The structure of the Aquifex aeolicus MATE family multidrug resistance transporter and sequence comparisons suggest the existence of a new subfamily

Multidrug and toxic compound extrusion (MATE) transporters are widespread in all domains of life. Bacterial MATE transporters confer multidrug resistance by utilizing an electrochemical gradient of H+ or Na+ to export xenobiotics across the membrane. Despite the availability of X-ray structures of several MATE transporters, a detailed understanding of the transport mechanism has remained elusive. Here we report the crystal structure of a MATE transporter from Aquifex aeolicus at 2.0-Å resolution. In light of its phylogenetic placement outside of the diversity of hitherto-described MATE transporters and the lack of conserved acidic residues, this protein may represent a subfamily of prokaryotic MATE transporters, which was proven by phylogenetic analysis. Furthermore, the crystal structure and substrate docking results indicate that the substrate binding site is located in the N bundle. The importance of residues surrounding this binding site was demonstrated by structure-based site-directed mutagenesis. We suggest that Aq_128 is functionally similar but structurally diverse from DinF subfamily transporters. Our results provide structural insights into the MATE transporter, which further advances our global understanding of this important transporter family.

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Molecular modelling and site-directed mutagenesis of the inositol 1,3,4,5-tetrakisphosphate-binding pleckstrin homology domain from the Ras GTPase-activating protein GAP1IP4BP

Biochemical Journal ◽

10.1042/bj3490333 ◽

2000 ◽

Vol 349 (1) ◽

pp. 333-342 ◽

Cited By ~ 5

Author(s):

Gyles COZIER ◽

Richard SESSIONS ◽

Joanna R. BOTTOMLEY ◽

Jon S. REYNOLDS ◽

Peter J. CULLEN

Keyword(s):

Crystal Structure ◽

Binding Site ◽

Inositol Phosphate ◽

Site Directed Mutagenesis ◽

Pleckstrin Homology Domain ◽

Directed Mutagenesis ◽

Ph Domain ◽

Pleckstrin Homology ◽

Gtpase Activating Protein ◽

Ras Gtpase

GAP1IP4BP is a Ras GTPase-activating protein (GAP) that in vitro is regulated by the cytosolic second messenger inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4]. We have studied Ins(1,3,4,5)P4 binding to GAP1IP4BP, and shown that the inositol phosphate specificity and binding affinity are similar to Ins(1,3,4,5)P4 binding to Bruton's tyrosine kinase (Btk), evidence which suggests a similar mechanism for Ins(1,3,4,5)P4 binding. The crystal structure of the Btk pleckstrin homology (PH) domain in complex with Ins(1,3,4,5)P4 has shown that the binding site is located in a partially buried pocket between the β1/β2- and β3/β4-loops. Many of the residues involved in the binding are conserved in GAP1IP4BP. Therefore we generated a model of the PH domain of GAP1IP4BP in complex with Ins(1,3,4,5)P4 based on the Btk-Ins(1,3,4,5)P4 complex crystal structure. This model had the typical PH domain fold, with the proposed binding site modelling well on the Btk structure. The model has been verified by site-directed mutagenesis of various residues in and around the proposed binding site. These mutations have markedly reduced affinity for Ins(1,3,4,5)P4, indicating a specific and tight fit for the substrate. The model can also be used to explain the specificity of inositol phosphate binding.

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Crystal structures of bacterial Small Multidrug Resistance transporter EmrE in complex with structurally diverse substrates

10.1101/2022.01.11.475788 ◽

2022 ◽

Author(s):

Ali A Kermani ◽

Olive E. Burata ◽

B Ben Koff ◽

Akiko Koide ◽

Shohei Koide ◽

...

Keyword(s):

Multidrug Resistance ◽

Binding Site ◽

Quaternary Ammonium ◽

Structural Information ◽

Structural Description ◽

Small Multidrug Resistance ◽

Novel Approach ◽

Backbone Structure ◽

Multidrug Resistance Transporter ◽

Ammonium Compounds

Proteins from the bacterial small multidrug resistance (SMR) family are proton-coupled exporters of diverse antiseptics and antimicrobials, including polyaromatic cations and quaternary ammonium compounds. The transport mechanism of the Escherichia coli transporter, EmrE, has been studied extensively, but a lack of high-resolution structural information has impeded a structural description of its molecular mechanism. Here we apply a novel approach, multipurpose crystallization chaperones, to solve several structures of EmrE, including a 2.9 Å structure at low pH without substrate. We report five additional structures in complex with structurally diverse transported substrates, including quaternary phosphonium, quaternary ammonium, and planar polyaromatic compounds. These structures show that binding site tryptophan and glutamate residues adopt different rotamers to conform to disparate structures without requiring major rearrangements of the backbone structure. Structural and functional comparison to Gdx-Clo, an SMR protein that transports a much narrower spectrum of substrates, suggests that in EmrE, a relatively sparse hydrogen bond network among binding site residues permits increased sidechain flexibility.

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Critical residues for ligand binding in blade 2 of the propeller domain of the integrin αIIb subunit

Thrombosis and Haemostasis ◽

10.1160/th03-06-0392 ◽

2004 ◽

Vol 91 (01) ◽

pp. 111-118 ◽

Cited By ~ 4

Author(s):

Tatsushiro Tamura ◽

Jun Yamanouchi ◽

Shigeru Fujita ◽

Takaaki Hato

Keyword(s):

Crystal Structure ◽

Ligand Binding ◽

Binding Site ◽

Thrombus Formation ◽

Direct Interaction ◽

Binding Pocket ◽

Crystal Structure Analysis ◽

Site Directed Mutagenesis ◽

Structural Basis ◽

Critical Residues

SummaryLigand binding to integrin αIIbβ3 is a key event of thrombus formation. The propeller domain of the αIIb subunit has been implicated in ligand binding. Recently, the ligand binding site of the αV propeller was determined by crystal structure analysis. However, the structural basis of ligand recognition by the αIIb propeller remains to be determined. In this study, we conducted site-directed mutagenesis of all residues located in the loops extending above blades 2 and 4 of the αIIb propeller, which are spatially close to, but distinct from, the loops that contain the binding site for an RGD ligand in the crystal structure of the αV propeller. Replacement by alanine of Q111, H112 or N114 in the loop within the blade 2 (the W2:2-3 loop in the propeller model) abolished binding of a ligand-mimetic antibody and fibrinogen to αIIbβ3 induced by different types of integrin activation including activation of αIIbβ3 by β3 cytoplasmic mutation. CHO cells stably expressing recombinant αIIbβ3 bearing Q111A, H112A or N114A mutation did not exhibit αIIbβ3mediated adhesion to fibrinogen. According to the crystal structure of αVβ3, the αV residue corresponding to αIIbN114 is exposed on the integrin surface and close to the RGD binding site. These results suggest that the Q111, H112 and N114 residues in the loop within blade 2 of the αIIb propeller are critical for ligand binding, possibly because of direct interaction with ligands or modulation of the RGD binding pocket.

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Insights into the Function of theN-Acetyltransferase SatA That Detoxifies Streptothricin inBacillus subtilisandBacillus anthracis

Applied and Environmental Microbiology ◽

10.1128/aem.03029-18 ◽

2019 ◽

Vol 85 (6) ◽

Cited By ~ 4

Author(s):

Rachel M. Burckhardt ◽

Jorge C. Escalante-Semerena

Keyword(s):

Binding Site ◽

Site Directed Mutagenesis ◽

Size Exclusion ◽

Titration Calorimetry ◽

Side Chains ◽

Missense Mutations ◽

Content Type ◽

C Terminus ◽

Exclusion Chromatography ◽

Domains Of Life

ABSTRACTAcylation of epsilon amino groups of lysyl side chains is a widespread modification of proteins and small molecules in cells of all three domains of life. Recently, we showed thatBacillus subtilisandBacillus anthracisencode the GCN5-relatedN-acetyltransferase (GNAT) SatA that can acetylate and inactivate streptothricin, which is a broad-spectrum antibiotic produced by actinomycetes in the soil. To determine functionally relevant residues ofB. subtilisSatA (BsSatA), a mutational screen was performed, highlighting the importance of a conserved area near the C terminus. Upon inspection of the crystal structure of theB. anthracisAmes SatA (BaSatA; PDB entry 3PP9), this area appears to form a pocket with multiple conserved aromatic residues; we hypothesized this region contains the streptothricin-binding site. Chemical and site-directed mutagenesis was used to introduce missense mutations intosatA, and the functionality of the variants was assessed using a heterologous host (Salmonella enterica). Results of isothermal titration calorimetry experiments showed that residue Y164 ofBaSatA was important for binding streptothricin. Results of size exclusion chromatography analyses showed that residue D160 was important for dimerization. Together, these data advance our understanding of how SatA interacts with streptothricin.IMPORTANCEThis work provides insights into how an abundant antibiotic found in soil is bound to the enzyme that inactivates it. This work identifies residues for the binding of the antibiotic and probes the contributions of substituting side chains for those in the native protein, providing information regarding hydrophobicity, size, and flexibility of the antibiotic binding site.

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Few Conserved Amino Acids in the Small Multidrug Resistance Transporter EmrE Influence Drug Polyselectivity

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00461-18 ◽

2018 ◽

Vol 62 (8) ◽

Cited By ~ 3

Author(s):

Marwah Saleh ◽

Denice C. Bay ◽

Raymond J. Turner

Keyword(s):

Amino Acids ◽

Multidrug Resistance ◽

Drug Binding ◽

Conserved Residues ◽

Content Type ◽

Small Multidrug Resistance ◽

Transporter Family ◽

Wide Range ◽

Multidrug Resistance Transporter ◽

Conserved Amino Acids

ABSTRACT EmrE is the archetypical member of the small multidrug resistance transporter family and confers resistance to a wide range of disinfectants and dyes known as quaternary cation compounds (QCCs). The aim of this study was to examine which conserved amino acids play an important role in substrate selectivity. On the basis of a previous analysis of EmrE homologues, a total of 33 conserved residues were targeted for cysteine or alanine replacement within E. coli EmrE. The antimicrobial resistance of each EmrE variant expressed in Escherichia coli strain JW0451 (lacking dominant pump acrB) to a collection of 16 different QCCs was tested using agar spot dilution plating to determine MIC values. The results determined that only a few conserved residues were drug polyselective, based on ≥4-fold decreases in MIC values: the active-site residue E14 (E14D and E14A) and 4 additional conserved residues (A10C, F44C, L47C, W63A). EmrE variants I11C, V15C, P32C, I62C, L93C, and S105C enhanced resistance to polyaromatic QCCs, while the remaining EmrE variants reduced resistance to one or more QCCs with shared chemical features: acylation, tri- and tetraphenylation, aromaticity, and dicationic charge. Mapping of EmrE variants onto transmembrane helical wheel projections using the highest resolved EmrE structure suggests that polyselective EmrE variants were located closest to the helical faces surrounding the predicted drug binding pocket, while EmrE variants with greater drug specificity mapped onto distal helical faces. This study reveals that few conserved residues are essential for drug polyselectivity and indicates that aromatic QCC selection involves a greater portion of conserved residues than that in other QCCs.

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Electrostatic lock in the transport cycle of the multidrug resistance transporter EmrE

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1722399115 ◽

2018 ◽

Vol 115 (32) ◽

pp. E7502-E7511 ◽

Cited By ~ 11

Author(s):

Josh V. Vermaas ◽

Susan B. Rempe ◽

Emad Tajkhorshid

Keyword(s):

Hydrogen Bond ◽

Multidrug Resistance ◽

Protein Conformation ◽

Water Dynamics ◽

Coupling Mechanism ◽

Transporter Family ◽

Secondary Transporter ◽

Transport Cycle ◽

Proton Transfer Reactions ◽

Multidrug Resistance Transporter

EmrE is a small, homodimeric membrane transporter that exploits the established electrochemical proton gradient across the Escherichia coli inner membrane to export toxic polyaromatic cations, prototypical of the wider small-multidrug resistance transporter family. While prior studies have established many fundamental aspects of the specificity and rate of substrate transport in EmrE, low resolution of available structures has hampered identification of the transport coupling mechanism. Here we present a complete, refined atomic structure of EmrE optimized against available cryo-electron microscopy (cryo-EM) data to delineate the critical interactions by which EmrE regulates its conformation during the transport process. With the model, we conduct molecular dynamics simulations of the transporter in explicit membranes to probe EmrE dynamics under different substrate loading and conformational states, representing different intermediates in the transport cycle. The refined model is stable under extended simulation. The water dynamics in simulation indicate that the hydrogen-bonding networks around a pair of solvent-exposed glutamate residues (E14) depend on the loading state of EmrE. One specific hydrogen bond from a tyrosine (Y60) on one monomer to a glutamate (E14) on the opposite monomer is especially critical, as it locks the protein conformation when the glutamate is deprotonated. The hydrogen bond provided by Y60 lowers the pKa of one glutamate relative to the other, suggesting both glutamates should be protonated for the hydrogen bond to break and a substrate-free transition to take place. These findings establish the molecular mechanism for the coupling between proton transfer reactions and protein conformation in this proton-coupled secondary transporter.

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Crystal structure of CotA laccase complexed with 2,2-azinobis-(3-ethylbenzothiazoline-6-sulfonate) at a novel binding site

Acta Crystallographica Section F Structural Biology Communications ◽

10.1107/s2053230x1600426x ◽

2016 ◽

Vol 72 (4) ◽

pp. 328-335 ◽

Cited By ~ 10

Author(s):

Zhongchuan Liu ◽

Tian Xie ◽

Qiuping Zhong ◽

Ganggang Wang

Keyword(s):

Crystal Structure ◽

Binding Site ◽

Binding Pocket ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

The Novel ◽

Wild Type ◽

Outer Coat ◽

Cota Laccase ◽

Key Residues

The CotA laccase fromBacillus subtilisis an abundant component of the spore outer coat and has been characterized as a typical laccase. The crystal structure of CotA complexed with 2,2-azinobis-(3-ethylbenzothiazoline-6-sulfonate) (ABTS) in a hole motif has been solved. The novel binding site was about 26 Å away from the T1 binding pocket. Comparison with known structures of other laccases revealed that the hole is a specific feature of CotA. The key residues Arg476 and Ser360 were directly bound to ABTS. Site-directed mutagenesis studies revealed that the residues Arg146, Arg429 and Arg476, which are located at the bottom of the novel binding site, are essential for the oxidation of ABTS and syringaldazine. Specially, a Thr480Phe variant was identified to be almost 3.5 times more specific for ABTS than for syringaldazine compared with the wild type. These results suggest this novel binding site for ABTS could be a potential target for protein engineering of CotA laccases.

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