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Crystal structures of the recombinant -factor XIIa protease with bound Thr-Arg and Pro-Arg substrate mimetics

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Autor(es):
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Pathak, Monika [1] ; Manna, Rosa [1] ; Li, Chan [1] ; Kaira, Bubacarr G. [1] ; Hamad, Badraldin Kareem [1] ; Belviso, Benny Danilo [1] ; Bonturi, Camila R. [2] ; Dreveny, Ingrid [1] ; Fischer, Peter M. [1] ; Dekker, Lodewijk V. [1] ; Vilela Oliva, Maria Luiza [2] ; Emsley, Jonas [1]
Número total de Autores: 12
Afiliação do(s) autor(es):
[1] Univ Nottingham, Sch Pharm, Ctr Biomol Sci, Nottingham NG7 2RD - England
[2] Univ Fed Sao Paulo, Biochem Dept, BR-04044020 Sao Paulo, SP - Brazil
Número total de Afiliações: 2
Tipo de documento: Artigo Científico
Fonte: ACTA CRYSTALLOGRAPHICA SECTION D-STRUCTURAL BIOLOGY; v. 75, n. 6, p. 578-591, JUN 2019.
Citações Web of Science: 0
Resumo

Coagulation factor XII (FXII) is a key initiator of the contact pathway, which contributes to inflammatory pathways. FXII circulates as a zymogen, which when auto-activated forms factor XIIa (FXIIa). Here, the production of the recombinant FXIIa protease domain (FXIIa(His)) with yields of approximate to 1-2mg per litre of insect-cell culture is reported. A second construct utilized an N-terminal maltose-binding protein (MBP) fusion (MBP-FXIIa(His)). Crystal structures were determined of MBP-FXIIa(His) in complex with the inhibitor d-Phe-Pro-Arg chloromethyl ketone (PPACK) and of FXIIa(His) in isolation. The FXIIa(His) structure revealed that the S2 and S1 pockets were occupied by Thr and Arg residues, respectively, from an adjacent molecule in the crystal. The Thr-Arg sequence mimics the P2-P1 FXIIa cleavage-site residues present in the natural substrates prekallikrein and FXII, and Pro-Arg (from PPACK) mimics the factor XI cleavage site. A comparison of the FXIIa(His) structure with the available crystal structure of the zymogen-like FXII protease revealed large conformational changes centred around the S1 pocket and an alternate conformation for the 99-loop, Tyr99 and the S2 pocket. Further comparison with activated protease structures of factors IXa and Xa, which also have the Tyr99 residue, reveals that a more open form of the S2 pocket only occurs in the presence of a substrate mimetic. The FXIIa inhibitors EcTI and infestin-4 have Pro-Arg and Phe-Arg P2-P1 sequences, respectively, and the interactions that these inhibitors make with FXIIa are also described. These structural studies of FXIIa provide insight into substrate and inhibitor recognition and establish a scaffold for the structure-guided drug design of novel antithrombotic and anti-inflammatory agents. (AU)

Processo FAPESP: 17/07972-9 - Desenvolvimento de compostos para profilaxia e tratamento de doenças cardiovasculares
Beneficiário:Maria Luiza Vilela Oliva
Linha de fomento: Auxílio à Pesquisa - Regular
Processo FAPESP: 17/06630-7 - Fragmentos derivados de proteínas com seletividade para inibição de enzimas de mamíferos e micro-organismos e seu papel como agente anti-inflamatório, antimicrobiano, antitrombótico e antitumoral: mecanismo de ação
Beneficiário:Maria Luiza Vilela Oliva
Linha de fomento: Auxílio à Pesquisa - Temático