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
= 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
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
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
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.
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.