The 2.1 Å Structure of a Cysteine Protease with Proline Specificity from Ginger Rhizome, Zingiber officinale
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- 作者：Kyung H. Choi ; Richard A. Laursen ; and Karen N. Allen
- 刊名：Biochemistry
- 出版年：1999
- 出版时间：September 7, 1999
- 年：1999
- 卷：38
- 期：36
- 页码：11624 - 11633
- 全文大小：340K
- 年卷期：v.38,no.36(September 7, 1999)
- ISSN：1520-4995
文摘
A cysteine protease from ginger rhizome (GP-II) cleaves peptides and proteins with proline atthe P2 position. The unusual specificity for proline makes GP-II an attractive tool for protein sequencingand identification of stably folded domains in proteins. The enzyme is a 221 amino acid glycoproteinpossessing two N-linked oligosaccharide chains (8% glycosylated by weight) at Asn99 and Asn156. Theavailability of the sequence of these glycosyl chains afforded the opportunity to observe their structureand impact on protein conformation. The three-dimensional structure of GP-II has been determined byX-ray crystallography to a resolution of 2.1 Å (overall R-factor = 0.214, free R = 0.248). The overallstructure of GP-II is similar to that of the homologous cysteine proteases papain, actinidin, and glycylendopeptidase, folding into two distinct domains of roughly equal size which are divided by a cleft. Theobserved N-linked glycosyl chains (half the total carbohydrate sequence) participate in both crystallographicand noncrystallographic contacts, tethering the proteins together via hydrogen bonds to the carbohydrateresidues without intervening ordered water molecules. The putative S2 binding pocket (the prolinerecognition site) was identified by superposition of the GP-II structure with structures of four previouslydetermined papain-inhibitor complexes. The particular enzymic amino acids forming the S2 pocket ofGP-II (Trp, Met, and Ala) are similar to those found in the proline binding pockets of the unrelatedenzymes 
-lytic protease and cyclophilin. However, there is no conserved three-dimensional arrangementof these residues between the three enzymes (i.e., no proline binding motif). Thus, the particular aminoacids found at S2 are consistent with a binding pocket for a moiety with the steric characteristics andcharge distribution of proline. Size exclusion is also a mechanism for selectivity compared to the S2binding pocket of papain. The S2 binding pocket of GP-II greatly restricts the size of the side chain whichcould be bound because of the occurrence of a tryptophan in place of the corresponding tyrosine in papain.In light of the nature of the binding pocket, the specificity of GP-II for proline over other small nonpolaramino acids may be attributed to a direct effect of proline on the substrate peptide backbone conformation.
-lytic protease and cyclophilin. However, there is no conserved three-dimensional arrangementof these residues between the three enzymes (i.e., no proline binding motif). Thus, the particular aminoacids found at S2 are consistent with a binding pocket for a moiety with the steric characteristics andcharge distribution of proline. Size exclusion is also a mechanism for selectivity compared to the S2binding pocket of papain. The S2 binding pocket of GP-II greatly restricts the size of the side chain whichcould be bound because of the occurrence of a tryptophan in place of the corresponding tyrosine in papain.In light of the nature of the binding pocket, the specificity of GP-II for proline over other small nonpolaramino acids may be attributed to a direct effect of proline on the substrate peptide backbone conformation.