Allosteric Signal Research Articles

Modulation of the conformational landscape of the PDZ3 domain by perturbation on a distal non-canonical α3 helix: decoding the microscopic mechanism of allostery in the PDZ3 domain.

While allosteric signal transduction is crucial for protein signaling and regulation, the dynamic process of allosteric communication remains poorly understood. The third PDZ domain (PDZ stands for the common structural domain shared by the postsynaptic density protein (PSD95), Drosophila disc large tumor suppressor (DlgA), and zonula occludens-1 protein (ZO-1)) serves as a classic example of a single-domain allosteric protein, demonstrating a long-range coupling between the C-terminal α helix (known as the α3 helix) and ligand binding. A molecular level understanding of how the α3 helix modulates the ligand binding affinity of the PDZ3 domain is still lacking. In this study, extensive molecular dynamics simulations corroborated with principal component analysis (PCA), ligand binding free energy calculations, energetic frustration analysis and Markov state model analysis are employed to uncover such molecular details. We demonstrate the definite presence of a binding competent closed-like state in the conformational landscape of wild-type PDZ3. The population modulations of this closed state and other binding incompetent states in the landscape due to α3-truncation/mutation of PDZ3 are explored. A correlation between the closed state population and calculated binding free energy is established, which supports the conformation selection mechanism. Covariance analysis identified the presence of correlated motion between two distant loops (β1-β2 and β2-β3) in the wild-type PDZ3 system, which weakened due to truncation/mutation in the distant α3 helix. It has also been observed that whenever the α3 helix was perturbed, the β2-β3 loop got further away from the binding groove and it is found to be correlated with the binding free energy values. Energetic frustration analysis of the PDZ3 domain also showed that the β2-β3 loop is highly frustrated. Finally, MSM analysis revealed a relevant timescale (closed to open state transition), which is similar to the observed experimental signal transduction timescale for the system. These observations led to the conclusion that the distantly located α3 helix plays a pivotal role in regulating the conformational landscape of the PDZ3 domain, determining the ligand binding affinity and resulting in allosteric behavior of the domain.

Elucidating the molecular mechanism of noncompetitive inhibition of acetylcholinesterase by an antidiabetic drug chlorpropamide: identification of new allosteric sites.

Acetylcholinesterase (AChE) has emerged as an important drug target for the treatment of neurodegenerative disorders such as Alzheimer's disease (AD). Recent experimental studies indicate that certain antidiabetic drugs can be repurposed as potent AChE inhibitors. Enzymatic kinetic assays suggest that the antidiabetic drug chlorpropamide (CPM) acts as a noncompetitive inhibitor, but the mechanism of action and the binding site(s) of interaction with AChE are not known. In this work, we have carried out molecular dynamics (MD) simulations to discover a new allosteric site in addition to the known peripheral anionic site (PAS) as a potential binding site of this noncompetitive inhibitor. We show that the conformational ensemble of the catalytic triad, particularly the HIS447, undergoes a significant population shift on ligand binding that is responsible for deactivation of the enzyme. We also elucidate the pathway of the allosteric signaling in terms of locally correlated domains of the inter-residue interaction network. Thus, our work identifies a new allosteric site for AChE inhibition and eludiates the underlying mechanistic principles. These results would be useful for the rational design of new noncompetitive inhibitors for AChE.

Computational analysis of long-range allosteric communications in CFTR.

Malfunction of the CFTR protein results in cystic fibrosis, one of the most common hereditary diseases. CFTR functions as an anion channel, the gating of which is controlled by long-range allosteric communications. Allostery also has direct bearings on CF treatment: the most effective CFTR drugs modulate its activity allosterically. Herein, we integrated Gaussian network model, transfer entropy, and anisotropic normal mode-Langevin dynamics and investigated the allosteric communications network of CFTR. The results are in remarkable agreement with experimental observations and mutational analysis and provide extensive novel insight. We identified residues that serve as pivotal allosteric sources and transducers, many of which correspond to disease-causing mutations. We find that in the ATP-free form, dynamic fluctuations of the residues that comprise the ATP-binding sites facilitate the initial binding of the nucleotide. Subsequent binding of ATP then brings to the fore and focuses on dynamic fluctuations that were present in a latent and diffuse form in the absence of ATP. We demonstrate that drugs that potentiate CFTR's conductance do so not by directly acting on the gating residues, but rather by mimicking the allosteric signal sent by the ATP-binding sites. We have also uncovered a previously undiscovered allosteric 'hotspot' located proximal to the docking site of the phosphorylated regulatory (R) domain, thereby establishing a molecular foundation for its phosphorylation-dependent excitatory role. This study unveils the molecular underpinnings of allosteric connectivity within CFTR and highlights a novel allosteric 'hotspot' that could serve as a promising target for the development of novel therapeutic interventions.

Computational analysis of long-range allosteric communications in CFTR

Malfunction of the CFTR protein results in cystic fibrosis, one of the most common hereditary diseases. CFTR functions as an anion channel, the gating of which is controlled by long-range allosteric communications. Allostery also has direct bearings on CF treatment: the most effective CFTR drugs modulate its activity allosterically. Herein, we integrated Gaussian network model, transfer entropy, and anisotropic normal mode-Langevin dynamics and investigated the allosteric communications network of CFTR. The results are in remarkable agreement with experimental observations and mutational analysis and provide extensive novel insight. We identified residues that serve as pivotal allosteric sources and transducers, many of which correspond to disease-causing mutations. We find that in the ATP-free form, dynamic fluctuations of the residues that comprise the ATP-binding sites facilitate the initial binding of the nucleotide. Subsequent binding of ATP then brings to the fore and focuses on dynamic fluctuations that were present in a latent and diffuse form in the absence of ATP. We demonstrate that drugs that potentiate CFTR’s conductance do so not by directly acting on the gating residues, but rather by mimicking the allosteric signal sent by the ATP-binding sites. We have also uncovered a previously undiscovered allosteric ‘hotspot’ located proximal to the docking site of the phosphorylated regulatory (R) domain, thereby establishing a molecular foundation for its phosphorylation-dependent excitatory role. This study unveils the molecular underpinnings of allosteric connectivity within CFTR and highlights a novel allosteric ‘hotspot’ that could serve as a promising target for the development of novel therapeutic interventions.

New insights into the structure and function of the complex between the Escherichia coli Hsp70, DnaK, and its nucleotide-exchange factor, GrpE

The 70 kDa heat shock proteins (Hsp70s) play a pivotal role in many cellular functions using allosteric communication between their nucleotide-binding domain (NBD) and substrate-binding domain, mediated by an interdomain linker, to modulate their affinity for protein clients. Critical to modulation of the Hsp70 allosteric cycle, nucleotide-exchange factors (NEFs) act by a conserved mechanism involving binding to the ADP-bound NBD and opening of the nucleotide-binding cleft to accelerate the release of ADP and binding of ATP. The crystal structure of the complex between the NBD of the Escherichia coli Hsp70, DnaK, and its NEF, GrpE, was reported previously, but the GrpE in the complex carried a point mutation (G122D). Both the functional impact of this mutation and its location on the NEF led us to revisit the DnaK NBD/GrpE complex structurally using AlphaFold modeling and validation by solution methods that report on protein conformation and mutagenesis. This work resulted in a new model for the DnaK NBD in complex with GrpE in which subdomain IIB of the NBD rotates more than in the crystal structure, resulting in an open conformation of the nucleotide-binding cleft, which now resembles more closely what is seen in other Hsp/NEF complexes. Moreover, the new model is consistent with the increased ADP off-rate accompanying GrpE binding. Excitingly, our findings point to an interdomain allosteric signal in DnaK triggered by GrpE binding.

Artificial Protein Crosstalk with a Molecule that Exchanges Binding Partners.

Drawing inspiration from allosteric signaling enzymes, whose catalytic and regulatory units are non-covalently linked, we have devised a method to establish unnatural, effector-mediated enzyme activation within native cells. The feasibility of this approach is demonstrated by introducing a synthetic regulatory unit (sRU) onto glycogen synthase kinase 3 (GSK-3) through non-covalent means. Our study reveals that this synthetic regulator mediates an unnatural crosstalk between GSK-3 and lactate dehydrogenase A (LDHA), whose expression is regulated by cellular oxygen levels. Specifically, with this approach, the constitutively active GSK-3 is transformed into an activable enzyme, whereas LDHA is repurposed as an unnatural effector protein that controls the activity of the kinase, making it unnaturally dependent on the cell's hypoxic response. These findings demonstrate a step toward imitating the function of effector-regulated cell-signaling enzymes, which play a key biological role in mediating the response of cells to changes in their environment. In addition, at the proof-of-principle level, our results indicate the potential to develop a new class of protein inhibitors whose inhibitory effect in cells is dictated by the cell's environment and consequent protein expression profile.

Artificial Protein Crosstalk with a Molecule that Exchanges Binding Partners

AbstractDrawing inspiration from allosteric signaling enzymes, whose catalytic and regulatory units are non‐covalently linked, we have devised a method to establish unnatural, effector‐mediated enzyme activation within native cells. The feasibility of this approach is demonstrated by introducing a synthetic regulatory unit (sRU) onto glycogen synthase kinase 3 (GSK‐3) through non‐covalent means. Our study reveals that this synthetic regulator mediates an unnatural crosstalk between GSK‐3 and lactate dehydrogenase A (LDHA), whose expression is regulated by cellular oxygen levels. Specifically, with this approach, the constitutively active GSK‐3 is transformed into an activable enzyme, whereas LDHA is repurposed as an unnatural effector protein that controls the activity of the kinase, making it unnaturally dependent on the cell‘s hypoxic response. These findings demonstrate a step toward imitating the function of effector‐regulated cell‐signaling enzymes, which play a key biological role in mediating the response of cells to changes in their environment. In addition, at the proof‐of‐principle level, our results indicate the potential to develop a new class of protein inhibitors whose inhibitory effect in cells is dictated by the cell‘s environment and consequent protein expression profile.

Intra-protein interacting collective modes in the terahertz frequency region.

Understanding how proteins work requires a thorough understanding of their internal dynamics. Proteins support a wide range of motions, from the femtoseconds to seconds time scale, relevant to crucial biological functions. In this context, the term "protein collective dynamics" refers to the complex patterns of coordinated motions of numerous atoms throughout the protein in the sub-picosecond time scale (terahertz frequency region). It is hypothesized that these dynamics have a substantial impact on the regulation of functional dynamical mechanisms, including ligand binding and allosteric signalling, charge transport direction, and the regulation of thermodynamic and thermal transport properties. Using the theoretical framework of hydrodynamics, the collective dynamics of proteins had previously been described in a manner akin to that of simple liquids, i.e. in terms of a single acoustic-like excitation, related to intra-protein vibrational motions. Here, we employ an interacting-mode model to analyse the results from molecular dynamics simulations and we unveil that the vibrational landscape of proteins is populated by multiple acoustic-like and low-frequency optic-like modes, with mixed symmetry and interfering with each other. We propose an interpretation at the molecular level of the observed scenario that we relate to the side-chains and the hydrogen-bonded networks dynamics. The present insights provide a perspective for understanding the molecular mechanisms underlying the energy redistribution processes in the interior of proteins.

The nuclear receptor LRH-1 discriminates between ligands using distinct allosteric signaling circuits.

Nuclear receptors (NRs) are transcription factors that regulate essential biological processes in response to cognate ligands. An important part of NR function involves ligand-induced conformational changes that recruit coregulator proteins to the activation function surface (AFS), ~15 Å away from the ligand binding pocket. Ligands must communicate with the AFS to recruit appropriate coregulators and elicit different transcriptional outcomes, but this communication is poorly understood. These studies illuminate allosteric communication networks underlying activation of liver receptor homolog-1 (LRH-1), a NR that regulates development, metabolism, cancer progression and intestinal inflammation. Using >100 microseconds of all-atom molecular dynamics simulations involving 74 LRH-1 complexes, we identify distinct signaling circuits used by active and inactive ligands for AFS communication. Inactive ligands communicate via strong, coordinated motions along paths through the receptor to the AFS. Activating ligands disrupt the "inactive" circuit and induce connectivity with a second allosteric site. Ligand-contacting residues in helix 7 help mediate the switch between circuits, suggesting new avenues for developing LRH-1-targeted therapeutics. We also elucidate aspects of coregulator signaling, showing that localized, destabilizing fluctuations are induced by inappropriate ligand-coregulator pairings. These studies have uncovered novel features of LRH-1 allostery, and the quantitative approach used to analyze many simulations provides a framework to study allosteric signaling in other receptors. This article is protected by copyright. All rights reserved.

Dynamic Insights into the Self-Activation Pathway and Allosteric Regulation of the Orphan G-Protein-Coupled Receptor GPR52.

Within over 800 members of G-protein-coupled receptors, there are numerous orphan receptors whose endogenous ligands are largely unknown, providing many opportunities for novel drug discovery. However, the lack of an in-depth understanding of the intrinsic working mechanism for orphan receptors severely limits the related rational drug design. The G-protein-coupled receptor 52 (GPR52) is a unique orphan receptor that constitutively increases cellular 5'-cyclic adenosine monophosphate (cAMP) levels without binding any exogenous agonists and has been identified as a promising therapeutic target for central nervous system disorders. Although recent structural biology studies have provided snapshots of both active and inactive states of GPR52, the mechanism of the conformational transition between these states remains unclear. Here, an acceptable self-activation pathway for GPR52 was proposed through 6 μs Gaussian accelerated molecular dynamics (GaMD) simulations, in which the receptor spontaneously transitions from the active state to that matching the inactive crystal structure. According to the three intermediate states of the receptor obtained by constructing a reweighted potential of mean force, how the allosteric regulation occurs between the extracellular orthosteric binding pocket and the intracellular G-protein-binding site is revealed. Combined with the independent gradient model, several important microswitch residues and the allosteric communication pathway that directly links the two regions are both identified. Transfer entropy calculations not only reveal the complex allosteric signaling within GPR52 but also confirm the unique role of ECL2 in allosteric regulation, which is mutually validated with the results of GaMD simulations. Overall, this work elucidates the allosteric mechanism of GPR52 at the atomic level, providing the most detailed information to date on the self-activation of the orphan receptor.

Connected Component Analysis of Dynamical Perturbation Contact Networks.

Describing protein dynamical networks through amino acid contacts is a powerful way to analyze complex biomolecular systems. However, due to the size of the systems, identifying the relevant features of protein-weighted graphs can be a difficult task. To address this issue, we present the connected component analysis (CCA) approach that allows for fast, robust, and unbiased analysis of dynamical perturbation contact networks (DPCNs). We first illustrate the CCA method as applied to a prototypical allosteric enzyme, the imidazoleglycerol phosphate synthase (IGPS) enzyme from Thermotoga maritima bacteria. This approach was shown to outperform the clustering methods applied to DPCNs, which could not capture the propagation of the allosteric signal within the protein graph. On the other hand, CCA reduced the DPCN size, providing connected components that nicely describe the allosteric propagation of the signal from the effector to the active sites of the protein. By applying the CCA to the IGPS enzyme in different conditions, i.e., at high temperature and from another organism (yeast IGPS), and to a different enzyme, i.e., a protein kinase, we demonstrated how CCA of DPCNs is an effective and transferable tool that facilitates the analysis of protein-weighted networks.

A critical inter-subunit interaction for the transmission of the allosteric signal in the Agrobacterium tumefaciens ADP-glucose pyrophosphorylase.

ADP-glucose pyrophosphorylase is a key regulatory enzyme involved in starch and glycogen synthesis in plants and bacteria, respectively. It has been hypothesized that inter-subunit communications are important for the allosteric effect in this enzyme. However, no specific interactions have been identified as part of the regulatory signal. The enzyme from Agrobacterium tumefaciens is a homotetramer allosterically regulated by fructose 6-phosphate and pyruvate. Three pairs of distinct subunit-subunit interfaces are present. Here we focus on an interface that features two symmetrical interactions between Arg11 and Asp141 from one subunit with residues Asp141 and Arg11 of the neighbor subunit, respectively. Previously, scanning mutagenesis showed that a mutation at the Arg11 position disrupted the activation of the enzyme. Considering the distance of these residues from the allosteric and catalytic sites, we hypothesized that the interaction between Arg11 and Asp141 is critical for allosteric signaling rather than effector binding. To prove our hypothesis, we mutated those two sites (D141A, D141E, D141N, D141R, R11D, and R11K) and performed kinetic and binding analysis. Mutations that altered the charge affected the regulation the most. To prove that the interaction per se (rather than the presence of specific residues) is critical, we partially rescued the R11D protein by introducing a second mutation (R11D/D141R). This could not restore the activator effect on kcat , but it did rescue the effect on substrate affinity. Our results indicate the critical functional role of Arg11 and Asp141 to relay the allosteric signal in this subunit interface.

Programming conformational cooperativity to regulate allosteric protein-oligonucleotide signal transduction

Conformational cooperativity is a universal molecular effect mechanism and plays a critical role in signaling pathways. However, it remains a challenge to develop artificial molecular networks regulated by conformational cooperativity, due to the difficulties in programming and controlling multiple structural interactions. Herein, we develop a cooperative strategy by programming multiple conformational signals, rather than chemical signals, to regulate protein-oligonucleotide signal transduction, taking advantage of the programmability of allosteric DNA constructs. We generate a cooperative regulation mechanism, by which increasing the loop lengths at two different structural modules induced the opposite effects manifesting as down- and up-regulation. We implement allosteric logic operations by using two different proteins. Further, in cell culture we demonstrate the feasibility of this strategy to cooperatively regulate gene expression of PLK1 to inhibit tumor cell proliferation, responding to orthogonal protein-signal stimulation. This programmable conformational cooperativity paradigm has potential applications in the related fields.

Markov State Models and Perturbation-Based Approaches Reveal Distinct Dynamic Signatures and Hidden Allosteric Pockets in the Emerging SARS-Cov-2 Spike Omicron Variant Complexes with the Host Receptor: The Interplay of Dynamics and Convergent Evolution Modulates Allostery and Functional Mechanisms.

The new generation of SARS-CoV-2 Omicron variants displayed a significant growth advantage and increased viral fitness by acquiring convergent mutations, suggesting that the immune pressure can promote convergent evolution leading to the sudden acceleration of SARS-CoV-2 evolution. In the current study, we combined structural modeling, microsecond molecular dynamics simulations, and Markov state models to characterize conformational landscapes and identify specific dynamic signatures of the SARS-CoV-2 spike complexes with the host receptor ACE2 for the recently emerged highly transmissible XBB.1, XBB.1.5, BQ.1, and BQ.1.1 Omicron variants. Microsecond simulations and Markovian modeling provided a detailed characterization of the functional conformational states and revealed the increased thermodynamic stabilization of the XBB.1.5 subvariant, which can be contrasted to more dynamic BQ.1 and BQ.1.1 subvariants. Despite considerable structural similarities, Omicron mutations can induce unique dynamic signatures and specific distributions of the conformational states. The results suggested that variant-specific changes of the conformational mobility in the functional interfacial loops of the receptor-binding domain in the SARS-CoV-2 spike protein can be fine-tuned through crosstalk between convergent mutations which could provide an evolutionary path for modulation of immune escape. By combining atomistic simulations and Markovian modeling analysis with perturbation-based approaches, we determined important complementary roles of convergent mutation sites as effectors and receivers of allosteric signaling involved in modulation of conformational plasticity and regulation of allosteric communications. This study also revealed hidden allosteric pockets and suggested that convergent mutation sites could control evolution and distribution of allosteric pockets through modulation of conformational plasticity in the flexible adaptable regions.

Class B1 GPCR activation by an intracellular agonist

G protein-coupled receptors (GPCRs) generally accommodate specific ligands in the orthosteric-binding pockets. Ligand binding triggers a receptor allosteric conformational change that leads to the activation of intracellular transducers, G proteins and β-arrestins. Because these signals often induce adverse effects, the selective activation mechanism for each transducer must be elucidated. Thus, many orthosteric-biased agonists have been developed, and intracellular-biased agonists have recently attracted broad interest. These agonists bind within the receptor intracellular cavity and preferentially tune the specific signalling pathway over other signalling pathways, without allosteric rearrangement of the receptor from the extracellular side1–3. However, only antagonist-bound structures are currently available1,4–6, and there is no evidence to support that biased agonist binding occurs within the intracellular cavity. This limits the comprehension of intracellular-biased agonism and potential drug development. Here we report the cryogenic electron microscopy structure of a complex of Gs and the human parathyroid hormone type 1 receptor (PTH1R) bound to a PTH1R agonist, PCO371. PCO371 binds within an intracellular pocket of PTH1R and directly interacts with Gs. The PCO371-binding mode rearranges the intracellular region towards the active conformation without extracellularly induced allosteric signal propagation. PCO371 stabilizes the significantly outward-bent conformation of transmembrane helix 6, which facilitates binding to G proteins rather than β-arrestins. Furthermore, PCO371 binds within the highly conserved intracellular pocket, activating 7 out of the 15 class B1 GPCRs. Our study identifies a new and conserved intracellular agonist-binding pocket and provides evidence of a biased signalling mechanism that targets the receptor–transducer interface.

Comprehensive Approach to Simulating Large Scale Conformational Changes in Biological Systems Utilizing a Path Collective Variable and New Barrier Restraint.

Conformational sampling of complex biomolecules is an emerging frontier in drug discovery. Advances in lab-based structural biology and related computational approaches like AlphaFold have made great strides in obtaining static protein structures for biologically relevant targets. However, biology is in constant motion, and many important biological processes rely on conformationally driven events. Conventional molecular dynamics (MD) simulations run on standard hardware are impractical for many drug design projects, where conformationally driven biological events can take microseconds to milliseconds or longer. An alternative approach is to focus the search on a limited region of conformational space defined by a putative reaction coordinate (i.e., path collective variable). The search space is typically limited by applying restraints, which can be guided by insights about the underlying biological process of interest. The challenge is striking a balance between the degree to which the system is constrained and still allowing for natural motions along the path. A plethora of restraints exist to limit the size of conformational search space, although each has drawbacks when simulating complex biological motions. In this work, we present a three-stage procedure to construct realistic path collective variables (PCVs) and introduce a new kind of barrier restraint that is particularly well suited for complex conformationally driven biological events, such as allosteric modulations and conformational signaling. The PCV presented here is all-atom (as opposed to C-alpha or backbone only) and is derived from all-atom MD trajectory frames. The new restraint relies on a barrier function (specifically, the scaled reciprocal function), which we show is particularly beneficial in the context of molecular dynamics, where near-hard-wall restraints are needed with zero tolerance to restraint violation. We have implemented our PCV and barrier restraint within a hybrid sampling framework that combines well-tempered metadynamics and extended-Lagrangian adaptive biasing force (meta-eABF). We use three particular examples of high pharmaceutical interest to demonstrate the value of this approach: (1) sampling the distance from ubiquitin to a protein of interest within the supramolecular cullin-RING ligase complex, (2) stabilizing the wild-type conformation of the oncogenic mutant JAK2-V617F pseudokinase domain, and (3) inducing an activated state of the stimulator of interferon genes (STING) protein observed upon ligand binding. For examples 2 and 3, we present statistical analysis of meta-eABF free energy estimates and, for each case, code for reproducing this work.

Thumb-domain dynamics modulate the functional repertoire of DNA-Polymerase IV (DinB).

In order to cope with the risk of stress-induced mutagenesis, cells in all kingdoms of life employ Y-family DNA polymerases to resolve resulting DNA lesions and thus maintaining the integrity of the genome. In Escherichia coli,the DNA polymerase IV, or DinB, plays this crucial role in coping with these type of mutations via the so-called translesion DNA synthesis. Despite the availability of several high-resolution crystal structures, important aspects of the functional repertoire of DinB remain elusive. In this study, we use advanced solution NMR spectroscopy methods in combination with biophysical characterization to elucidate the crucial role of the Thumb domain within DinB's functional cycle. We find that the inherent dynamics of this domain guide the recognition of double-stranded (ds) DNA buried within the interior of the DinB domain arrangement and trigger allosteric signals through the DinB protein. Subsequently, we characterized the RNA polymerase interaction with DinB, revealing an extended outside surface of DinB and thus not mutually excluding the DNA interaction. Altogether the obtained results lead to a refined model of the functional repertoire of DinB within the translesion DNA synthesis pathway.

Allosteric communication between ACE2 active site and binding interface with SARS-CoV-2.

SARS-CoV-2, the virus causing COVID-19, initiates cell invasion by deploying a receptor binding domain (RBD) to recognize the host transmembrane peptidase angiotensin-converting enzyme 2 (ACE2). Numerous experimental and theoretical studies have adopted high-throughput and structure-guided approaches to (i) understand how the RBD recognizes ACE2, (ii) rationalize, and (iii) predict the effect of viral mutations on the binding affinity. Here, we investigate the allosteric signal triggered by the dissociation of the ACE2-RBD complex. To this end, we construct an Elastic Network Model (ENM), and we use the Structural Perturbation Method (SPM). Our key result is that complex dissociation opens the ACE2 substrate-binding cleft located away from the interface and that fluctuations of the ACE2 binding cleft are facilitated by RBD binding. These and other observations provide a structural and dynamical basis for the influence of SARS-CoV-2 on ACE2 enzymatic activity. In addition, we identify a conserved glycine (G502 in SARS-CoV-2) as a key participant in complex disassembly.

Investigating the Activation Mechanism Differences between Human and Mouse cGAS by Molecular Dynamics Simulations.

Cyclic GMP-AMP synthase (cGAS) has been widely investigated as a drug target for its crucial role in innate immunity. However, the inhibitors designed using mouse model were often shown to be ineffective for humans. This outcome indicates that the activation mechanisms of human and mouse cGAS (mcGAS) are different. The cGAS activation is achieved by dimerization via binding to DNA, the detailed mechanism of which, however, is not entirely clear. To investigate these mechanisms, molecular dynamics (MD) simulations were performed on several states of four types of cGAS, namely, the mcGAS, the wild-type and A- and C-type mutations of human cGAS (hcGAS). We find that sequence differences between hcGAS and mcGAS can directly affect the protein structure stability, especially that of the siteB domain. The sequence and structural differences also contribute to DNA-binding differences. In addition, the conformational fluctuations of cGAS are found to correlate with the regulation of catalytic capacity. More importantly, we illustrate that dimerization enhances the correlation among distant residues and significantly reinforces the allosteric signal transmission among the DNA-binding interfaces and the catalytic pocket, which facilitates rapid immune response to cytosolic DNA. We conclude that siteB domain plays a prominent role in mcGAS activation, while siteA domain is key to hcGAS activation.

Structural Dynamics Predominantly Determine the Adaptability of Proteins to Amino Acid Deletions.

The insertion or deletion (indel) of amino acids has a variety of effects on protein function, ranging from disease-forming changes to gaining new functions. Despite their importance, indels have not been systematically characterized towards protein engineering or modification goals. In the present work, we focus on deletions composed of multiple contiguous amino acids (mAA-dels) and their effects on the protein (mutant) folding ability. Our analysis reveals that the mutant retains the native fold when the mAA-del obeys well-defined structural dynamics properties: localization in intrinsically flexible regions, showing low resistance to mechanical stress, and separation from allosteric signaling paths. Motivated by the possibility of distinguishing the features that underlie the adaptability of proteins to mAA-dels, and by the rapid evaluation of these features using elastic network models, we developed a positive-unlabeled learning-based classifier that can be adopted for protein design purposes. Trained on a consolidated set of features, including those reflecting the intrinsic dynamics of the regions where the mAA-dels occur, the new classifier yields a high recall of 84.3% for identifying mAA-dels that are stably tolerated by the protein. The comparative examination of the relative contribution of different features to the prediction reveals the dominant role of structural dynamics in enabling the adaptation of the mutant to mAA-del without disrupting the native fold.