Structural Modelling of the Lumenal Domain of Human GPAA1, the Metallo-Peptide Synthetase Subunit of the Transamidase Complex, Reveals Zinc-Binding Mode and Two Flaps Surrounding the Active Site

Abstract Background The transamidase complex is a molecular machine in the endoplasmic reticulum of eukaryotes that attaches a glycosylphosphatidylinositol (GPI) lipid anchor to substrate proteins after cleaving a C-terminal propeptide with a defined sequence signal. Its five subunits are very hydrophobic; thus, solubility, heterologous expression and complex reconstruction are difficult. Therefore, theoretical approaches are currently the main source of insight into details of 3D structure and of the catalytic process. Results In this work, we generated model 3D structures of the lumenal domain of human GPAA1, the M28-type metallo-peptide-synthetase subunit of the transamidase, including zinc ion and model substrate positions. In comparative molecular dynamics (MD) simulations of M28-type structures and our GPAA1 models, we estimated the metal ion binding energies with evolutionary conserved amino acid residues in the catalytic cleft. We find that canonical zinc binding sites 2 and 3 are strongest binders for Zn1 and, where a second zinc is available, sites 2 and 4 for Zn2. Zinc interaction of site 5 with Zn1 enhances upon substrate binding in structures with only one zinc. Whereas a previously studied glutaminyl cyclase structure, the best known homologue to GPAA1, binds only one zinc ion at the catalytic site, GPAA1 can sterically accommodate two. The M28-type metallopeptidases segregate into two independent branches with regard to one/two zinc ion binding modality in a phylogenetic tree where the GPAA1 family is closer to the joint origin of both groups. For GPAA1 models, MD studies revealed two large loops (flaps) surrounding the active site being involved in an anti-correlated, breathing-like dynamics. Conclusions In the light of combined sequence-analytic and phylogenetic arguments as well as 3D structural modelling results, GPAA1 is most likely a single zinc ion metallopeptidase. Two large flaps environ the catalytic site restricting access to large substrates. Reviewers This article was reviewed by Thomas Dandekar (MD) and Michael Gromiha.

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Crystal structure of the DENR-MCT-1 complex revealed zinc-binding site essential for heterodimer formation

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1809688116 ◽

2018 ◽

Vol 116 (2) ◽

pp. 528-533 ◽

Cited By ~ 6

Author(s):

Ivan B. Lomakin ◽

Sergey E. Dmitriev ◽

Thomas A. Steitz

Keyword(s):

Crystal Structure ◽

Binding Site ◽

Translation Initiation ◽

Zinc Ion ◽

Zinc Binding ◽

Ion Binding ◽

Binding Interface ◽

Zinc Binding Site ◽

Reading Frames ◽

Zinc Ion Binding

The density-regulated protein (DENR) and the malignant T cell-amplified sequence 1 (MCT-1/MCTS1) oncoprotein support noncanonical translation initiation, promote translation reinitiation on a specific set of mRNAs with short upstream reading frames, and regulate ribosome recycling. DENR and MCT-1 form a heterodimer, which binds to the ribosome. We determined the crystal structure of the heterodimer formed by human MCT-1 and the N-terminal domain of DENR at 2.0-Å resolution. The structure of the heterodimer reveals atomic details of the mechanism of DENR and MCT-1 interaction. Four conserved cysteine residues of DENR (C34, C37, C44, C53) form a classical tetrahedral zinc ion-binding site, which preserves the structure of the DENR’s MCT-1–binding interface that is essential for the dimerization. Substitution of all four cysteines by alanine abolished a heterodimer formation. Our findings elucidate further the mechanism of regulation of DENR-MCT-1 activities in unconventional translation initiation, reinitiation, and recycling.

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Dual-Function, Light Switchable Cobalt Catalysis via Active Site Metamorphosis

10.26434/chemrxiv.11353106.v1 ◽

2019 ◽

Author(s):

Enrico Bergamaschi ◽

Frédéric Beltran ◽

Christopher Teskey

Keyword(s):

Visible Light ◽

Active Site ◽

Catalytic Site ◽

Dual Function ◽

Catalytic Systems ◽

Hydride Complex ◽

Dual Functional ◽

Multiple Processes ◽

Olefin Migration

Switchable catalysis offers opportunities to control the rate or selectivity of a reaction via a stimulus such as pH or light. However, few examples of switchable catalytic systems that can facilitate multiple processes exist. Here we report a rare example of such dual-functional, switchable catalysis. Featuring an easily prepared, bench-stable cobalt(I) hydride complex in conjunction with pinacolborane, we can completely alter the reaction outcome between two widely employed transformations – olefin migration and hydroboration – with visible light as the sole trigger. This dichotomy arises from ligand photodissociation which leads to metamorphosis of the active catalytic site, resulting in divergent mechanistic pathways.

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Faculty Opinions recommendation of Investigation of indigoidine synthetase reveals conserved active site base residue of nonribosomal peptide synthetase oxidases.

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

10.3410/f.738101115.793575536 ◽

2020 ◽

Author(s):

Zixin Deng ◽

Xudong Qu

Keyword(s):

Active Site ◽

Nonribosomal Peptide Synthetase ◽

Nonribosomal Peptide ◽

Peptide Synthetase

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1.85 Å Resolution Structure of the ZincII β-Lactamase from Bacillus cereus

Acta Crystallographica Section D Biological Crystallography ◽

10.1107/s0907444997010627 ◽

1998 ◽

Vol 54 (3) ◽

pp. 313-323 ◽

Cited By ~ 80

Author(s):

Andrea Carfi ◽

Emile Duée ◽

Moreno Galleni ◽

Jean-Marie Frère ◽

Otto Dideberg

Keyword(s):

Bacillus Cereus ◽

Active Site ◽

Metal Ion ◽

Wide Spectrum ◽

Zinc Ion ◽

Beta Lactamases ◽

Carbonate Ion ◽

Class B ◽

Bivalent Metal Ions ◽

Using Data

Class B \beta-lactamases are wide spectrum enzymes which require bivalent metal ions for activity. The structure of the class B zinc-ion-dependent β-lactamase from Bacillus cereus (BCII) has been refined at 1.85 Å resolution using data collected on cryocooled crystals (100 K). The enzyme from B. cereus has a molecular mass of 24 946 Da and is folded into a \beta-sandwich structure with helices on the external faces. The active site is located in a groove running between the two \beta-sheets [Carfi et al. (1995). EMBO J. 14, 4914–4921]. The 100 K high-resolution BCII structure shows one fully and one partially occupied zinc site. The zinc ion in the fully occupied site (the catalytic zinc) is coordinated by three histidines and one water molecule. The second zinc ion is at 3.7 Å from the first one and is coordinated by one histidine, one cysteine, one aspartate and one unknown molecule (which is most likely to be a carbonate ion). In the B. cereus zinc \beta-lactamase the affinity for the second metal ion is low at the pH of crystallization (Kd = 25 mM, 293 K; [Baldwin et al. (1978). Biochem. J. 175, 441–447] and the dissociation constant of the second zinc ion thus apparently decreased at the cryogenic temperature. In addition, the structure of the apo enzyme was determined at 2.5 Å resolution. The removal of the zinc ion by chelating agents causes small changes in the active-site environment.

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Kinetics and thermodynamics of metal ion binding to synthetic peptides modelling the non-catalytic zinc binding sites of Hladh isozymes

Journal of Inorganic Biochemistry ◽

10.1016/0162-0134(95)97412-j ◽

1995 ◽

Vol 59 (2-3) ◽

pp. 311

Author(s):

H.W. Adolph ◽

A. Schneider ◽

M. Kiefer

Keyword(s):

Binding Sites ◽

Synthetic Peptides ◽

Metal Ion ◽

Zinc Binding ◽

Ion Binding ◽

Metal Ion Binding ◽

Kinetics And Thermodynamics ◽

Catalytic Zinc

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Models for protein-zinc ion binding sites. II. The catalytic sites

International Journal of Quantum Chemistry ◽

10.1002/qua.1207 ◽

2001 ◽

Vol 83 (3-4) ◽

pp. 150-165 ◽

Cited By ~ 24

Author(s):

David W. Deerfield ◽

Charles W. Carter ◽

Lee G. Pedersen

Keyword(s):

Binding Sites ◽

Zinc Ion ◽

Ion Binding ◽

Catalytic Sites ◽

Ion Binding Sites ◽

Zinc Ion Binding

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X-ray structure of a putative reaction intermediate of 5-aminolaevulinic acid dehydratase

Biochemical Journal ◽

10.1042/bj20030513 ◽

2003 ◽

Vol 373 (3) ◽

pp. 733-738 ◽

Cited By ~ 18

Author(s):

Peter T. ERSKINE ◽

Leighton COATES ◽

Danica BUTLER ◽

James H. YOUELL ◽

Amanda A. BRINDLEY ◽

...

Keyword(s):

Active Site ◽

Catalytic Mechanism ◽

Zinc Ion ◽

Side Chain ◽

Hydroxide Ion ◽

X Ray ◽

Aminolaevulinic Acid ◽

Acid Dehydratase ◽

Cyclic Intermediate ◽

Aminolaevulinic Acid Dehydratase

The X-ray structure of yeast 5-aminolaevulinic acid dehydratase, in which the catalytic site of the enzyme is complexed with a putative cyclic intermediate composed of both substrate moieties, has been solved at 0.16 nm (1.6 Å) resolution. The cyclic intermediate is bound covalently to Lys263 with the amino group of the aminomethyl side chain ligated to the active-site zinc ion in a position normally occupied by a catalytic hydroxide ion. The cyclic intermediate is catalytically competent, as shown by its turnover in the presence of added substrate to form porphobilinogen. The findings, combined with those of previous studies, are consistent with a catalytic mechanism in which the C–C bond linking both substrates in the intermediate is formed before the C–N bond.

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On the rate of F1-ATPase turnover during ATP hydrolysis by the single catalytic site Evidence that hydrolysis with a slow rate of product release does not occur at the alternating active site

FEBS Letters ◽

10.1016/0014-5793(87)80186-2 ◽

1987 ◽

Vol 222 (1) ◽

pp. 32-36 ◽

Cited By ~ 17

Author(s):

Ya.M. Milgrom ◽

M.B. Murataliev

Keyword(s):

Active Site ◽

Slow Rate ◽

Atp Hydrolysis ◽

Catalytic Site ◽

F1 Atpase ◽

Product Release

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Reversible autoinhibitory regulation ofEscherichia colimetallopeptidase BepA for selective β-barrel protein degradation

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2010301117 ◽

2020 ◽

Vol 117 (45) ◽

pp. 27989-27996

Author(s):

Yasushi Daimon ◽

Shin-ichiro Narita ◽

Ryoji Miyazaki ◽

Yohei Hizukuri ◽

Hiroyuki Mori ◽

...

Keyword(s):

Active Site ◽

Protease Activity ◽

Underlying Mechanism ◽

Zinc Ion ◽

Wild Type ◽

Protease Domain ◽

Loop Region ◽

Assembly Pathway

Escherichia coliperiplasmic zinc-metallopeptidase BepA normally functions by promoting maturation of LptD, a β-barrel outer-membrane protein involved in biogenesis of lipopolysaccharides, but degrades it when its membrane assembly is hampered. These processes should be properly regulated to ensure normal biogenesis of LptD. The underlying mechanism of regulation, however, remains to be elucidated. A recently solved BepA structure has revealed unique features: In particular, the active site is buried in the protease domain and conceivably inaccessible for substrate degradation. Additionally, the His-246 residue in the loop region containing helix α9 (α9/H246 loop), which has potential flexibility and covers the active site, coordinates the zinc ion as the fourth ligand to exclude a catalytic water molecule, thereby suggesting that the crystal structure of BepA represents a latent form. To examine the roles of the α9/H246 loop in the regulation of BepA activity, we constructed BepA mutants with a His-246 mutation or a deletion of the α9/H246 loop and analyzed their activities in vivo and in vitro. These mutants exhibited an elevated protease activity and, unlike the wild-type BepA, degraded LptD that is in the normal assembly pathway. In contrast, tethering of the α9/H246 loop repressed the LptD degradation, which suggests that the flexibility of this loop is important to the exhibition of protease activity. Based on these results, we propose that the α9/H246 loop undergoes a reversible structural change that enables His-246–mediated switching (histidine switch) of its protease activity, which is important for regulated degradation of stalled/misassembled LptD.

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