Crystal structure of acivicin-inhibited gamma-glutamyltranspeptidase reveals critical roles for its C-terminus in autoprocessing and catalysis

Abstract

Helicobacter pylori gamma-glutamyltranspeptidase (HpGT) is a general gamma-glutamyl hydrolase and a demonstrated virulence factor. The enzyme confers a growth advantage to the bacterium, providing essential amino acid precursors by initiating the degradation of extracellular glutathione and glutamine. HpGT is a member of the N-terminal nucleophile (Ntn) hydrolase superfamily and undergoes autoprocessing to generate the active form of the enzyme. Acivicin is a widely used gamma-glutamyltranspeptidase inhibitor that covalently modifies the enzyme, but its precise mechanism of action remains unclear. The time-dependent inactivation of HpGT exhibits a hyperbolic dependence on acivicin concentration with k(max) = 0.033 +/- 0.006 s(-1) and K(I) = 19.7 +/- 7.2 microM. Structure determination of acivicin-modified HpGT (1.7 A; R(factor) = 17.9%; R(free) = 20.8%) demonstrates that acivicin is accommodated within the gamma-glutamyl binding pocket of the enzyme. The hydroxyl group of Thr 380, the catalytic nucleophile in the autoprocessing and enzymatic reactions, displaces chloride from the acivicin ring to form the covalently linked complex. Within the acivicin-modified HpGT structure, the C-terminus of the protein becomes ordered with Phe 567 positioned over the active site. Substitution or deletion of Phe 567 leads to a >10-fold reduction in enzymatic activity, underscoring its importance in catalysis. The mobile C-terminus is positioned by several electrostatic interactions within the C-terminal region, most notably a salt bridge between Arg 475 and Glu 566. Mutational analysis reveals that Arg 475 is critical for the proper placement of the C-terminal region, the Tyr 433 containing loop, and the proposed oxyanion hole.

Figure 1

Comparison of glutathione and acivicin structures.

Figure 2. Inactivation of HpGT by acivicin

(A) HpGT (0.1 mg/ml) was incubated with 10 μM acivicin at pH 7.4 and 4 °C in the absence (squares) and presence (triangles) of 1 mM glutamate. The k_obs for the reactions were determined from the slope of ln(E_t/E_o) versus time, where E_o and E_t are the enzymatic activities at time 0 and time t respectively. (B) Dependence of the rate of HpGT inactivation on acivicin concentration. HpGT was incubated with various concentrations of acivicin and the rate of inactivation determined as in Panel A. (Inset) Double reciprocal plot used to calculate k_max and K_I.

Figure 3. Electron density corresponding to the site of acivicin modification

The refined model of acivicin-modified HpGT with pertinent active site residues is shown in stick representation and is superimposed with acivicin-modified E.coli γGT. Oxygen atoms are shown in red and nitrogen atoms in blue. Carbon atoms are colored green in HpGT, grey in acivicin, and blue in E.coli γGT. Residue numbers are noted in black (HpGT) and blue (E.coli γGT). Also shown are the calculated electron density maps after the initial round of refinement, prior to inclusion of Thr 380 and acivicin in the model. The relevant 2F_o-F_c electron density is contoured at 1.0 σ and illustrated in black. Positive and negative peaks in the difference map, contoured at 3.0 σ, are shown in blue and red respectively. Potential hydrogen bonds were identified with Chimera and are indicated as solid black lines.

Figure 4. Alternate conformations of Thr 380 following acivicin modification

Crystals of acivicin-modified HpGT contain a heterotetramer within the asymmetric unit, and provide two separate active site models. The active site regions of the refined structure are colored as in Figure 3. For reference, Thr 380 and bound glutamate from the previously determined HpGT-glutamate complex (blue) have been superimposed (20). The dihydroisoxazole ring of acivicin superimposes reasonably well with the glutamate side chain, and nearly identical placements of active site residues are observed with the exception of the slight displacement of Thr 380. (A) Within one active site, formed by the A and B subunits of the heterotetramer, Thr 380 adopts a conformation comparable to previous HpGT structures, with its α-amino group within hydrogen bond distance to the backbone carbonyl group of Asn 400, the hydroxyl group of Thr 398, and a tightly bound water molecule. (B) In the second active site comprised of subunits C and D, Thr 380 has rotated such that its α-amino group is within hydrogen bond distance to the backbone carbonyl group of Tyr 397 and the hydroxyl group of Thr 398. A nearly identical placement of acivicin is observed in both active sites.

Figure 5. The C-terminus is ordered in the acivicin-modified HpGT structure

A ribbon stereodiagram of HpGT is shown with the 40 kDa subunit colored in purple and the 20 kDa subunit in green. Relevant active site residues are shown in ball and stick representation. Carbon atoms are colored in green (20 kDa subunit), purple (40 kDa subunit), grey (acivicin) and yellow (the ordered C-terminus). Oxygen atoms are shown in red and nitrogen atoms in blue. In each of the previously determined HpGT structures, residues Lys 565, Glu 566, and Phe 567 were disordered. In the acivicin-modified HpGT structure, unambiguous electron density corresponding to the C-terminus of the protein was observed (not shown). Residues Arg 175 and Arg 475 appear to stabilize this region, positioning Phe 567 over the catalytic nucleophile, Thr 380.

Figure 6. The truncated C-terminus of HpGT is unique among

γ-glutamyltranspeptidases. (A) An alignment of representative γ-glutamyltranspeptidase sequences indicates that an aromatic residue is typically found at the C-terminal position. HpGT is considerably shorter than other γ-glutamyltranspeptidases, suggesting that the active site capping by Phe 567 observed in the acivicin-modified HpGT structure may be unique among γ-glutamyltranspeptidases. Sequences used in the alignment are H. pylori (O25743), E. coli (P18956), H. sapiens (P19440), D. rerio (Q7T2A1), D. melanogaster (Q9VWT3), C. elegans (Q9N5V4), A. thaliana (Q8VYW6), and S. cerevisiae (Q05902). (B) The superimposed structures of HpGT and E. coli γGT are shown in ribbon representation. HpGT is colored as in Figure 4 and the 40 kDa and 20 kDa subunits of E. coli γGT are colored in blue and grey respectively. Whereas the C-terminus of HpGT (yellow) extends over the active site, the C-terminus of E. coli γGT reverses and extends back to the surface of the protein, creating a more open active site pocket.

Figure 7. An extensive hydrogen bond network in the C-terminal region of the 20 kDa subunit of HpGT is required for autoprocessing and catalytic activity

In the stereodiagrams, a ribbon illustration of HpGT is presented with relevant residues shown in ball and stick representation. Atoms are colored as in Figure 4 and potential hydrogen bonds were identified with Chimera (indicated as solid black lines). Gly 472 and Gly 473, whose backbone amides form the proposed oxyanion hole, are highlighted as a red ribbon. (A) Arg 475 is critical for positioning of both the Tyr 433 loop and the Phe 567 C-terminal tail, as well as the backbone amides of Gly 472 and Gly 473. (B) The Glu 515/Arg 564 and the Asp 562/Arg 502 salt bridges stabilize the active site region and are critical for autoprocessing and enzymatic activity. Arg 513 also contributes to the overall integrity of the active site.