowest energy structures by design and miss these high energy but functionally important conformations. To address this problem, we have developed a computationally efficient ActiveGEnSeMBLE method that systematically predicts multiple conformations that are likely in the GPCR activation landscape, including multiple active- and inactive-state conformations. ActiveGEnSeMBLE starts with a systematic coarse grid sampling of helix tilts/rotations (~13 trillion transmembrane domain conformations) and identifies multiple potential active-state energy wells, using the TM3-TM6 intracellular distance as a surrogate activation coordinate. These energy wells are then sampled locally using a finer grid in conformational space to find a locally minimized conformation in each energy well, which can be further relaxed using molecular dynamics (MD) simulations. This method, combining homology modeling, hierarchical complete conformational sampling, and nanosecond scale MD, provides one of the very few computational methods that predict multiple candidates for active-state conformations and is one of the most computationally affordable.