A cysteine protease from ginger rhizome (GP-II) cleaves peptides and proteins with proline at the P2 position. The unusual specificity for proline makes GP-II an attractive tool for protein sequencing and identification of stably folded domains in proteins. The enzyme is a 221 amino acid glycoprotein possessing two N-linked oligosaccharide chains (8% glycosylated by weight) at Asn99 and Asn 156. The availability of the sequence of these glycosyl chains afforded the opportunity to observe their structure and impact on protein conformation. The three-dimensional structure of GP-II has been determined by X-ray crystallography to a resolution of 2.1 Å (overall R-factor = 0.214, free R = 0.248). The overall structure of GP-II is similar to that of the homologous cysteine proteases papain, actinidin, and glycyl endopeptidase, folding into two distinct domains of roughly equal size which are divided by a cleft. The observed N-linked glycosyl chains (half the total carbohydrate sequence) participate in both crystallographic and noncrystallographic contacts, tethering the proteins together via hydrogen bonds to the carbohydrate residues without intervening ordered water molecules. The putative S2 binding pocket (the proline recognition site) was identified by superposition of the GP-II structure with structures of four previously determined papain-inhibitor complexes. The particular enzymic amino acids forming the S2 pocket of GP-II (Trp, Met, and Ala) are similar to those found in the proline binding pockets of the unrelated enzymes α- lytic protease and cyclophilin. However, there is no conserved three- dimensional arrangement of these residues between the three enzymes (i.e., no proline binding motif). Thus, the particular amino acids found at S2 are consistent with a binding pocket for a moiety with the steric characteristics and charge distribution of proline. Size exclusion is also a mechanism for selectivity compared to the S2 binding pocket of papain. The S2 binding pocket of GP-II greatly restricts the size of the side chain which could 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 nonpolar amino acids may be attributed to a direct effect of proline on the substrate peptide backbone conformation.
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