RESUMO
Type II topoisomerases transiently cleave duplex DNA as part of a strand passage mechanism that helps control chromosomal organization and superstructure. Aberrant DNA cleavage can result in genomic instability, and how topoisomerase activity is controlled to prevent unwanted breaks is poorly understood. Using a genetic screen, we identified mutations in the beta isoform of human topoisomerase II (hTOP2ß) that render the enzyme hypersensitive to the chemotherapeutic agent etoposide. Several of these variants were unexpectedly found to display hypercleavage behavior in vitro and to be capable of inducing cell lethality in a DNA repair-deficient background; surprisingly, a subset of these mutations were also observed in TOP2B sequences from cancer genome databases. Using molecular dynamics simulations and computational network analyses, we found that many of the mutations obtained from the screen map to interfacial points between structurally coupled elements, and that dynamical modeling could be used to identify other damage-inducing TOP2B alleles present in cancer genome databases. This work establishes that there is an innate link between DNA cleavage predisposition and sensitivity to topoisomerase II poisons, and that certain sequence variants of human type II topoisomerases found in cancer cells can act as DNA-damaging agents. Our findings underscore the potential for hTOP2ß to function as a clastogen capable of generating DNA damage that may promote or support cellular transformation.
Assuntos
Mutagênicos , Neoplasias , Humanos , Inibidores da Topoisomerase II/farmacologia , Etoposídeo/farmacologia , DNA Topoisomerases Tipo II/genética , DNA Topoisomerases Tipo II/metabolismo , Dano ao DNA , DNARESUMO
Structure-based drug discovery efforts require knowledge of where drug-binding sites are located on target proteins. To address the challenge of finding druggable sites, we developed a machine-learning algorithm called TACTICS (trajectory-based analysis of conformations to identify cryptic sites), which uses an ensemble of molecular structures (such as molecular dynamics simulation data) as input. First, TACTICS uses k-means clustering to select a small number of conformations that represent the overall conformational heterogeneity of the data. Then, TACTICS uses a random forest model to identify potentially bindable residues in each selected conformation, based on protein motion and geometry. Lastly, residues in possible binding pockets are scored using fragment docking. As proof-of-principle, TACTICS was applied to the analysis of simulations of the SARS-CoV-2 main protease and methyltransferase and the Yersinia pestis aryl carrier protein. Our approach recapitulates known small-molecule binding sites and predicts the locations of sites not previously observed in experimentally determined structures. The TACTICS code is available at https://github.com/Albert-Lau-Lab/tactics_protein_analysis.
Assuntos
COVID-19 , SARS-CoV-2 , Sítios de Ligação , Humanos , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Conformação Proteica , ProteínasRESUMO
The earliest metazoan ancestors of humans include the ctenophore Mnemiopsis leidyi The genome of this comb jelly encodes homologs of vertebrate ionotropic glutamate receptors (iGluRs) that are distantly related to glycine-activated NMDA receptors and that bind glycine with unusually high affinity. Using ligand-binding domain (LBD) mutants for electrophysiological analysis, we demonstrate that perturbing a ctenophore-specific interdomain Arg-Glu salt bridge that is notably absent from vertebrate AMPA, kainate, and NMDA iGluRs greatly increases the rate of recovery from desensitization, while biochemical analysis reveals a large decrease in affinity for glycine. X-ray crystallographic analysis details rearrangements in the binding pocket stemming from the mutations, and molecular dynamics simulations suggest that the interdomain salt bridge acts as a steric barrier regulating ligand binding and that the free energy required to access open conformations in the glycine-bound LBD is largely responsible for differences in ligand affinity among the LBD variants.
Assuntos
Glicina/química , Glicina/metabolismo , Ligação Proteica , Receptores de N-Metil-D-Aspartato/química , Receptores de N-Metil-D-Aspartato/metabolismo , Animais , Sítios de Ligação , Ligação Competitiva , Cristalografia por Raios X , Ctenóforos/metabolismo , Dipeptídeos , Eletrofisiologia , Humanos , Ligação de Hidrogênio , Ligantes , Modelos Moleculares , Simulação de Dinâmica Molecular , Proteínas Mutantes , Mutação Puntual , Ligação Proteica/genética , Conformação Proteica , Receptores Ionotrópicos de Glutamato/metabolismo , Receptores de N-Metil-D-Aspartato/genéticaRESUMO
Ionotropic glutamate receptors are postsynaptic tetrameric ligand-gated channels whose activity mediates fast excitatory transmission. Glutamate binding to clamshell-shaped ligand binding domains (LBDs) triggers opening of the integral ion channel, but how the four LBDs orchestrate receptor activation is unknown. Here, we present a high-resolution x-ray crystal structure displaying two tetrameric LBD arrangements fully bound to glutamate. Using a series of engineered metal ion trapping mutants, we showed that the more compact of the two assemblies corresponds to an arrangement populated during activation of full-length receptors. State-dependent cross-linking of the mutants identified zinc bridges between the canonical active LBD dimers that formed when the tetramer was either fully or partially bound by glutamate. These bridges also stabilized the resting state, consistent with the recently published full-length apo structure. Our results provide insight into the activation mechanism of glutamate receptors and the complex conformational space that the LBD layer can sample.
Assuntos
Receptores de AMPA/química , Receptores de AMPA/metabolismo , Animais , Apoproteínas/química , Apoproteínas/genética , Apoproteínas/metabolismo , Cristalografia por Raios X , Glutamatos/metabolismo , Ligantes , Modelos Moleculares , Mutação , Domínios Proteicos , Multimerização Proteica , Ratos , Receptores de AMPA/genética , Zinco/metabolismoRESUMO
Excitatory neurotransmission is principally mediated by α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-subtype ionotropic glutamate receptors (AMPARs). Negative allosteric modulators are therapeutic candidates that inhibit AMPAR activation and can compete with positive modulators to control AMPAR function through unresolved mechanisms. Here we show that allosteric inhibition pushes AMPARs into a distinct state that prevents both activation and positive allosteric modulation. We used cryo-electron microscopy to capture AMPARs bound to glutamate, while a negative allosteric modulator, GYKI-52466, and positive allosteric modulator, cyclothiazide, compete for control of the AMPARs. GYKI-52466 binds in the ion channel collar and inhibits AMPARs by decoupling the ligand-binding domains from the ion channel. The rearrangement of the ligand-binding domains ruptures the cyclothiazide site, preventing positive modulation. Our data provide a framework for understanding allostery of AMPARs and for rational design of therapeutics targeting AMPARs in neurological diseases.
RESUMO
The Sodium/Iodide Symporter (NIS), a 13-helix transmembrane protein found in the thyroid and other tissues, transports iodide, a required constituent of thyroid hormones T3 and T4. Despite extensive experimental information and clinical data, structural details of the intermediate microstates comprising the conformational transition of NIS between its inwardly and outwardly open states remain unresolved. We present data from a combination of enhanced sampling and transition path molecular dynamics (MD) simulations that elucidate the principal intermediate states comprising the inwardly to outwardly open transition of fully bound and apo NIS under an enforced ionic gradient. Our findings suggest that in both the absence and presence of bound physiological ions, NIS principally occupies a proximally inward to inwardly open state. When fully bound, NIS is also found to occupy a rare "inwardly occluded" state. The results of this work provide novel insight into the populations of NIS intermediates and the free energy landscape comprising the conformational transition, adding to a mechanistic understanding of NIS ion transport. Moreover, the knowledge gained from this approach can serve as a basis for studies of NIS mutants to target therapeutic interventions.
Assuntos
Iodetos , Simportadores , Simportadores/metabolismo , Glândula Tireoide/química , Glândula Tireoide/metabolismo , Termodinâmica , Sódio/metabolismoRESUMO
Ubiquitin phosphorylation at Ser65 increases the population of a rare C-terminally retracted (CR) conformation. Transition between the Major and CR ubiquitin conformations is critical for promoting mitochondrial degradation. The mechanisms by which the Major and CR conformations of Ser65-phosphorylated (pSer65) ubiquitin interconvert, however, remain unresolved. Here, we perform all-atom molecular dynamics simulations using the string method with swarms of trajectories to calculate the lowest free-energy path between these two conformers. Our analysis reveals the existence of a Bent intermediate in which the C-terminal residues of the ß5 strand shift to resemble the CR conformation, while pSer65 retains contacts resembling the Major conformation. This stable intermediate was reproduced in well-tempered metadynamics calculations but was less stable for a Gln2Ala mutant that disrupts contacts with pSer65. Lastly, dynamical network modeling reveals that the transition from the Major to CR conformations involves a decoupling of residues near pSer65 from the adjacent ß1 strand.
Assuntos
Simulação de Dinâmica Molecular , Ubiquitina , Fosforilação , Ubiquitina/metabolismo , Conformação Molecular , Ubiquitina-Proteína Ligases/química , Conformação ProteicaRESUMO
Excitatory neurotransmission is principally mediated by AMPA-subtype ionotropic glutamate receptors (AMPARs). Dysregulation of AMPARs is the cause of many neurological disorders and how therapeutic candidates such as negative allosteric modulators inhibit AMPARs is unclear. Here, we show that non-competitive inhibition desensitizes AMPARs to activation and prevents positive allosteric modulation. We dissected the noncompetitive inhibition mechanism of action by capturing AMPARs bound to glutamate and the prototypical negative allosteric modulator, GYKI-52466, with cryo-electron microscopy. Noncompetitive inhibition by GYKI-52466, which binds in the transmembrane collar region surrounding the ion channel, negatively modulates AMPARs by decoupling glutamate binding in the ligand binding domain from the ion channel. Furthermore, during allosteric competition between negative and positive modulators, negative allosteric modulation by GKYI-52466 outcompetes positive allosteric modulators to control AMPAR function. Our data provide a new framework for understanding allostery of AMPARs and foundations for rational design of therapeutics targeting AMPARs in neurological diseases.
RESUMO
The bacterial divisome is a macromolecular machine composed of more than 30 proteins that controls cell wall constriction during division. Here, we present a model of the structure and dynamics of the core complex of the E. coli divisome, supported by a combination of structure prediction, molecular dynamics simulation, single-molecule imaging, and mutagenesis. We focus on the septal cell wall synthase complex formed by FtsW and FtsI, and its regulators FtsQ, FtsL, FtsB, and FtsN. The results indicate extensive interactions in four regions in the periplasmic domains of the complex. FtsQ, FtsL, and FtsB support FtsI in an extended conformation, with the FtsI transpeptidase domain lifted away from the membrane through interactions among the C-terminal domains. FtsN binds between FtsI and FtsL in a region rich in residues with superfission (activating) and dominant negative (inhibitory) mutations. Mutagenesis experiments and simulations suggest that the essential domain of FtsN links FtsI and FtsL together, potentially modulating interactions between the anchor-loop of FtsI and the putative catalytic cavity of FtsW, thus suggesting a mechanism of how FtsN activates the cell wall synthesis activities of FtsW and FtsI.
Assuntos
Proteínas de Escherichia coli , Escherichia coli , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Divisão Celular , Proteínas de Ciclo Celular/metabolismo , Proteínas de Membrana/metabolismo , Parede Celular/metabolismo , Proteínas de Bactérias/metabolismoRESUMO
Photoswitched tethered ligands (PTLs) can be used to remotely control protein function with light. We have studied the geometric and conformational factors that determine the efficacy of PTL gating in the ionotropic glutamate receptor iGluR6 using a family of photoiosomerizable MAG (maleimide-azobenzene-glutamate) PTLs that covalently attach to the clamshell ligand-binding domain. Experiments and molecular dynamics simulations of the modified proteins show that optical switching depends on 2 factors: (i) the relative occupancy of the binding pocket in the 2 photoisomers of MAG and (ii) the degree of clamshell closure that is possible given the disposition of the MAG linker. A synthesized short version of MAG turns the channel on in either the cis or trans state, depending on the point of attachment. This yin/yang optical control makes it possible for 1 wavelength of light to elicit action potentials in one set of neurons, while deexciting a second set of neurons in the same preparation, whereas a second wavelength has the opposite effect. The ability to generate opposite responses with a single PTL and 2 versions of a target channel, which can be expressed in different cell types, paves the way for engineering opponency in neurons that mediate opposing functions.
Assuntos
Luz , Nanotecnologia/métodos , Receptores de Glutamato/metabolismo , Animais , Compostos Azo/metabolismo , Linhagem Celular , Simulação por Computador , Ácido Glutâmico/metabolismo , Humanos , Ativação do Canal Iônico , Ligantes , Maleimidas/metabolismo , Modelos Moleculares , Neurônios/metabolismo , Técnicas de Patch-Clamp , Conformação Proteica , Quinoxalinas , Ratos , Receptores de Glutamato/química , Estereoisomerismo , TitulometriaRESUMO
N-methyl-D-aspartate receptors (NMDARs) uniquely require binding of two different neurotransmitter agonists for synaptic transmission. D-serine and glycine bind to one subunit, GluN1, while glutamate binds to the other, GluN2. These agonists bind to the receptor's bi-lobed ligand-binding domains (LBDs), which close around the agonist during receptor activation. To better understand the unexplored mechanisms by which D-serine contributes to receptor activation, we performed multi-microsecond molecular dynamics simulations of the GluN1/GluN2A LBD dimer with free D-serine and glutamate agonists. Surprisingly, we observed D-serine binding to both GluN1 and GluN2A LBDs, suggesting that D-serine competes with glutamate for binding to GluN2A. This mechanism is confirmed by our electrophysiology experiments, which show that D-serine is indeed inhibitory at high concentrations. Although free energy calculations indicate that D-serine stabilizes the closed GluN2A LBD, its inhibitory behavior suggests that it either does not remain bound long enough or does not generate sufficient force for ion channel gating. We developed a workflow using pathway similarity analysis to identify groups of residues working together to promote binding. These conformation-dependent pathways were not significantly impacted by the presence of N-linked glycans, which act primarily by interacting with the LBD bottom lobe to stabilize the closed LBD.
Assuntos
Ácido Glutâmico , Receptores de N-Metil-D-Aspartato , Receptores de N-Metil-D-Aspartato/metabolismo , Ácido Glutâmico/metabolismo , Conformação Molecular , Simulação de Dinâmica Molecular , SerinaRESUMO
N-methyl-D-aspartate receptors (NMDARs) are critically involved in basic brain functions and neurodegeneration as well as tumor invasiveness. Targeting specific subtypes of NMDARs with distinct activities has been considered an effective therapeutic strategy for neurological disorders and diseases. However, complete elimination of off-target effects of small chemical compounds has been challenging and thus, there is a need to explore alternative strategies for targeting NMDAR subtypes. Here we report identification of a functional antibody that specifically targets the GluN1-GluN2B NMDAR subtype and allosterically down-regulates ion channel activity as assessed by electrophysiology. Through biochemical analysis, x-ray crystallography, single-particle electron cryomicroscopy, and molecular dynamics simulations, we show that this inhibitory antibody recognizes the amino terminal domain of the GluN2B subunit and increases the population of the non-active conformational state. The current study demonstrates that antibodies may serve as specific reagents to regulate NMDAR functions for basic research and therapeutic objectives.
Assuntos
Anticorpos Monoclonais/farmacologia , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , Animais , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/isolamento & purificação , Anticorpos Monoclonais/ultraestrutura , Clonagem Molecular , Microscopia Crioeletrônica , Cristalografia por Raios X , Fragmentos Fab das Imunoglobulinas/genética , Fragmentos Fab das Imunoglobulinas/isolamento & purificação , Fragmentos Fab das Imunoglobulinas/farmacologia , Fragmentos Fab das Imunoglobulinas/ultraestrutura , Região Variável de Imunoglobulina/genética , Região Variável de Imunoglobulina/isolamento & purificação , Região Variável de Imunoglobulina/farmacologia , Região Variável de Imunoglobulina/ultraestrutura , Simulação de Dinâmica Molecular , Oócitos , Ratos , Receptores de N-Metil-D-Aspartato/genética , Receptores de N-Metil-D-Aspartato/isolamento & purificação , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/farmacologia , Proteínas Recombinantes/ultraestrutura , Células Sf9 , Spodoptera , Xenopus laevisRESUMO
Glutamate delta (GluD) receptors are a functionally enigmatic subfamily of ionotropic glutamate receptors. Despite sharing similar sequences and structures with AMPA, NMDA, and kainate receptors, GluD receptors do not bind glutamate nor function as ligand-gated ion channels. Binding d-serine and engaging in transsynaptic protein-protein interactions, GluD receptors are thought to undergo complex conformational rearrangements for non-ionotropic signaling that regulates synaptic plasticity. Recent structural, biochemical, and computational studies have elucidated multiple conformational and thermodynamic factors governing the unique properties of GluD receptors. Here, we review advances in biophysical insights into GluD receptors and discuss the structural thermodynamic relationships that underpin their neurobiological functions.
Assuntos
Receptores Ionotrópicos de Glutamato/química , Receptores Ionotrópicos de Glutamato/metabolismo , Termodinâmica , Animais , Ácido Glutâmico , Humanos , Canais Iônicos de Abertura Ativada por LiganteRESUMO
Inflammasomes are filamentous signaling platforms integral to innate immunity. Currently, little is known about how these structurally similar filaments recognize and distinguish one another. A cryo-EM structure of the AIM2PYD filament reveals that the architecture of the upstream filament is essentially identical to that of the adaptor ASCPYD filament. In silico simulations using Rosetta and molecular dynamics followed by biochemical and cellular experiments consistently demonstrate that individual filaments assemble bidirectionally. By contrast, the recognition between AIM2 and ASC requires at least one to be oligomeric and occurs in a head-to-tail manner. Using in silico mutagenesis as a guide, we also identify specific axial and lateral interfaces that dictate the recognition and distinction between AIM2 and ASC filaments. Together, the results here provide a robust framework for delineating the signaling specificity and order of inflammasomes.
Assuntos
Proteínas Adaptadoras de Sinalização CARD/metabolismo , Proteínas de Ligação a DNA/metabolismo , Imunidade Inata/fisiologia , Inflamassomos/metabolismo , Proteínas Adaptadoras de Sinalização CARD/genética , Microscopia Crioeletrônica , Proteínas de Ligação a DNA/genética , Células HEK293 , Humanos , Simulação de Dinâmica Molecular , Mutação/genética , Estrutura Secundária de Proteína , Transdução de Sinais/fisiologiaRESUMO
For ion channels, the transmembrane potential plays a critical role by acting as a driving force for permeant ions. At the microscopic level, the transmembrane potential is thought to decay nonlinearly across the ion permeation pathway because of the irregular three-dimensional shape of the channel's pore. By taking advantage of the current structural and functional understanding of cyclic nucleotide-gated channels, in this study we experimentally explore the transmembrane potential's distribution across the open pore. As a readout for the voltage drop, we engineered cysteine residues along the selectivity filter and scanned the sensitivity of their modification rates by Ag(+) to the transmembrane potential. The experimental data, which indicate that the majority of the electric field drops across the selectivity filter, are in good agreement with continuum electrostatic calculations using a homology model of an open CNG channel. By focusing the transmembrane potential across the selectivity filter, the electromotive driving force is coupled with the movement of permeant ions in the filter, maximizing the efficiency of this process.
Assuntos
Canais de Cátion Regulados por Nucleotídeos Cíclicos/metabolismo , Substituição de Aminoácidos , Animais , Fenômenos Biofísicos , Canais de Cátion Regulados por Nucleotídeos Cíclicos/química , Canais de Cátion Regulados por Nucleotídeos Cíclicos/genética , Cisteína/química , Feminino , Técnicas In Vitro , Cinética , Potenciais da Membrana , Modelos Moleculares , Mutagênese Sítio-Dirigida , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Oócitos/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Eletricidade Estática , XenopusRESUMO
Despite their classification as ionotropic glutamate receptors, GluD receptors are not functional ligand-gated ion channels and do not bind glutamate. GluD2 receptors bind D-serine and coordinate transsynaptic complexes that regulate synaptic plasticity. Instead of opening the ion channel pore, mechanical tension produced from closure of GluD2 ligand-binding domains (LBDs) drives conformational rearrangements for non-ionotropic signaling. We report computed conformational free energy landscapes for the GluD2 LBD in apo and D-serine-bound states. Unexpectedly, the conformational free energy associated with GluD2 LBD closure upon D-serine binding is greater than that for AMPA, NMDA, and kainate receptor LBDs upon agonist binding. This excludes insufficient force generation as an explanation for lack of ion channel activity in GluD2 receptors and suggests that non-ionotropic conformational rearrangements do more work than pore opening. We also report free energy landscapes for GluD2 LBD harboring a neurodegenerative mutation and demonstrate selective stabilization of closed conformations in the apo state.
Assuntos
Receptores de Glutamato/química , Receptores de Glutamato/metabolismo , Serina/metabolismo , Sítios de Ligação , Ligantes , Simulação de Dinâmica Molecular , Mutação , Domínios Proteicos , Receptores de Glutamato/genética , Serina/química , TermodinâmicaRESUMO
Ionotropic glutamate receptors are ligand-gated transmembrane ion channels activated by the binding of glutamate. The free energy landscapes governing the opening/closing of the GluR2 S1S2 ligand-binding domain in the apo, DNQX-, and glutamate-bound forms are computed by using all-atom molecular dynamics simulations with explicit solvent, in conjunction with an umbrella sampling strategy. The apo S1S2 easily accesses low-energy conformations that are more open than observed in X-ray crystal structures. A free energy of 9-12 kcal/mol becomes available upon glutamate binding for driving conformational changes in S1S2 associated with receptor activation. Small-angle X-ray scattering profiles calculated from computed ensemble averages agree better with experimental results than profiles calculated from static X-ray crystal structures. Water molecules in the cleft may contribute to stabilizing the apo S1S2 in open conformations. Free energy landscapes were also computed for the glutamate-bound T686A and T686S S1S2 mutants, and the results elaborate on findings from experimental functional studies.
Assuntos
Receptores de AMPA/química , Termodinâmica , Apoproteínas/química , Apoproteínas/metabolismo , Sítios de Ligação , Simulação por Computador , Ácido Glutâmico/química , Ácido Glutâmico/metabolismo , Ligantes , Modelos Moleculares , Mutação , Conformação Proteica , Estrutura Terciária de Proteína , Quinoxalinas/química , Quinoxalinas/metabolismo , Receptores de AMPA/genética , Receptores de AMPA/metabolismo , Espalhamento a Baixo Ângulo , Difração de Raios XRESUMO
The majority of excitatory synaptic transmission in the central nervous system is mediated by ionotropic glutamate receptors (iGluRs). These membrane-bound protein assemblies consist of modular domains that can be genetically isolated and expressed, which has resulted in a plethora of crystal structures of individual domains in different conformations bound to different ligands. These structures have presented opportunities for molecular dynamics (MD) simulation studies. To examine the free energies that govern molecular behavior, simulation strategies and algorithms have been developed, collectively called enhanced sampling methods This review focuses on the use of enhanced sampling MD simulations of isolated iGluR ligand-binding domains to characterize thermodynamic properties important to receptor function.
Assuntos
Ligantes , Receptores Ionotrópicos de Glutamato/química , Animais , Humanos , Simulação de Dinâmica Molecular , Ligação Proteica , Domínios Proteicos , Receptores de AMPA/química , Receptores Ionotrópicos de Glutamato/genética , Receptores de N-Metil-D-Aspartato/química , Transmissão Sináptica , TermodinâmicaRESUMO
The kainate family of ionotropic glutamate receptors (iGluRs) mediates pre- and postsynaptic neurotransmission. Previously computed conformational potentials of mean force (PMFs) for iGluR ligand-binding domains (LBDs) revealed subtype-dependent conformational differences between α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and N-methyl-D-aspartic acid (NMDA) iGluR subfamilies. Here we report PMFs for the kainate receptor GluK2 in apo and glutamate-bound states. Apo and glutamate-bound GluK2 LBDs preferentially access closed-cleft conformations. Apo GluK2 exhibits a surprisingly high degree of conformational flexibility, accessing open and closed states. Comparing across iGluR subtypes, these results are similar to glycine-binding GluN1 and GluN3A NMDA subunits and differ from glutamate-binding GluA2 and GluN2A subunits. To test the contribution of cross-lobe interactions on closed-cleft LBD stability, we computed PMFs for two GluK2 mutants, D462A and D656S. D462A, but not D656S, weakens closed-cleft conformations of the glutamate-bound LBD. Theoretical Boltzmann-weighted small-angle X-ray scattering profiles improve agreement with experimental results compared with calculations from the LBD crystal structure alone.