Crystal structure of a human neuronal nAChR extracellular domain in pentameric assembly: Ligand-bound α2 homopentamer

In this study we report the X-ray crystal structure of the extracellular domain (ECD) of the human neuronal α2 nicotinic acetylcholine receptor (nAChR) subunit in complex with the agonist epibatidine at 3.2 Å. Interestingly, α2 was crystallized as a pentamer, revealing the intersubunit interactions in a wild type neuronal nAChR ECD and the full ligand binding pocket conferred by two adjacent α subunits. The pentameric assembly presents the conserved structural scaffold observed in homologous proteins, as well as distinctive features, providing unique structural information of the binding site between principal and complementary faces. Structure-guided mutagenesis and electrophysiological data confirmed the presence of the α2(+)/α2(−) binding site on the heteromeric low sensitivity α2β2 nAChR and validated the functional importance of specific residues in α2 and β2 nAChR subunits. Given the pathological importance of the α2 nAChR subunit and the high sequence identity with α4 (78%) and other neuronal nAChR subunits, our findings offer valuable information for modeling several nAChRs and ultimately for structure-based design of subtype specific drugs against the nAChR associated diseases.

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Characterization of the Acetate Binding Pocket in the Methanosarcina thermophila Acetate Kinase

Journal of Bacteriology ◽

10.1128/jb.187.7.2386-2394.2005 ◽

2005 ◽

Vol 187 (7) ◽

pp. 2386-2394 ◽

Cited By ~ 36

Author(s):

Cheryl Ingram-Smith ◽

Andrea Gorrell ◽

Sarah H. Lawrence ◽

Prabha Iyer ◽

Kerry Smith ◽

...

Keyword(s):

Crystal Structure ◽

Binding Site ◽

Binding Pocket ◽

Acetate Kinase ◽

Phosphoryl Group ◽

Acetyl Phosphate ◽

Hydrophobic Pocket ◽

Methanosarcina Thermophila ◽

Methyl Group ◽

Substrate Binding Sites

ABSTRACT Acetate kinase catalyzes the reversible magnesium-dependent synthesis of acetyl phosphate by transfer of the ATP γ-phosphoryl group to acetate. Inspection of the crystal structure of the Methanosarcina thermophila enzyme containing only ADP revealed a solvent-accessible hydrophobic pocket formed by residues Val93, Leu122, Phe179, and Pro232 in the active site cleft, which identified a potential acetate binding site. The hypothesis that this was a binding site was further supported by alignment of all acetate kinase sequences available from databases, which showed strict conservation of all four residues, and the recent crystal structure of the M. thermophila enzyme with acetate bound in this pocket. Replacement of each residue in the pocket produced variants with Km values for acetate that were 7- to 26-fold greater than that of the wild type, and perturbations of this binding pocket also altered the specificity for longer-chain carboxylic acids and acetyl phosphate. The kinetic analyses of variants combined with structural modeling indicated that the pocket has roles in binding the methyl group of acetate, influencing substrate specificity, and orienting the carboxyl group. The kinetic analyses also indicated that binding of acetyl phosphate is more dependent on interactions of the phosphate group with an unidentified residue than on interactions between the methyl group and the hydrophobic pocket. The analyses also indicated that Phe179 is essential for catalysis, possibly for domain closure. Alignments of acetate kinase, propionate kinase, and butyrate kinase sequences obtained from databases suggested that these enzymes have similar catalytic mechanisms and carboxylic acid substrate binding sites.

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THE APPLICABILITY OF THE CRYSTAL STRUCTURE OF TERMOTOGA MARITIMA 4--GLUCANOTRANSFERASE AS THE TEMPLATE FOR SULOCHRIN AS -GLUCOSIDASE INHIBITORS

Indonesian Journal of Chemistry ◽

10.22146/ijc.21520 ◽

2010 ◽

Vol 9 (3) ◽

pp. 479-486

Author(s):

Rizna Triana Dewi ◽

Yulia Anita ◽

Enade Perdana Istyastono ◽

Akhmad Darmawan ◽

Muhamad Hanafi

Keyword(s):

Crystal Structure ◽

Saccharomyces Cerevisiae ◽

Thermotoga Maritima ◽

Binding Pocket ◽

Operating Environment ◽

High Sequence Identity ◽

Glucosidase Inhibitor ◽

Glucosidase Inhibitors ◽

Docking Method ◽

Binding Residues

Interaction of sulochrin to active site of glucosidase enzyme of Termotoga maritime has been studied by employing docking method using Molecular Operating Environment (MOE), in comparison with those are reports of established inhibitor α-glucosidase such as acarbose, miglitol and voglibose, and salicinol, as reference compounds. The crystal structure T. maritima α-glucanotransferase (PDB code: 1LWJ) can be employed to serve as the template in the virtual screening of S. cerevisiae α-glucosidase. The comparison between the binding pocket residues of Thermotoga maritima α-glucanotransferase and Saccharomyces cerevisiae α-glucosidase show a high sequence identity and similarity. The result showed that sulochrin could be located in the binding pocket and formed some interactions with the binding residues. The ligands showed proper predicted binding energy (-6.74 - -4.13 kcal/mol) and predicted Ki values (0.011 - 0.939 mM). Sulochrin has a possibility to serve as a lead compound in the development of new α-glucosidase inhibitor. Keywords: Docking, sulochrin, α-glucosidase Inhibitor, Thermotoga maritime α-glucotransferase, Saccharomyces cerevisiae α-glucosidase, MOE

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Identification of the site of oxidase substrate binding inScytalidium thermophilumcatalase

Acta Crystallographica Section D Structural Biology ◽

10.1107/s2059798318010628 ◽

2018 ◽

Vol 74 (10) ◽

pp. 979-985 ◽

Cited By ~ 2

Author(s):

Yonca Yuzugullu Karakus ◽

Gunce Goc ◽

Sinem Balci ◽

Briony A. Yorke ◽

Chi H. Trinh ◽

...

Keyword(s):

Crystal Structure ◽

Kinetic Analysis ◽

Binding Site ◽

Active Site ◽

Oxidase Activity ◽

Substrate Binding ◽

Binding Pocket ◽

Scytalidium Thermophilum ◽

Lateral Channel

The catalase fromScytalidium thermophilumis a homotetramer containing a hemedin each active site. Although the enzyme has a classical monofunctional catalase fold, it also possesses oxidase activity towards a number of small organics, including catechol and phenol. In order to further investigate this, the crystal structure of the complex of the catalase with the classical catalase inhibitor 3-amino-1,2,4-triazole (3TR) was determined at 1.95 Å resolution. Surprisingly, no binding to the heme site was observed; instead, 3TR occupies a binding site corresponding to the NADPH-binding pocket in mammalian catalases at the entrance to a lateral channel leading to the heme. Kinetic analysis of site-directed mutants supports the assignment of this pocket as the binding site for oxidase substrates.

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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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ATP-binding Cassette Exporters: Structure and Mechanism with a Focus on P-glycoprotein and MRP1

Current Medicinal Chemistry ◽

10.2174/0929867324666171012105143 ◽

2019 ◽

Vol 26 (7) ◽

pp. 1062-1078 ◽

Cited By ~ 7

Author(s):

Maite Rocío Arana ◽

Guillermo Alejandro Altenberg

Keyword(s):

Binding Site ◽

Conformational Changes ◽

Structural Information ◽

Nucleotide Binding ◽

Binding Pocket ◽

Atp Binding ◽

Binding Domains ◽

P Glycoprotein ◽

And Function ◽

Atp Binding Cassette

Background:Proteins that belong to the ATP-binding cassette superfamily include transporters that mediate the efflux of substrates from cells. Among these exporters, P-glycoprotein and MRP1 are involved in cancer multidrug resistance, protection from endo and xenobiotics, determination of drug pharmacokinetics, and the pathophysiology of a variety of disorders. Objective:To review the information available on ATP-binding cassette exporters, with a focus on Pglycoprotein, MRP1 and related proteins. We describe tissue localization and function of these transporters in health and disease, and discuss the mechanisms of substrate transport. We also correlate recent structural information with the function of the exporters, and discuss details of their molecular mechanism with a focus on the nucleotide-binding domains. Methods: Evaluation of selected publications on the structure and function of ATP-binding cassette proteins. Conclusions:Conformational changes on the nucleotide-binding domains side of the exporters switch the accessibility of the substrate-binding pocket between the inside and outside, which is coupled to substrate efflux. However, there is no agreement on the magnitude and nature of the changes at the nucleotide- binding domains side that drive the alternate-accessibility. Comparison of the structures of Pglycoprotein and MRP1 helps explain differences in substrate selectivity and the bases for polyspecificity. P-glycoprotein substrates are hydrophobic and/or weak bases, and polyspecificity is explained by a flexible hydrophobic multi-binding site that has a few acidic patches. MRP1 substrates are mostly organic acids, and its polyspecificity is due to a single bipartite binding site that is flexible and displays positive charge.

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Prediction and Validation of a Druggable Site on Virulence Factor of Drug Resistant Burkholderia Cenocepacia

10.26434/chemrxiv.13006382.v1 ◽

2020 ◽

Author(s):

Kanhaya Lal ◽

Rafael Bermo ◽

Jonathan Cramer ◽

Francesca Vasile ◽

Beat Ernst ◽

...

Keyword(s):

Crystal Structure ◽

Virtual Screening ◽

Binding Site ◽

Virulence Factor ◽

Opportunistic Pathogen ◽

Binding Pocket ◽

Burkholderia Cenocepacia ◽

Drug Resistant ◽

Novel Approach ◽

Virtual Screening Approach

We use a virtual screening approach to explore the binding pocket close to the fucose binding site in a lectin from opportunistic pathogen. This is a novel approach for designing anti-adhesive drugs, and it has been very successful since we were able to obtain leads with sub millimolar affinity. Furthermore, the crystal structure of the target protein complexed with the fragment validated the existence of this secondary binding site, opening route for new design of inhibitors

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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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Prediction and Validation of a Druggable Site on Virulence Factor of Drug Resistant Burkholderia Cenocepacia

10.26434/chemrxiv.13006382 ◽

2020 ◽

Author(s):

Kanhaya Lal ◽

Rafael Bermo ◽

Jonathan Cramer ◽

Francesca Vasile ◽

Beat Ernst ◽

...

Keyword(s):

Crystal Structure ◽

Virtual Screening ◽

Binding Site ◽

Virulence Factor ◽

Opportunistic Pathogen ◽

Binding Pocket ◽

Burkholderia Cenocepacia ◽

Drug Resistant ◽

Novel Approach ◽

Virtual Screening Approach

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A closed-homodimer structure of human importin-α1

Acta Crystallographica Section A Foundations and Advances ◽

10.1107/s2053273314095011 ◽

2014 ◽

Vol 70 (a1) ◽

pp. C498-C498

Author(s):

Hideyuki Miyatake ◽

Akira Sanjoh ◽

Go Matsuda ◽

Yuko Tatsumi ◽

Yoichi Miyamoto ◽

...

Keyword(s):

Crystal Structure ◽

Binding Site ◽

Binding Affinity ◽

Binding Sites ◽

Binding Pocket ◽

Dimer Formation ◽

Functional Aspects ◽

Physicochemical Studies

We determined the crystal structure of N-terminal importin-β-binding (IBB) domain truncated human importin-α1 (ΔIBB-importin-α1) at 2.63Å resolution. The crystal structure of ΔIBB-importin-α1 is a novel closed-homodimer. The homodimer exists in an autoinhibition state in which both of the major and minor NLS-binding sites are completely buried in the homodimerization interface to avoid NLS binding. Importin-α1 is in dimer-monomer equilibration in solution. In the dimerization sate, the P1'-binding pocket in the minor NLS binding site plays a role to stabilize the dimer formation. The external K108 binds into the P1'-binding pocket that results in the autoinhibition of the NLS binding. The present closed-homodimer of ΔIBB-importin-α1 conjured the functional aspects of multimerization of importin-α1. The further physicochemical studies using full- and ΔIBB- importin-α1 reveal that the IBB domain is involved in the monomer-dimer equilibration; thereby the NLS binding affinity is kept even in the higher concentration of importin-α1. Owing to the multimerization property, importin-αs can autoinhibition the NLS binding, that may result in a variety of NLS recognition way.

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The Active Site of O-Acetylserine Sulfhydrylase Is the Anchor Point for Bienzyme Complex Formation with Serine Acetyltransferase

Journal of Bacteriology ◽

10.1128/jb.187.9.3201-3205.2005 ◽

2005 ◽

Vol 187 (9) ◽

pp. 3201-3205 ◽

Cited By ~ 62

Author(s):

Bin Huang ◽

Matthew W. Vetting ◽

Steven L. Roderick

Keyword(s):

Crystal Structure ◽

Complex Formation ◽

Binding Site ◽

Active Site ◽

Binding Pocket ◽

Anion Binding ◽

Carboxyl Group ◽

Serine Acetyltransferase ◽

Cysteine Synthase

ABSTRACT The biosynthesis of cysteine in bacteria and plants is carried out by a two-step pathway, catalyzed by serine acetyltransferase (SAT) and O-acetylserine sulfhydrylase (OASS; O-acetylserine [thiol] lyase). The aerobic form of OASS forms a tight bienzyme complex with SAT in vivo, termed cysteine synthase. We have determined the crystal structure of OASS in complex with a C-terminal peptide of SAT required for bienzyme complex formation. The binding site of the peptide is at the active site of OASS, and its C-terminal carboxyl group occupies the same anion binding pocket as the α-carboxylate of the O-acetylserine substrate of OASS. These results explain the partial inhibition of OASS by SAT on complex formation as well as the competitive dissociation of the complex by O-acetylserine.

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