Crystal Structure of Dinitrogenase Reductase-activating Glycohydrolase (DRAG) Reveals Conservation in the ADP-Ribosylhydrolase Fold and Specific Features in the ADP-Ribose-binding Pocket

AbstractSteroid hormones are essential in stress response, immune system regulation, and reproduction in mammals. Steroids with 3-oxo-Δ4 structure, such as testosterone or progesterone, are catalyzed by steroid 5α-reductases (SRD5As) to generate their corresponding 3-oxo-5α steroids, which are essential for multiple physiological and pathological processes. SRD5A2 is already a target of clinically relevant drugs. However, the detailed mechanism of SRD5A-mediated reduction remains elusive. Here we report the crystal structure of PbSRD5A from Proteobacteria bacterium, a homolog of both SRD5A1 and SRD5A2, in complex with the cofactor NADPH at 2.0 Å resolution. PbSRD5A exists as a monomer comprised of seven transmembrane segments (TMs). The TM1-4 enclose a hydrophobic substrate binding cavity, whereas TM5-7 coordinate cofactor NADPH through extensive hydrogen bonds network. Homology-based structural models of HsSRD5A1 and -2, together with biochemical characterization, define the substrate binding pocket of SRD5As, explain the properties of disease-related mutants and provide an important framework for further understanding of the mechanism of NADPH mediated steroids 3-oxo-Δ4 reduction. Based on these analyses, the design of therapeutic molecules targeting SRD5As with improved specificity and therapeutic efficacy would be possible.

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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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Crystal structure of microtubule affinity-regulating kinase 4 catalytic domain in complex with a pyrazolopyrimidine inhibitor

Acta Crystallographica Section F Structural Biology Communications ◽

10.1107/s2053230x15024747 ◽

2016 ◽

Vol 72 (2) ◽

pp. 129-134 ◽

Cited By ~ 22

Author(s):

John S. Sack ◽

Mian Gao ◽

Susan E. Kiefer ◽

Joseph E. Myers ◽

John A. Newitt ◽

...

Keyword(s):

Crystal Structure ◽

Catalytic Domain ◽

Neurofibrillary Tangle ◽

Binding Pocket ◽

Atp Binding ◽

Activation Loop ◽

Serine Threonine Kinase ◽

Atp Binding Site ◽

Threonine Kinase ◽

Catalytic Loop

Microtubule-associated protein/microtubule affinity-regulating kinase 4 (MARK4) is a serine/threonine kinase involved in the phosphorylation of MAP proteins that regulate microtubule dynamics. Abnormal activity of MARK4 has been proposed to contribute to neurofibrillary tangle formation in Alzheimer's disease. The crystal structure of the catalytic and ubiquitin-associated domains of MARK4 with a potent pyrazolopyrimidine inhibitor has been determined to 2.8 Å resolution with anRworkof 22.8%. The overall structure of MARK4 is similar to those of the other known MARK isoforms. The inhibitor is located in the ATP-binding site, with the pyrazolopyrimidine group interacting with the inter-lobe hinge region while the aminocyclohexane moiety interacts with the catalytic loop and the DFG motif, forcing the activation loop out of the ATP-binding pocket.

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Crystal structure of the human NK1 tachykinin receptor

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1812717115 ◽

2018 ◽

Vol 115 (52) ◽

pp. 13264-13269 ◽

Cited By ~ 9

Author(s):

Jie Yin ◽

Karen Chapman ◽

Lindsay D. Clark ◽

Zhenhua Shao ◽

Dominika Borek ◽

...

Keyword(s):

Crystal Structure ◽

Substance P ◽

Binding Pocket ◽

Computational Docking ◽

Tachykinin Receptor ◽

Approved Drugs ◽

Dynamics Simulations ◽

Receptor Modulators ◽

Further Development ◽

G Protein Coupled

The NK1 tachykinin G-protein–coupled receptor (GPCR) binds substance P, the first neuropeptide to be discovered in mammals. Through activation of NK1R, substance P modulates a wide variety of physiological and disease processes including nociception, inflammation, and depression. Human NK1R (hNK1R) modulators have shown promise in clinical trials for migraine, depression, and emesis. However, the only currently approved drugs targeting hNK1R are inhibitors for chemotherapy-induced nausea and vomiting (CINV). To better understand the molecular basis of ligand recognition and selectivity, we solved the crystal structure of hNK1R bound to the inhibitor L760735, a close analog of the drug aprepitant. Our crystal structure reveals the basis for antagonist interaction in the deep and narrow orthosteric pocket of the receptor. We used our structure as a template for computational docking and molecular-dynamics simulations to dissect the energetic importance of binding pocket interactions and model the binding of aprepitant. The structure of hNK1R is a valuable tool in the further development of tachykinin receptor modulators for multiple clinical applications.

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A covalent adduct of MbtN, an acyl-ACP dehydrogenase fromMycobacterium tuberculosis, reveals an unusual acyl-binding pocket

Acta Crystallographica Section D Biological Crystallography ◽

10.1107/s1399004715001650 ◽

2015 ◽

Vol 71 (4) ◽

pp. 862-872 ◽

Cited By ~ 2

Author(s):

Ai-Fen Chai ◽

Esther M. M. Bulloch ◽

Genevieve L. Evans ◽

J. Shaun Lott ◽

Edward N. Baker ◽

...

Keyword(s):

Crystal Structure ◽

Double Bond ◽

Acyl Carrier Protein ◽

Acyl Chain ◽

Binding Pocket ◽

Carrier Protein ◽

Strongly Correlated ◽

Steady State Kinetics ◽

Expected Length ◽

Acyl Chains

Mycobacterium tuberculosis(Mtb) is the causative agent of tuberculosis. Access to iron in host macrophages depends on iron-chelating siderophores called mycobactins and is strongly correlated withMtbvirulence. Here, the crystal structure of anMtbenzyme involved in mycobactin biosynthesis, MbtN, in complex with its FAD cofactor is presented at 2.30 Å resolution. The polypeptide fold of MbtN conforms to that of the acyl-CoA dehydrogenase (ACAD) family, consistent with its predicted role of introducing a double bond into the acyl chain of mycobactin. Structural comparisons and the presence of an acyl carrier protein, MbtL, in the same gene locus suggest that MbtN acts on an acyl-(acyl carrier protein) rather than an acyl-CoA. A notable feature of the crystal structure is the tubular density projecting from N(5) of FAD. This was interpreted as a covalently bound polyethylene glycol (PEG) fragment and resides in a hydrophobic pocket where the substrate acyl group is likely to bind. The pocket could accommodate an acyl chain of 14–21 C atoms, consistent with the expected length of the mycobactin acyl chain. Supporting this, steady-state kinetics show that MbtN has ACAD activity, preferring acyl chains of at least 16 C atoms. The acyl-binding pocket adopts a different orientation (relative to the FAD) to other structurally characterized ACADs. This difference may be correlated with the apparent ability of MbtN to catalyse the formation of an unusualcisdouble bond in the mycobactin acyl chain.

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Crystal structure of the adenosine A2Areceptor bound to an antagonist reveals a potential allosteric pocket

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1621423114 ◽

2017 ◽

Vol 114 (8) ◽

pp. 2066-2071 ◽

Cited By ~ 69

Author(s):

Bingfa Sun ◽

Priti Bachhawat ◽

Matthew Ling-Hon Chu ◽

Martyn Wood ◽

Tom Ceska ◽

...

Keyword(s):

Crystal Structure ◽

Parkinson’S Disease ◽

Parkinson's Disease ◽

Protein Structures ◽

Binding Pocket ◽

Cubic Phase ◽

Receptor Subtype ◽

Type I ◽

Intracellular Loop ◽

Drug Candidates

The adenosine A2Areceptor (A2AR) has long been implicated in cardiovascular disorders. As more selective A2AR ligands are being identified, its roles in other disorders, such as Parkinson’s disease, are starting to emerge, and A2AR antagonists are important drug candidates for nondopaminergic anti-Parkinson treatment. Here we report the crystal structure of A2Areceptor bound to compound 1 (Cmpd-1), a novel A2AR/N-methyld-aspartate receptor subtype 2B (NR2B) dual antagonist and potential anti-Parkinson candidate compound, at 3.5 Å resolution. The A2Areceptor with a cytochrome b562-RIL (BRIL) fusion (A2AR–BRIL) in the intracellular loop 3 (ICL3) was crystallized in detergent micelles using vapor-phase diffusion. Whereas A2AR–BRIL bound to the antagonist ZM241385 has previously been crystallized in lipidic cubic phase (LCP), structural differences in the Cmpd-1–bound A2AR–BRIL prevented formation of the lattice observed with the ZM241385–bound receptor. The crystals grew with a type II crystal lattice in contrast to the typical type I packing seen from membrane protein structures crystallized in LCP. Cmpd-1 binds in a position that overlaps with the native ligand adenosine, but its methoxyphenyl group extends to an exosite not previously observed in other A2AR structures. Structural analysis revealed that Cmpd-1 binding results in the unique conformations of two tyrosine residues, Tyr91.35and Tyr2717.36, which are critical for the formation of the exosite. The structure reveals insights into antagonist binding that are not observed in other A2AR structures, highlighting flexibility in the binding pocket that may facilitate the development of A2AR-selective compounds for the treatment of Parkinson’s disease.

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Structure of the regulatory domain of the LysR family regulator NMB2055 (MetR-like protein) fromNeisseria meningitidis

Acta Crystallographica Section F Structural Biology and Crystallization Communications ◽

10.1107/s1744309112010603 ◽

2012 ◽

Vol 68 (7) ◽

pp. 730-737 ◽

Cited By ~ 1

Author(s):

Sarah Sainsbury ◽

Jingshan Ren ◽

Nigel J. Saunders ◽

David I. Stuart ◽

Raymond J. Owens

Keyword(s):

Crystal Structure ◽

Transcription Factors ◽

Neisseria Meningitidis ◽

Disulfide Bond ◽

Binding Pocket ◽

Cysteine Residues ◽

Regulatory Domain ◽

High Degree ◽

Effector Binding ◽

Lysr Family

The crystal structure of the regulatory domain of NMB2055, a putative MetR regulator fromNeisseria meningitidis, is reported at 2.5 Å resolution. The structure revealed that there is a disulfide bond inside the predicted effector-binding pocket of the regulatory domain. Mutation of the cysteines (Cys103 and Cys106) that form the disulfide bond to serines resulted in significant changes to the structure of the effector pocket. Taken together with the high degree of conservation of these cysteine residues within MetR-related transcription factors, it is suggested that the Cys103 and Cys106 residues play an important role in the function of MetR regulators.

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Crystal structure and mechanism of human carboxypeptidase O: Insights into its specific activity for acidic residues

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1803685115 ◽

2018 ◽

Vol 115 (17) ◽

pp. E3932-E3939 ◽

Cited By ~ 4

Author(s):

Maria C. Garcia-Guerrero ◽

Javier Garcia-Pardo ◽

Esther Berenguer ◽

Roberto Fernandez-Alvarez ◽

Gifty B. Barfi ◽

...

Keyword(s):

Crystal Structure ◽

Specific Activity ◽

Dominant Role ◽

Substrate Binding ◽

Digestive Enzyme ◽

Binding Pocket ◽

Site Directed Mutagenesis ◽

Enzyme Specificity ◽

Substrate Binding Pocket ◽

Acidic Residues

Human metallocarboxypeptidase O (hCPO) is a recently discovered digestive enzyme localized to the apical membrane of intestinal epithelial cells. Unlike pancreatic metallocarboxypeptidases, hCPO is glycosylated and produced as an active enzyme with distinctive substrate specificity toward C-terminal (C-t) acidic residues. Here we present the crystal structure of hCPO at 1.85-Å resolution, both alone and in complex with a carboxypeptidase inhibitor (NvCI) from the marine snail Nerita versicolor. The structure provides detailed information regarding determinants of enzyme specificity, in particular Arg275, placed at the bottom of the substrate-binding pocket. This residue, located at “canonical” position 255, where it is Ile in human pancreatic carboxypeptidases A1 (hCPA1) and A2 (hCPA2) and Asp in B (hCPB), plays a dominant role in determining the preference of hCPO for acidic C-t residues. Site-directed mutagenesis to Asp and Ala changes the specificity to C-t basic and hydrophobic residues, respectively. The single-site mutants thus faithfully mimic the enzymatic properties of CPB and CPA, respectively. hCPO also shows a preference for Glu over Asp, probably as a consequence of a tighter fitting of the Glu side chain in its S1′ substrate-binding pocket. This unique preference of hCPO, together with hCPA1, hCPA2, and hCPB, completes the array of C-t cleavages enabling the digestion of the dietary proteins within the intestine. Finally, in addition to activity toward small synthetic substrates and peptides, hCPO can also trim C-t extensions of proteins, such as epidermal growth factor, suggesting a role in the maturation and degradation of growth factors and bioactive peptides.

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