High-resolution Crystal Structures Research Articles

Probing Short-Range Interactions in Isostructural Hydrate and Perhydrate of Dabrafenib by Magic-Angle Spinning Solid-State NMR.

Dabrafenib (DBF), an anticancer drug, exhibits isostructural properties in its hydrate (DBF⊃H2O) and perhydrate (DBF⊃H2O2) forms, as revealed by single-crystal X-ray diffraction. Despite the H2O and H2O2 solvent molecules occupying identical locations, the two polymorphs have different thermal behaviors. In general, determination of stoichiometry of H2O in the perhydrate crystals is difficult due to the presence of both H2O and H2O2 in the same crystal voids. This study utilizes magic-angle spinning (MAS) solid-state NMR (SSNMR) combined with gauge-included projector augmented wave calculations to characterize the influence of solvent molecules on the local environment in pseudopolymorphs. SSNMR experiments were employed to assign 1H, 13C, and 15N peaks and identify spectral differences in the isostructural pseudopolymorphs. Proton spectroscopy at fast MAS was used to identify and quantify H2O2/H2O in DBF⊃H2O2 (mixed hydrate/perhydrate). 1H-1H dipolar-coupling-based experiments were recruited to confirm the 3D molecular packing of solvent molecules in DBF⊃H2O and DBF⊃H2O2. Homonuclear (1H-1H) and heteronuclear (1H-14N) distance measurements, in conjunction with diffraction structures and optimized hydrogen atom positions by density functional theory, helped decipher local interactions of H2O2 with DBF and their geometry in DBF⊃H2O2. This integrated X-ray structure, quantum chemical calculations, and NMR study of pseudopolymorphs offer a practical approach to scrutinizing crystallized solvent interactions in the crystal lattice even without high-resolution crystal structures or artificial sample enrichment.

Design of substituted tetrahydrofuran derivatives for HIV-1 protease inhibitors: synthesis, biological evaluation, and X-ray structural studies.

Substituted tetrahydrofuran derivatives were designed and synthesized to serve as the P2 ligand for a series of potent HIV-1 protease inhibitors. Both enantiomers of the tetrahydrofuran derivatives were synthesized stereoselectivity in optically active forms using lipase-PS catalyzed enzymatic resolution as the key step. These tetrahydrofuran derivatives are designed to promote hydrogen bonding and van der Waals interactions with the backbone atoms in the S2 subsite of the HIV-1 protease active site. Several inhibitors displayed very potent HIV-1 protease inhibitory activity. A high-resolution X-ray crystal structure of an inhibitor-bound HIV-1 protease provided important insight into the ligand binding site interactions in the active site.

Structure-Guided Design of Potent Coronavirus Inhibitors with a 2-Pyrrolidone Scaffold: Biochemical, Crystallographic, and Virological Studies.

Zoonotic coronaviruses are known to produce severe infections in humans and have been the cause of significant morbidity and mortality worldwide. SARS-CoV-2 was the largest and latest contributor of fatal cases, even though MERS-CoV has the highest case-fatality ratio among zoonotic coronaviruses. These infections pose a high risk to public health worldwide warranting efforts for the expeditious discovery of antivirals. Hence, we hereby describe a novel series of inhibitors of coronavirus 3CLpro embodying an N-substituted 2-pyrrolidone scaffold envisaged to exploit favorable interactions with the S3-S4 subsites and connected to an invariant Leu-Gln P2-P1 recognition element. Several inhibitors showed nanomolar antiviral activity in enzyme and cell-based assays, with no significant cytotoxicity. High-resolution crystal structures of inhibitors bound to the 3CLpro were determined to probe and identify the molecular determinants associated with binding, to inform the structure-guided optimization of the inhibitors, and to confirm the mechanism of action of the inhibitors.

Structural basis of the dehydratase module (hDH) of human fatty acid synthase.

The human fatty acid synthase (hFASN) produces fatty acids for cellar membrane construction, energy storage, biomolecule modifications and signal transduction. Abnormal expression and functions of hFASN highly associate with numerous human diseases such as obesity, diabetes, and cancers, and thereby it has been considered as a valuable potential drug target. So far, the structural and catalytic mechanisms of most of the hFASN enzymatic modules have been extensively studied, except the key dehydratase module (hDH). Here we presented the enzymatic characterization and the high-resolution crystal structure of hDH. We demonstrated that the hDH preferentially catalyzes the acyl substrates with short lengths between 4 to 8-carbons, and exhibits much lower enzymatic activity on longer substrates. Subsequent structural study showed that hDH displays a pseudo-dimeric organization with a single L-shaped composite hydrophobic catalytic tunnel as well as an atypical ACP binding site nearby, indicating that hDH achieves distinct substrate recognition and dehydration mechanisms compared to the conventional bacterial fatty acid dehydratases identified. Our findings laid the foundation for understanding the biological and pathogenic functions of hFASN, and may facilitate therapeutical drug development against diseases with abnormal functionality of hFASN.

Nonclinical and clinical characterization of MAU868, a novel human-derived monoclonal neutralizing antibody targeting Brenner-Karlsruhe polyomavirus viral protein 1

Reactivation of Brenner-Karlsruhe polyomavirus (human polyomavirus 1) (BKPyV) can cause significant kidney and bladder disease in immunocompromised patients. There are currently no effective, BKPyV-specific therapies. MAU868 is a novel, human immunoglobulin G1 monoclonal antibody that binds the major capsid protein, viral protein (VP) 1, of BKPyV with picomolar affinity, neutralizes infection by the 4 major BKPyV genotypes (EC50 ranging from 0.009-0.093 μg/mL; EC90 ranging from 0.102-4.160 μg/mL), and has comparable activity against variants with highly prevalent VP1 polymorphisms. No resistance-associated variants were identified in long-term selection studies, indicating a high in vitro barrier-to-resistance. The high-resolution crystal structure of MAU868 in complex with VP1 pentamer identified 3 key contact residues in VP1 (Y169, R170, and K172). To our knowledge, the first-in-human study was conducted to assess the safety, tolerability, and pharmacokinetics of MAU868 following intravenous and subcutaneous administration to healthy adults in a randomized, placebo-controlled, double-blinded, single ascending dose design. MAU868 was safe and well-tolerated. All adverse events were grade 1 and resolved. The pharmacokinetics of MAU868 was typical of a human immunoglobulin G, with dose-proportional systemic exposure and an elimination half-life ranging between 23 and 30 days. These results demonstrate the potential of MAU868 as a first-in-class therapeutic agent for the treatment or prevention of BKPyV disease.

TAAM refinement on high-resolution experimental and simulated 3D ED/MicroED data for organic molecules.

3D electron diffraction (3D ED), or microcrystal electron diffraction (MicroED), has become an alternative technique for determining the high-resolution crystal structures of compounds from sub-micron-sized crystals. Here, we considered L-alanine, α-glycine and urea, which are known to form good-quality crystals, and collected high-resolution 3D ED data on our in-house TEM instrument. In this study, we present a comparison of independent atom model (IAM) and transferable aspherical atom model (TAAM) kinematical refinement against experimental and simulated data. TAAM refinement on both experimental and simulated data clearly improves the model fitting statistics (R factors and residual electrostatic potential) compared to IAM refinement. This shows that TAAM better represents the experimental electrostatic potential of organic crystals than IAM. Furthermore, we compared the geometrical parameters and atomic displacement parameters (ADPs) resulting from the experimental refinements with the simulated refinements, with the periodic density functional theory (DFT) calculations and with published X-ray and neutron crystal structures. The TAAM refinements on the 3D ED data did not improve the accuracy of the bond lengths between the non-H atoms. The experimental 3D ED data provided more accurate H-atom positions than the IAM refinements on the X-ray diffraction data. The IAM refinements against 3D ED data had a tendency to lead to slightly longer X-H bond lengths than TAAM, but the difference was statistically insignificant. Atomic displacement parameters were too large by tens of percent for L-alanine and α-glycine. Most probably, other unmodelled effects were causing this behaviour, such as radiation damage or dynamical scattering.

A structural role for tryptophan in proteins, and the ubiquitous Trp Cδ1-H...O=C (backbone) hydrogen bond.

Tryptophan is the most prominent amino acid found in proteins, with multiple functional roles. Its side chain is made up of the hydrophobic indole moiety, with two groups that act as donors in hydrogen bonds: the Nϵ-H group, which is a potent donor in canonical hydrogen bonds, and a polarized Cδ1-H group, which is capable of forming weaker, noncanonical hydrogen bonds. Due to adjacent electron-withdrawing moieties, C-H...O hydrogen bonds are ubiquitous in macromolecules, albeit contingent on the polarization of the donor C-H group. Consequently, Cα-H groups (adjacent to the carbonyl and amino groups of flanking peptide bonds), as well as the Cϵ1-H and Cδ2-H groups of histidines (adjacent to imidazole N atoms), are known to serve as donors in hydrogen bonds, for example stabilizing parallel and antiparallel β-sheets. However, the nature and the functional role of interactions involving the Cδ1-H group of the indole ring of tryptophan are not well characterized. Here, data mining of high-resolution (r ≤ 1.5 Å) crystal structures from the Protein Data Bank was performed and ubiquitous close contacts between the Cδ1-H groups of tryptophan and a range of electronegative acceptors were identified, specifically main-chain carbonyl O atoms immediately upstream and downstream in the polypeptide chain. The stereochemical analysis shows that most of the interactions bear all of the hallmarks of proper hydrogen bonds. At the same time, their cohesive nature is confirmed by quantum-chemical calculations, which reveal interaction energies of 1.5-3.0 kcal mol-1, depending on the specific stereochemistry.

Structural characterization of ligand binding and pH-specific enzymatic activity of mouse Acidic Mammalian Chitinase.

Chitin is an abundant biopolymer and pathogen-associated molecular pattern that stimulates a host innate immune response. Mammals express chitin-binding and chitin-degrading proteins to remove chitin from the body. One of these proteins, Acidic Mammalian Chitinase (AMCase), is an enzyme known for its ability to function under acidic conditions in the stomach but is also active in tissues with more neutral pHs, such as the lung. Here, we used a combination of biochemical, structural, and computational modeling approaches to examine how the mouse homolog (mAMCase) can act in both acidic and neutral environments. We measured kinetic properties of mAMCase activity across a broad pH range, quantifying its unusual dual activity optima at pH 2 and 7. We also solved high-resolution crystal structures of mAMCase in complex with oligomeric GlcNAcn, the building block of chitin, where we identified extensive conformational ligand heterogeneity. Leveraging these data, we conducted molecular dynamics simulations that suggest how a key catalytic residue could be protonated via distinct mechanisms in each of the two environmental pH ranges. These results integrate structural, biochemical, and computational approaches to deliver a more complete understanding of the catalytic mechanism governing mAMCase activity at different pH. Engineering proteins with tunable pH optima may provide new opportunities to develop improved enzyme variants, including AMCase, for therapeutic purposes in chitin degradation.

Nature-Inspired Gallinamides Are Potent Antischistosomal Agents: Inhibition of the Cathepsin B1 Protease Target and Binding Mode Analysis.

Schistosomiasis, caused by a parasitic blood fluke of the genus Schistosoma, is a global health problem for which new chemotherapeutic options are needed. We explored the scaffold of gallinamide A, a natural peptidic metabolite of marine cyanobacteria that has previously been shown to inhibit cathepsin L-type proteases. We screened a library of 19 synthetic gallinamide A analogs and identified nanomolar inhibitors of the cathepsin B-type protease SmCB1, which is a drug target for the treatment of schistosomiasis mansoni. Against cultured S. mansoni schistosomula and adult worms, many of the gallinamides generated a range of deleterious phenotypic responses. Imaging with a fluorescent-activity-based probe derived from gallinamide A demonstrated that SmCB1 is the primary target for gallinamides in the parasite. Furthermore, we solved the high-resolution crystal structures of SmCB1 in complex with gallinamide A and its two analogs and describe the acrylamide covalent warhead and binding mode in the active site. Quantum chemical calculations evaluated the contribution of individual positions in the peptidomimetic scaffold to the inhibition of the target and demonstrated the importance of the P1' and P2 positions. Our study introduces gallinamides as a powerful chemotype that can be exploited for the development of novel antischistosomal chemotherapeutics.

Michaelis-like complex of mouse ketohexokinase isoform C.

Over the past forty years there has been a drastic increase in fructose-related diseases, including obesity, heart disease and diabetes. Ketohexokinase (KHK), the first enzyme in the liver fructolysis pathway, catalyzes the ATP-dependent phosphorylation of fructose to fructose 1-phosphate. Understanding the role of KHK in disease-related processes is crucial for the management and prevention of this growing epidemic. Molecular insight into the structure-function relationship in ligand binding and catalysis by KHK is needed for the design of therapeutic inhibitory ligands. Ketohexokinase has two isoforms: ketohexokinase A (KHK-A) is produced ubiquitously at low levels, whereas ketohexokinase C (KHK-C) is found at much higher levels, specifically in the liver, kidneys and intestines. Structures of the unliganded and liganded human isoforms KHK-A and KHK-C are known, as well as structures of unliganded and inhibitor-bound mouse KHK-C (mKHK-C), which shares 90% sequence identity with human KHK-C. Here, a high-resolution X-ray crystal structure of mKHK-C refined to 1.79 Å resolution is presented. The structure was determined in a complex with both the substrate fructose and the product of catalysis, ADP, providing a view of the Michaelis-like complex of the mouse ortholog. Comparison to unliganded structures suggests that KHK undergoes a conformational change upon binding of substrates that places the enzyme in a catalytically competent form in which the β-sheet domain from one subunit rotates by 16.2°, acting as a lid for the opposing active site. Similar kinetic parameters were calculated for the mouse and human enzymes and indicate that mice may be a suitable animal model for the study of fructose-related diseases. Knowledge of the similarity between the mouse and human enzymes is important for understanding preclinical efforts towards targeting this enzyme, and this ground-state, Michaelis-like complex suggests that a conformational change plays a role in the catalytic function of KHK-C.

Cytokinin oxidase/dehydrogenase inhibitors: progress towards agricultural practice.

Cytokinin oxidase/dehydrogenase (CKX) inhibitors reduce the degradation of cytokinins in plants and thereby may improve the efficiency of agriculture and plant tissue culture-based practices. Here, we report a synthesis and structure-activity relationship study of novel urea derivatives concerning their CKX inhibitory activity. The best compounds showed sub-nanomolar IC50 values with maize ZmCKX1, the lowest value yet documented. Other CKX isoforms of maize (Zea mays) and Arabidopsis were also inhibited very effectively. The binding mode of four compounds was characterized based on high-resolution crystal complex structures. Using the soil nematode Caenorhabditis elegans, and human skin fibroblasts, key CKX inhibitors with low toxicity were identified. These compounds enhanced the shoot regeneration of Lobelia, Drosera, and Plectranthus, as well as the growth of Arabidopsis and Brassica napus. At the same time, a key compound (namely 82), activated a cytokinin primary response gene ARR5:GUS and cytokinin sensor TCSv2:GUS, without activating the Arabidopsis cytokinin receptors AHK3 and AHK4. This strongly implies that the effect of compound 82 is due to the upregulation of cytokinin signalling. Overall, this work presents highly effective and easily prepared CKX inhibitors with a low risk of environmental toxicity for further investigation of their potential in agriculture and biotechnology.

Structure and mechanism of the human CTDNEP1–NEP1R1 membrane protein phosphatase complex necessary to maintain ER membrane morphology

C-terminal Domain Nuclear Envelope Phosphatase 1 (CTDNEP1) is a noncanonical protein serine/threonine phosphatase that has a conserved role in regulating ER membrane biogenesis. Inactivating mutations in CTDNEP1 correlate with the development of medulloblastoma, an aggressive childhood cancer. The transmembrane protein Nuclear Envelope Phosphatase 1 Regulatory Subunit 1 (NEP1R1) binds CTDNEP1, but the molecular details by which NEP1R1 regulates CTDNEP1 function are unclear. Here, we find that knockdown of NEP1R1 generates identical phenotypes to reported loss of CTDNEP1 in mammalian cells, establishing CTDNEP1-NEP1R1 as an evolutionarily conserved membrane protein phosphatase complex that restricts ER expansion. Mechanistically, NEP1R1 acts as an activating regulatory subunit that directly binds and increases the phosphatase activity of CTDNEP1. By defining a minimal NEP1R1 domain sufficient to activate CTDNEP1, we determine high-resolution crystal structures of the CTDNEP1-NEP1R1 complex bound to a peptide sequence acting as a pseudosubstrate. Structurally, NEP1R1 engages CTDNEP1 at a site distant from the active site to stabilize and allosterically activate CTDNEP1. Substrate recognition is facilitated by a conserved Arg residue in CTDNEP1 that binds and orients the substrate peptide in the active site. Together, this reveals mechanisms for how NEP1R1 regulates CTDNEP1 and explains how cancer-associated mutations inactivate CTDNEP1.

AIDA-1/ANKS1B Binds to the SynGAP Family RasGAPs with High Affinity and Specificity

AIDA-1, encoded by ANKS1B, is an abundant postsynaptic scaffold protein essential for brain development. Mutations of ANKS1B are closely associated with various psychiatric disorders. However, very little is known regarding the molecular mechanisms underlying AIDA-1′s involvements under physiological and pathophysiological conditions. Here, we discovered an interaction between AIDA-1 and the SynGAP family Ras-GTPase activating protein (GAP) via affinity purification using AIDA-1d as the bait. Biochemical studies showed that the PTB domain of AIDA-1 binds to an extended NPx[F/Y]-motif of the SynGAP family proteins with high affinities. The high-resolution crystal structure of AIDA-1 PTB domain in complex with the SynGAP NPxF-motif revealed the molecular mechanism governing the specific interaction between AIDA-1 and SynGAP. Our study not only explains why patients with ANKS1B or SYNGAP1 mutations share overlapping clinical phenotypes, but also allows identification of new AIDA-1 binding targets such as Ras and Rab interactors.

Structural and mechanistic insights into Quinolone Synthase to address its functional promiscuity

Quinolone synthase from Aegle marmelos (AmQNS) is a type III polyketide synthase that yields therapeutically effective quinolone and acridone compounds. Addressing the structural and molecular underpinnings of AmQNS and its substrate interaction in terms of its high selectivity and specificity can aid in the development of numerous novel compounds. This paper presents a high-resolution AmQNS crystal structure and explains its mechanistic role in synthetic selectivity. Additionally, we provide a model framework to comprehend structural constraints on ketide insertion and postulate that AmQNS’s steric and electrostatic selectivity plays a role in its ability to bind to various core substrates, resulting in its synthetic diversity. AmQNS prefers quinolone synthesis and can accommodate large substrates because of its wide active site entrance. However, our research suggests that acridone is exclusively synthesized in the presence of high malonyl-CoA concentrations. Potential implications of functionally relevant residue mutations were also investigated, which will assist in harnessing the benefits of mutations for targeted polyketide production. The pharmaceutical industry stands to gain from these findings as they expand the pool of potential drug candidates, and these methodologies can also be applied to additional promising enzymes.

Conformational Preferences of the Non-Canonical Amino Acids (2S,4S)-5-Fluoroleucine, (2S,4R)-5-Fluoroleucine, and 5,5'-Difluoroleucine in a Protein.

Proteins produced with leucine analogues, where CH2F groups substitute specific methyl groups, can readily be probed by 19F NMR spectroscopy. As CF and CH groups are similar in hydrophobicity and size, fluorinated leucines are expected to cause minimal structural perturbation, but the impact of fluorine on the rotational freedom of CH2F groups is unclear. We present high-resolution crystal structures of Escherichia coli peptidyl-prolyl cis-trans isomerase B (PpiB) prepared with uniform high-level substitution of leucine by (2S,4S)-5-fluoroleucine, (2S,4R)-5-fluoroleucine, or 5,5'-difluoroleucine. Apart from the fluorinated leucine residues, the structures show complete structural conservation of the protein backbone and the amino acid side chains except for a single isoleucine side chain located next to a fluorine atom in the hydrophobic core of the protein. The carbon skeletons of the fluorinated leucine side chains are also mostly conserved. The CH2F groups show a strong preference for staggered rotamers and often appear locked into single rotamers. Substitution of leucine CH3 groups for CH2F groups is thus readily tolerated in the three-dimensional (3D) structure of a protein, and the rotation of CH2F groups can be halted at cryogenic temperatures.

The three-dimensional structure of DapE from Enterococcus faecium reveals new insights into DapE/ArgE subfamily ligand specificity

DapE is a Zn2+-metallohydrolase recognized as a drug target for bacterial control. It is a homodimer that requires the exchange of interface strands by an induced fit essential for catalysis. Identifying novel anti-DapE agents requires greater structural details. Most of the characterized DapEs are from the Gram-negative group. Here, two high-resolution DapE crystal structures from Enterococcus faecium are presented for the first time with novel aspects. A loosened enzyme intermediate between the open and closed conformations is observed. Substrates may bind to loose state, subsequently it closes, where hydrolysis occurs, and finally, the change to the open state leads to the release of the products. Mutation of His352 suggests a role, along with His194, in the oxyanion stabilization in the mono-metalated Zn2+ isoform, while in the di-metalated isoform, the metal center 2 complements it function. An aromatic-π box potentially involved in the interaction of DapE with other proteins, and a peptide flip could determine the specificity in the Gram-positive ArgE/DapE group. Finally, details of two extra-catalytic cavities whose geometry changes depending on the conformational state of the enzyme are presented. These cavities could be a target for developing non-competitive agents that trap the enzyme in an inactive state.

Structure and non-reactive dynamics of the dimeric catalytic domain of human carbonic anhydrase IX

Despite the availability of high resolution crystal structure of the catalytic domain of human carbonic anhydrase IX (denoted as HCA IX-c), its solution structure and functional dynamics are mostly unknown. In this article, we report classical molecular dynamics (MD) simulation studies on the structure and non-reactive dynamics of dimeric HCA IX-c (d-HCA IX-c, with chains A and B) in water and compare them to each chain simulated in water as a monomeric solute (mA/mB). While the average solution structures appear to be largely similar in all systems, d-HCA IX-c shows significant deviations from mA and mB in terms of non-reactive reorganization of the active site needed to initiate the rate determining proton transfer step between a zinc-bound water and one of the sidechain N-atoms of a distal histidine residue (His-64). First, 2−6 water molecules are found to form fluctuating hydrogen bonded networks that would serve as proton paths across the active site. A minimally frustrated core of each monomer in the dimeric protein is found to be associated with a significantly higher likelihood of forming complete proton transfer pathways mediated by 2 water molecules synchronously at the two active sites hosted by the monomeric chains. The relative stability and kinetics of transition between two major sidechain rotamers of His-64 are also affected by dimerization. The sidechain rotation of His-64 is found to involve at least one transition as slow as 105s−1 in chain A of d-HCA IX-c thereby rendering it slower than the actual rate determining proton transfer step. This observation indicates an important deviation from the widely accepted mechanism of catalysis by HCAs.

High Resolution Crystal Structure of the Pyruvate Kinase Tetramer in Complex with the Allosteric Activator Mitapivat/AG-348

High Resolution Crystal Structure of the Pyruvate Kinase Tetramer in Complex with the Allosteric Activator Mitapivat/AG-348

Structural basis of chorismate isomerization by Arabidopsis ISOCHORISMATE SYNTHASE1.

Salicylic acid (SA) plays a crucial role in plant defense against biotrophic and semi-biotrophic pathogens. In Arabidopsis (Arabidopsis thaliana), isochorismate synthase 1 (AtICS1) is a key enzyme for the pathogen-induced biosynthesis of SA via catalytic conversion of chorismate into isochorismate, an essential precursor for SA synthesis. Despite the extensive knowledge of ICS1-related menaquinone, siderophore, tryptophan (MST) enzymes in bacteria, the structural mechanisms for substrate binding and catalysis in plant isochorismate synthase (ICS) enzymes are unknown. This study reveals that plant ICS enzymes catalyze the isomerization of chorismate through a magnesium-dependent mechanism, with AtICS1 exhibiting the most substantial catalytic activity. Additionally, we present high-resolution crystal structures of apo AtICS1 and its complex with chorismate, offering detailed insights into the mechanisms of substrate recognition and catalysis. Importantly, our investigation indicates the existence of a potential substrate entrance channel and a gating mechanism regulating substrate into the catalytic site. Structural comparisons of AtICS1 with MST enzymes suggest a shared structural framework with conserved gating and catalytic mechanisms. This work provides valuable insights into the structural and regulatory mechanisms governing substrate delivery and catalysis in AtICS1, as well as other plant ICS enzymes.

Structural insights in to the atypical type-I ABC Glucose-6-phosphate importer VCA0625-27 of Vibrio cholerae

Sugar phosphates are potential sources of carbon and phosphate for bacteria. Despite that the process of internalization of Glucose-6-Phosphate (G6P) through plasma membrane remained elusive in several bacteria. VCA0625-27, made of periplasmic ligand binding protein (PLBP) VCA0625, an atypical monomeric permease VCA0626, and a cytosolic ATPase VCA0627, recently emerged as hexose-6-phosphate uptake system of Vibrio cholerae. Here we report high resolution crystal structure of VCA0625 in G6P bound state that largely resembles AfuA of Actinobacillus pleuropneumoniae. MD simulations on VCA0625 in apo and G6P bound states unraveled an ‘open to close’ and swinging bi-lobal motions, which are diminished upon G6P binding. Mutagenesis followed by biochemical assays on VCA0625 underscored that R34 works as gateway to bind G6P. Although VCA0627 binds ATP, it is ATPase deficient in the absence of VCA0625 and VCA0626, which is a signature phenomenon of type-I ABC importer. Further, modeling, docking and systematic sequence analysis allowed us to envisage the existence of similar atypical type-I G6P importer with fused monomeric permease in 27 other gram-negative bacteria.