Gaussian Accelerated Molecular Dynamics Research Articles

Pathway and mechanism of drug binding to chemokine receptors revealed by accelerated molecular simulations.

Background: Chemokine GPCRsplay key roles in biology and medicine. Particularly, CXCR4 promotes cancer metastasis and facilitate HIV entry into host cells. Plerixafor (PLX) is a CXCR4 drug, but the pathway and binding site of PLX in CXCR4 remain unknown. Results &methodology: We have performed molecular docking and all-atom simulations using Gaussian accelerated molecular dynamics (GaMD), which are consistent with previous mutation experiments, suggesting that PLX binds to the orthosteric site of CXCR4 as an antagonist. The GaMD simulations further revealed an intermediate allosteric binding site at the extracellular mouth of CXCR4. Conclusion: The newly identified allosteric site can be targeted for novel drug design targeting CXCR4 and other chemokine receptors.

Computational modeling of cyclic peptide inhibitor–MDM2/MDMX binding through global docking and Gaussian accelerated molecular dynamics simulations

MDM2 and MDMX are potential targets for p53-dependent cancer therapy. Peptides are key in cellular immunology and oncology, and cyclic peptides generally have higher half-life than their linear counterparts. However, prediction of cyclic peptide–protein binding is challenging with normal molecular simulation approaches because of high peptide flexibility. Here, we used global peptide docking, normal molecular dynamics, Gaussian accelerated molecular dynamics (GaMD), two-dimensional (2D) potential of mean force (PMF) profiles, and solvated interaction energy (SIE) techniques to investigate the interactions of MDM2/MDMX with three N-to-C-terminal cyclic peptide–based inhibitors. We determined the possible cyclic peptide–MDM2/MDMX complex structures via 2D PMF profiles and SIE calculations. Our findings increase the accuracy of peptide–protein structural prediction, which may facilitate cyclic peptide drug design. Advancements in the computational methods and computing power may further aid in addressing the challenges in cyclic peptide drug design. Communicated by Ramaswamy H. Sarma

Mechanisms of γ-Secretase Activation and Substrate Processing.

Amyloid β-peptide, the principal component of characteristic cerebral plaques of Alzheimer’s disease (AD), is produced through intramembrane proteolysis of the amyloid precursor protein (APP) by γ-secretase. Despite the importance in the pathogenesis of AD, the mechanisms of intramembrane proteolysis and substrate processing by γ-secretase remain poorly understood. Here, complementary all-atom simulations using a robust Gaussian accelerated molecular dynamics (GaMD) method and biochemical experiments were combined to investigate substrate processing of wildtype and mutant APP by γ-secretase. The GaMD simulations captured spontaneous activation of γ-secretase, with hydrogen bonded catalytic aspartates and water poised for proteolysis of APP at the ε cleavage site. Furthermore, GaMD simulations revealed that familial AD mutations I45F and T48P enhanced the initial ε cleavage between residues Leu49–Val50, while M51F mutation shifted the ε cleavage site to the amide bond between Thr48–Leu49. Detailed analysis of the GaMD simulations allowed us to identify distinct low-energy conformational states of γ-secretase, different secondary structures of the wildtype and mutant APP substrate, and important active-site subpockets for catalytic function of the enzyme. The simulation findings were highly consistent with experimental analyses of APP proteolytic products using mass spectrometry and Western blotting. Taken together, the GaMD simulations and biochemical experiments have enabled us to elucidate the mechanisms of γ-secretase activation and substrate processing, which should facilitate rational computer-aided drug design targeting this functionally important enzyme.

Enhanced Sampling of Peptide Binding to Proteins through Gaussian Accelerated Molecular Dynamics Simulations

Peptides mediate up to 40% of known protein-protein interactions in higher eukaryotes and play an important role in cellular signaling. However, it is challenging to sufficiently sample peptide-protein interactions through conventional molecular dynamics simulations, due to long timescales and extremely high system flexibility. Here, all-atom simulations using a robust Gaussian accelerated molecular dynamics (GaMD) method have been applied for enhanced sampling of peptide binding to proteins. The GaMD simulations have identified important low-energy conformational states for three model peptide-protein complexes.

Dynamics and Location of the Allosteric Midazolam Site in Cytochrome P4503A4 in Lipid Nanodiscs

Promiscuous and allosteric drug interactions with cytochrome P450 3A4 (CYP3A4) are ubiquitous but incompletely understood at the molecular level. A classic allosteric CYP3A4 drug interaction includes the benzodiazepine midazolam (MDZ). MDZ exhibits homotropic and heterotropic allostery when metabolized to 1'-hydroxy and 4-hydroxy metabolites in varying ratios. The combination of hydrogen-deuterium exchange mass spectrometry (HDX-MS) and Gaussian accelerated molecular dynamics (GaMD) simulations of CYP3A4 in lipid nanodiscs and in a lipid bilayer, respectively, reveals MDZ-dependent changes in dynamics in a membrane environment. The F-, G-, and intervening helices, as well as the loop preceding the β1-sheets, display the largest observed changes in HDX. The GaMD suggests a potential allosteric binding site for MDZ in the F'- and G'-regions, which undergo significant increases in HDX at near-saturating MDZ concentrations. The HDX-MS and GaMD results confirm that changes in dynamics are most significant near the developing consensus allosteric site, and these changes are distinct from those observed previously with the nonallosteric inhibitor ketoconazole. The results suggest that the allosteric MDZ remains mobile in its binding site at the Phe-cluster. The results further suggest that this binding site remains dynamic or changes the depth of insertion in the membrane.

Allosteric Motions of the CRISPR-Cas9 HNH Nuclease Probed by NMR and Molecular Dynamics.

CRISPR-Cas9 is a widely employed genome-editing tool with functionality reliant on the ability of the Cas9 endonuclease to introduce site-specific breaks in double-stranded DNA. In this system, an intriguing allosteric communication has been suggested to control its DNA cleavage activity through flexibility of the catalytic HNH domain. Here, solution NMR experiments and a novel Gaussian-accelerated molecular dynamics (GaMD) simulation method are used to capture the structural and dynamic determinants of allosteric signaling within the HNH domain. We reveal the existence of a millisecond time scale dynamic pathway that spans HNH from the region interfacing the adjacent RuvC nuclease and propagates up to the DNA recognition lobe in full-length CRISPR-Cas9. These findings reveal a potential route of signal transduction within the CRISPR-Cas9 HNH nuclease, advancing our understanding of the allosteric pathway of activation. Further, considering the role of allosteric signaling in the specificity of CRISPR-Cas9, this work poses the mechanistic basis for novel engineering efforts aimed at improving its genome-editing capability.

The mechanism of high affinity pentasaccharide binding to antithrombin, insights from Gaussian accelerated molecular dynamics simulations

The activity of antithrombin (AT), a serpin protease inhibitor, is enhanced by heparin and heparin analogs against its target proteases, mainly thrombin, factors Xa and IXa. Considerable amount of information is available on the multistep mechanism of the heparin pentasaccharide binding and conformational activation. However, much of the details were inferred from ‘static’ structures obtained by X-ray diffraction. Moreover, limited information is available for the early steps of binding mechanism other than kinetic studies with various ligands. To gain insights into these processes, we performed enhanced sampling molecular dynamics (MD) simulations using the Gaussian Accelerated Molecular Dynamics (GAMD) method, applied previously in drug binding studies. We were able to observe the binding of the pentasaccharide idraparinux to a ‘non-activated’ AT conformation in two separate trajectories with low root mean square deviation (RMSD) values compared to X-ray structures of the bound state. These trajectories along with further simulations of the AT-pentasaccharide complex provided insights into the mechanisms of multiple conformational transitions, including the expulsion of the hinge region, the extension of helix D and the conformational behavior of the reactive center loop (RCL). We could also confirm the high stability of helix P in non-activated AT conformations, such states might play an important role in heparin binding. ‘Generalized correlation’ matrices revealed possible paths of allosteric signal propagation to the binding sites for the target proteases, factors Xa and IXa. Enhanced MD simulations of ligand binding to AT may assist the design of new anticoagulant drugs.Communicated by Ramaswamy H. Sarma

Improved Modeling of Peptide-Protein Binding Through Global Docking and Accelerated Molecular Dynamics Simulations.

Peptides mediate up to 40% of known protein-protein interactions in higher eukaryotes and play a key role in cellular signaling, protein trafficking, immunology, and oncology. However, it is challenging to predict peptide-protein binding with conventional computational modeling approaches, due to slow dynamics and high peptide flexibility. Here, we present a prototype of the approach which combines global peptide docking using ClusPro PeptiDock and all-atom enhanced simulations using Gaussian accelerated molecular dynamics (GaMD). For three distinct model peptides, the lowest backbone root-mean-square deviations (RMSDs) of their bound conformations relative to X-ray structures obtained from PeptiDock were 3.3–4.8 Å, being medium quality predictions according to the Critical Assessment of PRediction of Interactions (CAPRI) criteria. GaMD simulations refined the peptide-protein complex structures with significantly reduced peptide backbone RMSDs of 0.6–2.7 Å, yielding two high quality (sub-angstrom) and one medium quality models. Furthermore, the GaMD simulations identified important low-energy conformational states and revealed the mechanism of peptide binding to the target proteins. Therefore, PeptiDock+GaMD is a promising approach for exploring peptide-protein interactions.

G-Protein-Coupled Receptor-Membrane Interactions Depend on the Receptor Activation State.

G-protein-coupled receptors (GPCRs) are the largest family of human membrane proteins and serve as primary targets of approximately one-third of currently marketed drugs. In particular, adenosine A1 receptor (A1 AR) is an important therapeutic target for treating cardiac ischemia-reperfusion injuries, neuropathic pain, and renal diseases. As a prototypical GPCR, the A1 AR is located within a phospholipid membrane bilayer and transmits cellular signals by changing between different conformational states. It is important to elucidate the lipid-protein interactions in order to understand the functional mechanism of GPCRs. Here, all-atom simulations using a robust Gaussian accelerated molecular dynamics (GaMD) method were performed on both the inactive (antagonist bound) and active (agonist and G-protein bound) A1 AR, which was embedded in a 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC) lipid bilayer. In the GaMD simulations, the membrane lipids played a key role in stabilizing different conformational states of the A1 AR. Our simulations further identified important regions of the receptor that interacted distinctly with the lipids in highly correlated manner. Activation of the A1 AR led to differential dynamics in the upper and lower leaflets of the lipid bilayer. In summary, GaMD enhanced simulations have revealed strongly coupled dynamics of the GPCR and lipids that depend on the receptor activation state. © 2019 Wiley Periodicals, Inc.

Replica-Exchange Umbrella Sampling Combined with Gaussian Accelerated Molecular Dynamics for Free-Energy Calculation of Biomolecules.

We have developed an enhanced conformational sampling method combining replica-exchange umbrella sampling (REUS) with Gaussian accelerated molecular dynamics (GaMD). REUS enhances the sampling along predefined reaction coordinates, while GaMD accelerates the conformational dynamics by adding a boost potential to the system energy. The method, which we call GaREUS (Gaussian accelerated replica-exchange umbrella sampling), enhances the sampling more efficiently than REUS or GaMD, while the computational resource for GaREUS is the same as that required for REUS. The two-step reweighting procedure using the multistate Bennett acceptance ratio method and the cumulant expansion for the exponential average is applied to the simulation trajectories for obtaining the unbiased free-energy landscapes. We apply GaREUS to the calculations of free-energy landscapes for three different cases: conformational equilibria of N-glycan, folding of chignolin, and conformational change of adenyl kinase. We show that GaREUS speeds up the convergences of free-energy calculations using the same amount of computational resources as REUS. The free-energy landscapes reweighted from the trajectories of GaREUS agree with previously reported ones. GaREUS is applicable to free-energy calculations of various biomolecular dynamics and functions with reasonable computational costs.

Mechanistic Insights into Specific G Protein Interactions with Adenosine Receptors.

Coupling between G-protein-coupled receptors (GPCRs) and the G proteins is a key step in cellular signaling. Despite extensive experimental and computational studies, the mechanism of specific GPCR-G protein coupling remains poorly understood. This has greatly hindered effective drug design of GPCRs that are primary targets of ∼1/3 of currently marketed drugs. Here, we have employed all-atom simulations using a robust Gaussian accelerated molecular dynamics (GaMD) method to decipher the mechanism of the GPCR-G protein interactions. Adenosine receptors (ARs) were used as model systems based on very recently determined cryo-EM structures of the A1AR and A2AAR coupled with the Gi and Gs proteins, respectively. Changing the Gi protein to the Gs led to increased fluctuations in the A1AR and agonist adenosine (ADO), while agonist 5'-N-ethylcarboxamidoadenosine (NECA) binding in the A2AAR could be still stabilized upon changing the Gs protein to the Gi. Free energy calculations identified one stable low-energy conformation for each of the A1AR-Gi and A2AAR-Gs complexes as in the cryo-EM structures, similarly for the A2AAR-Gi complex. In contrast, the ADO agonist and Gs protein sampled multiple conformations in the A1AR-Gs system. GaMD simulations thus indicated that the A1AR preferred to couple with the Gi protein to the Gs, while the A2AAR could couple with both the Gs and Gi proteins, being highly consistent with experimental findings of the ARs. More importantly, detailed analysis of the atomic simulations showed that the specific AR-G protein coupling resulted from remarkably complementary residue interactions at the protein interface, involving mainly the receptor transmembrane 6 helix and the Gα α5 helix and α4-β6 loop. In summary, the GaMD simulations have provided unprecedented insights into the dynamic mechanism of specific GPCR-G protein interactions at an atomistic level.

A Computational Approach to the Study of the Binding Mode of S1P1R Agonists Based on the Active-Like Receptor Model.

Sphingosine-1-phosphate receptor 1 (S1P1R), a member of the G protein-coupled receptor (GPCR) family, is an attractive protein target for the treatment of autoimmune diseases, and a diverse array of S1P1R agonists have been developed. Rational drug design based on S1P1R remains challenging due to the limited information available on the binding mode between S1P1R and its agonists. In this work, the active-like state of S1P1R was modeled via Gaussian accelerated molecular dynamics (GaMD) based on its inactive form, which was further validated by docking studies with two representative S1P1R agonists. Moreover, with the usage of the induced active-like state, the binding mode between S1P1R and its agonists was studied through molecular dynamics simulations and MM-GBSA calculations. The results of those studies indicated that four groups of binding site residues were the major contributors to the ligand and receptor interactions. In addition, this model was verified by five chemically similar compounds synthesized in-house and 1145 known S1P1R agonists collected from the BindingDB database. The elucidation of the key binding characteristics will further complete the cognition of S1P1R, which can guide the rational design of novel S1P1R agonists.

Deciphering Off-Target Effects in CRISPR-Cas9 through Accelerated Molecular Dynamics.

CRISPR-Cas9 is the state-of-the-art technology for editing and manipulating nucleic acids. However, the occurrence of off-target mutations can limit its applicability. Here, all-atom enhanced molecular dynamics (MD) simulations—using Gaussian accelerated MD (GaMD)—are used to decipher the mechanism of off-target binding at the molecular level. GaMD reveals that base pair mismatches in the target DNA at distal sites with respect to the protospacer adjacent motif (PAM) can induce an extended opening of the RNA:DNA heteroduplex, which leads to newly formed interactions between the unwound DNA and the L2 loop of the catalytic HNH domain. These conserved interactions constitute a “lock” effectively decreasing the conformational freedom of the HNH domain and hampering its activation for cleavage. Remarkably, depending on their positions at PAM distal sites, DNA mismatches responsible for off-target cleavages are unable to “lock” the HNH domain, thereby leading to the unselective cleavage of DNA sequences. In consistency with the available experimental data, the ability to “lock” the catalytic HNH domain in an inactive “conformational checkpoint” is shown to be a key determinant in the onset of off-target effects. This mechanistic rationale contributes in clarifying a long lasting open issue in the CRISPR-Cas9 function and poses the foundation for designing novel and more specific Cas9 variants, which could be obtained by magnifying the “locking” interactions between HNH and the target DNA in the presence of any incorrect off-target sequence, thus preventing undesired cleavages.

Molecular Mechanism of Off-Target Effects in CRISPR-Cas9

Author(s): Ricci, Clarisse; Chen, Janice; Miao, Yinglong; Jinek, Martin; Doudna, Jennifer; McCammon, Andrew; Palermo, Giulia | Abstract: Abstract CRISPR-Cas9 is the state-of-the-art technology for editing and manipulating nucleic acids. However, the occurrence of off-target mutations can limit its applicability. Here, all-atom enhanced molecular dynamics (MD) simulations – using Gaussian accelerated MD (GaMD) – are used to decipher the mechanism of off-target binding at the molecular level. GaMD reveals that base pair mismatches in the target DNA at specific distal sites with respect to the Protospacer Adjacent Motif (PAM) induce an extended opening of the RNA:DNA heteroduplex, which leads to newly discovered interactions between the unwound nucleic acids and the protein counterpart. The conserved interactions between the target DNA strand and the L2 loop of the catalytic HNH domain constitute a “lock” effectively decreasing the conformational freedom of the HNH domain and its activation for cleavage. Remarkably, depending on their position at PAM distal sites, DNA mismatches leading to off-target cleavages are unable to “lock” the HNH domain, thereby identifying the ability to “lock” HNH as a key determinant. Consistently, off-target sequences hampering the catalysis have been shown to “trap” somehow the HNH domain in an inactive “conformational checkpoint” state (Dagdas et al. Sci Adv, 2017). As such, this mechanism identifies the molecular basis underlying off-target cleavages and contributes in clarifying a long-lasting open issue of the CRISPR-Cas9 function. It also poses the foundation for designing novel and more specific Cas9 variants, which could be obtained by magnifying the “locking” interactions between HNH and the target DNA in the presence of any incorrect off-target sequence, thus preventing undesired cleavages.

Mechanism of Specific G Protein Coupling to Adenosine Receptors

Four subtypes of adenosine receptors (ARs), the A1AR, A2AAR, A2BAR and A3AR, mediate a broad range of physiological functions. They have emerged as important therapeutic targets for treating human diseases including cardiac ischemia, neuropathic pain and cancer. The odd-numbered subtypes prefer to couple with the Gi/o proteins, while the even-numbered subtypes predominantly couple with the Gs proteins. Despite extensive experimental and computational studies, the exact determinants of ARs-G protein coupling specificity remain unclear. Here, all-atom enhanced sampling simulations using a robust Gaussian accelerated molecular dynamics (GaMD) method have been applied to determine the mechanism of specific coupling between the A1AR and A2AAR and the Gi and Gs proteins, respectively. The GaMD simulations have revealed differentiated fluctuations and distinct low-energy conformational states of the ARs upon binding of decoy versus cognate G proteins. Important motifs that are responsible for the receptor-G protein coupling specificity have been also identified. In addition, our GaMD simulation findings are consistent with recent experimental data. The GaMD simulations have provided important insights into the mechanism of specific coupling between ARs and the G proteins at an atomistic level, which will facilitate future computer-aided design of selective drug molecules targeting the ARs.

Molecular mechanism of point mutation-induced Monopolar spindle 1 (Mps1/TTK) inhibitor resistance revealed by a comprehensive molecular modeling study.

BackgroundMonopolar spindle 1 (Mps1/TTK) is an apical dual-specificity protein kinase in the spindle assembly checkpoint (SAC) that guarantees accurate segregation of chromosomes during mitosis. High levels of Mps1 are found in various types of human malignancies, such as glioblastoma, osteosarcoma, hepatocellular carcinoma, and breast cancer. Several potent inhibitors of Mps1 exist, and exhibit promising activity in many cell cultures and xenograft models. However, resistance due to point mutations in the kinase domain of Mps1 limits the therapeutic effects of these inhibitors. Understanding the detailed resistance mechanism induced by Mps1 point mutations is therefore vital for the development of novel inhibitors against malignancies.MethodsIn this study, conventional molecular dynamics (MD) simulation and Gaussian accelerated MD (GaMD) simulation were performed to elucidate the resistance mechanisms of Cpd-5, a potent Mps1 inhibitor, induced by the four representative mutations I531M, I598F, C604Y, S611R.ResultsOur results from conventional MD simulation combined with structural analysis and free energy calculation indicated that the four mutations weaken the binding affinity of Cpd-5 and the major variations in structural were the conformational changes of the P-loop, A-loop and αC-helix. Energetic differences of per-residue between the WT system and the mutant systems indicated the mutations may allosterically regulate the conformational ensemble and the major variations were residues of Ile-663 and Gln-683, which located in the key loops of catalytic loop and A-loop, respectively. The large conformational and energetic differences were further supported by the GaMD simulations. Overall, these obtained molecular mechanisms will aid rational design of novel Mps1 inhibitors to combat inhibitor resistance.

Replica-Exchange Methods for Biomolecular Simulations.

In this study, a replica-exchange method was developed to overcome conformational sampling difficulties in computer simulations of spin glass or other systems with rugged free-energy landscapes. This method was then applied to the protein-folding problem in combination with molecular dynamics (MD) simulation. Owing to its simplicity and sampling efficiency, the replica-exchange method has been applied to many other biological problems and has been continuously improved. The method has often been combined with other sampling techniques, such as umbrella sampling, free-energy perturbation, metadynamics, and Gaussian accelerated MD (GaMD). In this chapter, we first summarize the original replica-exchange molecular dynamics (REMD) method and discuss how new algorithms related to the original method are implemented to add new features. Heterogeneous and flexible structures of an N-glycan in a solution are simulated as an example of applications by REMD, replica exchange with solute tempering, and GaMD. The sampling efficiency of these methods on the N-glycan system and the convergence of the free-energy changes are compared. REMD simulation protocols and trajectory analysis using the GENESIS software are provided to facilitate the practical use of advanced simulation methods.

The effect of dimerization on the activation and conformational dynamics of adenosine A1 receptor.

The adenosine A1 receptor (A1R) is one of four adenosine receptors in humans, which are involved in the function of the cardiovascular, respiratory and central nervous systems. Experimental results indicate that A1R can form a homodimer and that the protomer-protomer interaction in the A1R dimer is related to certain pharmacological characteristics of A1R activation. In this work, we performed docking, metadynamics simulation, conventional molecular dynamics simulations, Gaussian-accelerated molecular dynamics simulations, potential of mean force calculations, dynamic cross-correlation motions analysis and community network analysis to study the binding mode of 5'-N-ethylcarboxamidoadenosine (NECA) to A1R and the effect of dimerization on the activation of A1R. Our results show that NECA binds to A1R in a similar mode to adenosine in the A1R crystal structure and NECA in the A2AR crystal structure. The A1R homodimer can be activated by one or two agonists with NECA occupying its orthosteric pockets in one (which we call the NECA-A1R system) or both protomers (which we call the dNECA-A1R system). In the NECA-A1R system, activation is predicated in the protomer without NECA bound. In the dNECA-A1R system, only one protomer achieves the active state. These findings suggest an asymmetrical activation mechanism of the homodimer and a negative cooperativity between the two protomers. We envision that our results may further facilitate the drug development of A1R.

Structural Basis for Binding of Allosteric Drug Leads in the Adenosine A1 Receptor

Despite intense interest in designing positive allosteric modulators (PAMs) as selective drugs of the adenosine A1 receptor (A1AR), structural binding modes of the receptor PAMs remain unknown. Using the first X-ray structure of the A1AR, we have performed all-atom simulations using a robust Gaussian accelerated molecular dynamics (GaMD) technique to determine binding modes of the A1AR allosteric drug leads. Two prototypical PAMs, PD81723 and VCP171, were selected. Each PAM was initially placed at least 20 Å away from the receptor. Extensive GaMD simulations using the AMBER and NAMD simulation packages at different acceleration levels captured spontaneous binding of PAMs to the A1AR. The simulations allowed us to identify low-energy binding modes of the PAMs at an allosteric site formed by the receptor extracellular loop 2 (ECL2), which are highly consistent with mutagenesis experimental data. Furthermore, the PAMs stabilized agonist binding in the receptor. In the absence of PAMs at the ECL2 allosteric site, the agonist sampled a significantly larger conformational space and even dissociated from the A1AR alone. In summary, the GaMD simulations elucidated structural binding modes of the PAMs and provided important insights into allostery in the A1AR, which will greatly facilitate the receptor structure-based drug design.

Identification of SLAC1 anion channel residues required for CO2/bicarbonate sensing and regulation of stomatal movements

Increases in CO2 concentration in plant leaves due to respiration in the dark and the continuing atmospheric [CO2] rise cause closing of stomatal pores, thus affecting plant-water relations globally. However, the underlying CO2/bicarbonate (CO2/HCO3-) sensing mechanisms remain unknown. [CO2] elevation in leaves triggers stomatal closure by anion efflux mediated via the SLAC1 anion channel localized in the plasma membrane of guard cells. Previous reconstitution analysis has suggested that intracellular bicarbonate ions might directly up-regulate SLAC1 channel activity. However, whether such a CO2/HCO3- regulation of SLAC1 is relevant for CO2 control of stomatal movements in planta remains unknown. Here, we computationally probe for candidate bicarbonate-interacting sites within the SLAC1 anion channel via long-timescale Gaussian accelerated molecular dynamics (GaMD) simulations. Mutations of two putative bicarbonate-interacting residues, R256 and R321, impaired the enhancement of the SLAC1 anion channel activity by CO2/HCO3- in Xenopus oocytes. Mutations of the neighboring charged amino acid K255 and residue R432 and the predicted gate residue F450 did not affect HCO3- regulation of SLAC1. Notably, gas-exchange experiments with slac1-transformed plants expressing mutated SLAC1 proteins revealed that the SLAC1 residue R256 is required for CO2 regulation of stomatal movements in planta, but not for abscisic acid (ABA)-induced stomatal closing. Patch clamp analyses of guard cells show that activation of S-type anion channels by CO2/HCO3-, but not by ABA, was impaired, indicating the relevance of R256 for CO2 signal transduction. Together, these analyses suggest that the SLAC1 anion channel is one of the physiologically relevant CO2/HCO3- sensors in guard cells.