Extracellular Cation Binding Pocket Is Essential For Ion Conduction Of OsHKT2;2

AbstractHKT channels mediate sodium uniport or sodium and potassium symport in plants. Monocotyledons express a higher number of HKT proteins than dicotyledons, and it is only within this clade of HKT channels that cation symport mechanisms are found. The prevailing ion composition in the extracellular medium affects the transport abilities of various HKT channels by changing their selectivity or ion transport rates. How this mutual effect is achieved at the molecular level is still unknown. Here, we built a homology model of the monocotyledonous OsHKT2;2, which shows sodium and potassium symport activity, and performed molecular dynamics simulations in the presence of sodium and potassium ions. By analyzing ion-protein interactions, we identified a cation binding pocket on the extracellular protein surface, which is formed by residues P71, D75, D501 and K504. Proline and the two aspartate residues coordinate cations, while K504 forms salt bridges with D75 and D501 and may be involved in the forwarding of cations towards the pore entrance. Functional validation via electrophysiological experiments confirmed the biological relevance of the predicted ion binding pocket and identified K504 as a central key residue. Mutation of the cation coordinating residues affected the functionality of HKT only slightly. Additional in silico mutants and simulations of K504 supported experimental results.

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l-Malate (−2) Protonation State is Required for Efficient Decarboxylation to l-Lactate by the Malolactic Enzyme of Oenococcus oeni

Molecules ◽

10.3390/molecules25153431 ◽

2020 ◽

Vol 25 (15) ◽

pp. 3431

Author(s):

Waldo Acevedo ◽

Pablo Cañón ◽

Felipe Gómez-Alvear ◽

Jaime Huerta ◽

Daniel Aguayo ◽

...

Keyword(s):

Molecular Dynamics ◽

Malic Acid ◽

Sequence Similarity ◽

Isothermal Calorimetry ◽

Homology Model ◽

Binding Pocket ◽

Oenococcus Oeni ◽

Ligand Docking ◽

Malolactic Enzyme ◽

Dynamics Simulations

Malolactic fermentation (MLF) is responsible for the decarboxylation of l-malic into lactic acid in most red wines and some white wines. It reduces the acidity of wine, improves flavor complexity and microbiological stability. Despite its industrial interest, the MLF mechanism is not fully understood. The objective of this study was to provide new insights into the role of pH on the binding of malic acid to the malolactic enzyme (MLE) of Oenococcus oeni. To this end, sequence similarity networks and phylogenetic analysis were used to generate an MLE homology model, which was further refined by molecular dynamics simulations. The resulting model, together with quantum polarized ligand docking (QPLD), was used to describe the MLE binding pocket and pose of l-malic acid (MAL) and its l-malate (−1) and (−2) protonation states (MAL− and MAL2−, respectively). MAL2− has the lowest ∆Gbinding, followed by MAL− and MAL, with values of −23.8, −19.6, and −14.6 kJ/mol, respectively, consistent with those obtained by isothermal calorimetry thermodynamic (ITC) assays. Furthermore, molecular dynamics and MM/GBSA results suggest that only MAL2− displays an extended open conformation at the binding pocket, satisfying the geometrical requirements for Mn2+ coordination, a critical component of MLE activity. These results are consistent with the intracellular pH conditions of O. oeni cells—ranging from pH 5.8 to 6.1—where the enzymatic decarboxylation of malate occurs.

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Structural insights on the effects of mutation of a charged binding pocket residue on phosphopeptide binding to 14-3-3 ζ Protein

10.1101/2021.09.27.461903 ◽

2021 ◽

Author(s):

Sreevidya T S ◽

Somavally Dalvi ◽

Prasanna Venkatraman ◽

Satyavani Vemparala

Keyword(s):

Electrostatic Interactions ◽

Structural Changes ◽

Structural Integrity ◽

Peptide Binding ◽

Limited Proteolysis ◽

Binding Pocket ◽

Mutant Protein ◽

Amino Acid Residues ◽

Salt Bridges ◽

Dynamics Simulations

Mutation of an invariant aspartate residue in the binding pocket of 14-3-3ζ isoform to alanine dramatically reduced phosphopeptide binding and induced opening of the binding pocket. Here we use extensive molecular dynamics simulations to understand the role of D124 residue in ligand binding. The simulations show that in the absence of phosphopeptide, the D124A mutation leads to binding pocket reorganization including widening up of the binding pocket at the major groove and repositioning of N173, a key residue that interacts with the main chain of phosphopeptide. These structural changes would interfere with the efficient binding of the peptide, corroborating the experimental observations. Both gain and loss of electrostatic interactions in the form of salt bridges strongly indicate a rearrangement of the network of interactions within the binding pocket. Limited proteolysis coupled mass spectrometry (lip-MS) of the apo and holo forms of WT and mutant protein shows a peptide binding helix otherwise buried in the WT protein was particularly accessible to trypsin in the apo form of the mutant protein and the region was mapped to 158-186 amino acid residues of 14-3-3ζ. These results further confirm the dynamic nature of D124A mutant. Unlike other basic residues, the invariant D124 facilitates peptide binding by maintaining the geometry of interacting residues and by enforcing the structural integrity of amphipathic pocket.

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Pharmacophore-Based Screening & Modification of Amiloride Analogs for targeting the NhaP-type Cation-Proton Antiporter in Vibrio cholerae

Canadian Journal of Microbiology ◽

10.1139/cjm-2021-0074 ◽

2021 ◽

Author(s):

Muntahi Mourin ◽

Arittra Bhattacharjee ◽

Alvan Wai ◽

Georg Hausner ◽

Joe O'Neil ◽

...

Keyword(s):

Molecular Dynamics ◽

Vibrio Cholerae ◽

Mutational Analysis ◽

Md Simulations ◽

Binding Pocket ◽

Cation Binding ◽

Important Amino Acid ◽

Loop Region ◽

Dynamics Simulations ◽

Amiloride Analogs

Structural and mutational analysis of Vc-NhaP2 identified a putative cation binding pocket formed by antiparallel extended regions of two transmembrane segments (TMSs V/XII) along with TMS VI. Molecular Dynamics (MD) simulations suggested that the flexibility of TMS-V/XII is crucial for the intra-molecular conformational events in Vc-NhaP2. In this study, we developed some putative Vc-NhaP2 inhibitors from Amiloride analogs (AAs). Molecular docking of the modified AAs revealed promising binding. The four selected drugs potentially interacted with functionally important amino acid residues located on the cytoplasmic side of TMS VI, the extended chain region of TMS V and TMS XII and the loop region between TMSs VIIII and IX. Molecular dynamics simulations revealed that binding of the selected drugs can potentially destabilize the Vc-NhaP2 and alters the flexibility of the functionally important TMS VI. The work presents the utility of in silico approaches for the rational identification of potential targets and drugs that could target NhaP2 cation proton antiporter to control Vibrio cholerae. The goal is to identify potential drugs that can be validated in future experiments.

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Physiological, Structural, and Functional Analysis of the Paralogous Cation–Proton Antiporters of NhaP Type from Vibrio cholerae

International Journal of Molecular Sciences ◽

10.3390/ijms20102572 ◽

2019 ◽

Vol 20 (10) ◽

pp. 2572 ◽

Cited By ~ 1

Author(s):

Muntahi Mourin ◽

Alvan Wai ◽

Joe O’Neil ◽

Georg Hausner ◽

Pavel Dibrov

Keyword(s):

Vibrio Cholerae ◽

Ion Selectivity ◽

Acid Tolerance ◽

Binding Pocket ◽

Cation Binding ◽

Transmembrane Segments ◽

Functional Regions ◽

Promising Target ◽

Tolerance Response ◽

Dynamics Simulations

The transmembrane K+/H+ antiporters of NhaP type of Vibrio cholerae (Vc-NhaP1, 2, and 3) are critical for maintenance of K+ homeostasis in the cytoplasm. The entire functional NhaP group is indispensable for the survival of V. cholerae at low pHs suggesting their possible role in the acid tolerance response (ATR) of V. cholerae. Our findings suggest that the Vc-NhaP123 group, and especially its major component, Vc-NhaP2, might be a promising target for the development of novel antimicrobials by narrowly targeting V. cholerae and other NhaP-expressing pathogens. On the basis of Vc-NhaP2 in silico structure modeling, Molecular Dynamics Simulations, and extensive mutagenesis studies, we suggest that the ion-motive module of Vc-NhaP2 is comprised of two functional regions: (i) a putative cation-binding pocket that is formed by antiparallel unfolded regions of two transmembrane segments (TMSs V/XII) crossing each other in the middle of the membrane, known as the NhaA fold; and (ii) a cluster of amino acids determining the ion selectivity.

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Faculty Opinions recommendation of Coupling between side chain interactions and binding pocket flexibility in HLA-B*44:02 molecules investigated by molecular dynamics simulations.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.718542946.793528448 ◽

2017 ◽

Author(s):

Tim Elliott ◽

Andy van Hateren

Keyword(s):

Molecular Dynamics ◽

Molecular Dynamics Simulations ◽

Binding Pocket ◽

Side Chain ◽

Dynamics Simulations

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IDENTIFICATION OF SELETIVE CLAUDIN CATION CHANNEL BLOCKERS

Inflammatory Bowel Diseases ◽

10.1093/ibd/izaa347.057 ◽

2021 ◽

Vol 27 (Supplement_1) ◽

pp. S25-S26

Author(s):

Jingjing Ma ◽

Emma Wu ◽

Ye Li ◽

William Seibel ◽

Le Shen ◽

...

Keyword(s):

Small Molecules ◽

Homology Model ◽

Docking Studies ◽

Channel Blockers ◽

Binding Modes ◽

Barrier Dysfunction ◽

Epithelial Barrier Function ◽

Dynamics Simulations ◽

In Silico Models

Abstract Compromised epithelial barrier function is known to be associated with inflammatory bowel disease (IBD) and may contribute to disease development. One mechanism of barrier dysfunction is increased expression of paracellular tight junction ion and water channels formed by claudins. Claudin-2 and -15 are two such channels. We hypothesize that blocking these channels could be a viable therapeutic approach to treat diarrhea. In an effort to develop blockers of these channels, we turn to our previously developed and validated in silico models of claudin-15 (Samanta et al. 2018). We reasoned that compounds that can bind with the interior of claudin pores can limit paracellular water and ion flux. Thus, we used docking algorithms to search for putative small molecules that bind in the claudin-15 pore. AutoDock Vina was initially used to assess rigid docking using small compound databases. The small molecules were analyzed based on binding affinity to the pore and visualized using VMD for their potential blockage of the channel. Clusters of binding modes were identified based on the prominent interacting residues of the protein with the small molecules. We initially screened 10,500 compounds from within the UIC Centre for Drug Discovery and a cross-section of 10,000 compounds from the NCI open compound repository. This initial screen allowed us to identify 2 first-in-class selective claudin-15 blockers with efficacy in MDCK monolayers induced to express claudin-15 and in wildtype duodenum. Next, we screened the entire NCI open compound repository for additional molecules structurally related to our best initially identified molecule and this has allowed us to identify 13 additional molecules that increase TER of claudin-15 expressing MDCK monolayers by 90–160%. Additionally, these molecules possess similar structural components that will be collected in a fragment library and explored through molecular dynamics simulations. We also developed a claudin-2 homology model on which we are performing docking studies and in vitro measurements, which we expect will result in similar candidate ligands for blocking claudin-2. Our study will provide important insight into the role of claudin-dependent cation permeability in fundamental physiology, which we believe will lead to the utility of claudin blockers as a novel and much needed approach to treat diseases such as IBD.

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Structural and Genetic Determinants of Convergence in the Drosophila tRNA Structure–Function Map

Journal of Molecular Evolution ◽

10.1007/s00239-021-09995-z ◽

2021 ◽

Vol 89 (1-2) ◽

pp. 103-116

Author(s):

Julie Baker Phillips ◽

David H. Ardell

Keyword(s):

Structure Function ◽

Metal Ion ◽

Binding Pocket ◽

Trna Synthetase ◽

Heterologous Gene ◽

Multigene Families ◽

Ion Binding ◽

Trna Genes ◽

Structural Factors ◽

Trna Structure

AbstractThe evolution of tRNA multigene families remains poorly understood, exhibiting unusual phenomena such as functional conversions of tRNA genes through anticodon shift substitutions. We improved FlyBase tRNA gene annotations from twelve Drosophila species, incorporating previously identified ortholog sets to compare substitution rates across tRNA bodies at single-site and base-pair resolution. All rapidly evolving sites fell within the same metal ion-binding pocket that lies at the interface of the two major stacked helical domains. We applied our tRNA Structure–Function Mapper (tSFM) method independently to each Drosophila species and one outgroup species Musca domestica and found that, although predicted tRNA structure–function maps are generally highly conserved in flies, one tRNA Class-Informative Feature (CIF) within the rapidly evolving ion-binding pocket—Cytosine 17 (C17), ancestrally informative for lysylation identity—independently gained asparaginylation identity and substituted in parallel across tRNAAsn paralogs at least once, possibly multiple times, during evolution of the genus. In D. melanogaster, most tRNALys and tRNAAsn genes are co-arrayed in one large heterologous gene cluster, suggesting that heterologous gene conversion as well as structural similarities of tRNA-binding interfaces in the closely related asparaginyl-tRNA synthetase (AsnRS) and lysyl-tRNA synthetase (LysRS) proteins may have played a role in these changes. A previously identified Asn-to-Lys anticodon shift substitution in D. ananassae may have arisen to compensate for the convergent and parallel gains of C17 in tRNAAsn paralogs in that lineage. Our results underscore the functional and evolutionary relevance of our tRNA structure–function map predictions and illuminate multiple genomic and structural factors contributing to rapid, parallel and compensatory evolution of tRNA multigene families.

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X-ray crystallography reveals molecular recognition mechanism for sugar binding in a melibiose transporter MelB

Communications Biology ◽

10.1038/s42003-021-02462-x ◽

2021 ◽

Vol 4 (1) ◽

Author(s):

Lan Guan ◽

Parameswaran Hariharan

Keyword(s):

Molecular Recognition ◽

Binding Pocket ◽

Cation Binding ◽

Recognition Mechanism ◽

X Ray ◽

X Ray Crystallography ◽

Sugar Binding ◽

Sugar Specificity ◽

Major Facilitator ◽

Mfs Transporters

AbstractMajor facilitator superfamily_2 transporters are widely found from bacteria to mammals. The melibiose transporter MelB, which catalyzes melibiose symport with either Na+, Li+, or H+, is a prototype of the Na+-coupled MFS transporters, but its sugar recognition mechanism has been a long-unsolved puzzle. Two high-resolution X-ray crystal structures of a Salmonella typhimurium MelB mutant with a bound ligand, either nitrophenyl-α-d-galactoside or dodecyl-β-d-melibioside, were refined to a resolution of 3.05 or 3.15 Å, respectively. In the substrate-binding site, the interaction of both galactosyl moieties on the two ligands with MelBSt are virturally same, so the sugar specificity determinant pocket can be recognized, and hence the molecular recognition mechanism for sugar binding in MelB has been deciphered. The conserved cation-binding pocket is also proposed, which directly connects to the sugar specificity pocket. These key structural findings have laid a solid foundation for our understanding of the cooperative binding and symport mechanisms in Na+-coupled MFS transporters, including eukaryotic transporters such as MFSD2A.

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