- A papain-like enzyme at work: Native and acyl–enzyme intermediate structures in phytochelatin synthesis

Overall Structure. We determined the crystal structures of NsPCS in its native form and as a complex with GSH, its natural substrate. The structure of the selenomethionine-labeled NsPCS in its native form was first solved by MAD and then refined to 2-Å resolution with an R-factor of 20.%0 (R free = 25.7%; see Table 1). The protein–GSH complex was trapped and crystallized at noncatalytically active pH values (≈3). The resulting structure, solved by molecular replacement and refined to 1.4-Å resolution (R = 17.4%; R free = 18.7%), corresponds to an acyl–enzyme complex. In both the native and the acyl–enzyme crystal forms, the asymmetric unit contains two monomers. With an rms deviation <0.5 Å, these monomers are almost identical; the few structural differences will be detailed below. Each monomer has an overall “crescent” shape (with approximate dimensions of 50 × 30 × 30 Å3) with α/β fold containing eight α-helices and six β-strands (Fig. 1A). A structural homology search with dali (30) revealed that the NsPCS monomer is structurally most similar to staphopain [z = 11.5 (31)], IdeS [z = 10.8 (32)], and bleomycin hydrolase [z = 10.4 (33)]. As a result, and despite very low sequence similarity (sequence identity is in all cases <10%), NsPCS fold definitely shows that it belongs to the papain superfamily of cysteine proteases. Moreover, the main catalytic residues responsible for the protease activity are structurally conserved.

In the two different crystal forms that we solved, two NsPCS molecules are clamped as a dimer (Fig. 1 A). The homodimerization is mediated mainly by the two N-terminal helices, which are absent from the papain fold and therefore represent a specificity of the PCS family. Upon dimerization, ≈1,130 Å2 are buried, corresponding to 12% of the solvent-accessible surface of each monomer. Although 13 intermolecular hydrogen bonds are observed at the dimer interface, 71% of the interacting surface is nonpolar with numerous Van der Waals contacts.

The Active Site. Detection and analysis of the residues involved in the catalysis benefit from previous mutagenesis studies (17) as well as from the considerable work done on other cysteine proteases and notably on papain-like proteins (34). The active site of NsPCS is formed by the so-called catalytic triad composed of residues Cys-70 (equivalent to Cys-25 in papain), His-183 (His-159 in papain), and Asp-201 (Asn-175 in papain) (Fig. 1B). Cys-70 is the catalytic cysteine, and Asp-201, with its side chain involved in a hydrogen bond with His-183 Nδ1, is responsible for both the correct orientation and polarization of the imidazolium group of His-183. Overall, the rms deviation between all atoms of the catalytic triad in NsPCS and papain is 0.65 Å. Furthermore, Gln-64 in NsPCS occupies an analogous position to Gln-19 in papain, whose side-chain amide group, together with the main-chain amide group of Cys-25, define the so-called “oxyanion hole.” This oxyanion hole is known to polarize the carbonyl group of the substrate scissile bond. The large structural similarities observed between the active site of both papain-like proteins and NsPCS have functional consequences and suggest a catalytic mechanism of the same kind. The main differences between the two families around the active site concern the other residues in interaction with Asp-201. In typical cysteine proteases, Asp-201 is shielded by an aromatic residue (Trp-177 in papain). In NsPCS, a short residue, Ser-203, takes this position, which results in an open space filled by the side chain of Arg-173 that is hydrogen bonded via its guanidine group to Ser-203 (OH) and Asp-201 (Oδ1). This unusual geometry might be important for NsPCS catalysis, as will be discussed below.

The Acyl–Enzyme Complex. The structure of the acyl–enzyme complex shows that, as expected in papain-like proteins, the carbonyl oxygen of the thioester points toward the oxyanion hole formed by Gln-64 Nε2 and Cys-70 N (Fig. 2A). The γ-EC is also held in place by other numerous hydrogen bonds involving the main chain of Gly-182 and Met-123 and the side chain of Arg-232 and Asp-225. The γ-EC orientation is such that the sulfur atom of the substrate points toward the solvent medium. A significant residual density is linked to this sulfur atom and could be due to its partial oxidation (Fig. 2 C and D). The carbonyl oxygen of the thioester linkage is distorted from planarity by 37° in molecule A and 30° in molecule B. These angles are intermediate between the planarity observed in the elastase/β-casomorphine-7 acyl–enzyme complex and the tetrahedral intermediate resulting from a nucleophilic attack on the thioester bond (35, 36). The catalytic water molecule W153, found in only one molecule of the dimer (molecule A), is ideally placed for this nucleophilic attack toward the synthesis of free γ-EC. W153, which is 2.70 Å away from the Nε2 of the imidazole ring of His-183 and 3.15 Å from the thioester carbon, forms indeed an angle of 103° relative to the carbonyl bond, a value close to the expected Bürgi's angle of 105° (37).

Structural Changes Accompanying Acylation Reaction. Apart from a small reorientation of the two monomers relative to each other, two main structural rearrangements accompany the acylation reaction. First, in the native form, the B-loop 1 is in a “closed” conformation, with the side chain of Gln-67 hydrogen bonded to the side chain of the catalytic histidine (His-183). Instead, in the acyl–enzyme complex, the B-loop 1 is in an “open” configuration and reveals an open space close to the active site (Fig. 2B). Interestingly, in the native protein the Oε1 of Gln-67 takes the same position as the catalytic water molecule in the acyl–enzyme complex. The second main structural change is the destabilization of the protruding loop in the acyl–enzyme complex. Indeed, two segments and a total of 11 residues in this loop are not resolved in the electron-density map calculated at a 1.4-Å resolution. Because part of this loop is in the vicinity of the above-mentioned B-loop 1, the two structural changes may be connected.

There are some subtle but significant changes when comparing the active site of molecules A and B of the dimer in the acyl–enzyme structure (Fig. 2 C and D). First, as already mentioned, the catalytic water molecule W153 is present only in molecule A. Instead, a small but contiguous electron density is present in molecule B. This density could tentatively be ascribed to a glycine (a product of the reaction) or an ethylene glycol (used for the cryoprotection of the crystals). However, in both cases the refinement of these compounds was not satisfactory and we therefore left this density unassigned. Second, the electron-density map around Arg-173 clearly shows that its side chain adopts two conformations in molecule A. Of these two orientations, one is similar to what is found in the native structure and the other is similar to that found in molecule B. Another disparity visible from Fig. 2 C and D is the dynamics of the protruding loop, which appears to be different in molecule A, where residues 87–94 are disordered, as compared with molecule B, where the disorder occurs from residue 88 to residue 91. This difference is located relatively close to the active site and may thus influence the reactivity of the enzyme.



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