Structures of human Golgi-resident glutaminyl cyclase and its complexes with inhibitors reveal a large loop movement upon inhibitor binding

Abstract

Aberrant pyroglutamate formation at the N terminus of certain peptides and proteins, catalyzed by glutaminyl cyclases (QCs), is linked to some pathological conditions, such as Alzheimer disease. Recently, a glutaminyl cyclase (QC) inhibitor, PBD150, was shown to be able to reduce the deposition of pyroglutamate-modified amyloid-β peptides in brain of transgenic mouse models of Alzheimer disease, leading to a significant improvement of learning and memory in those transgenic animals. Here, we report the 1.05-1.40 Å resolution structures, solved by the sulfur single-wavelength anomalous dispersion phasing method, of the Golgi-luminal catalytic domain of the recently identified Golgi-resident QC (gQC) and its complex with PBD150. We also describe the high-resolution structures of secretory QC (sQC)-PBD150 complex and two other gQC-inhibitor complexes. gQC structure has a scaffold similar to that of sQC but with a relatively wider and negatively charged active site, suggesting a distinct substrate specificity from sQC. Upon binding to PBD150, a large loop movement in gQC allows the inhibitor to be tightly held in its active site primarily by hydrophobic interactions. Further comparisons of the inhibitor-bound structures revealed distinct interactions of the inhibitors with gQC and sQC, which are consistent with the results from our inhibitor assays reported here. Because gQC and sQC may play different biological roles in vivo, the different inhibitor binding modes allow the design of specific inhibitors toward gQC and sQC.

FIGURE 1.

Overall structure and domain organization of gQC. The structure of the Golgi-luminal catalytic domain of gQC is shown as a ribbon diagram. The amino acid sequences corresponding to the nine α-helices (α1–α9), six β-strands (β1–β6), and three 3₁₀-helices can also be seen in Fig. 2. The catalytic zinc ion is shown as a gray ball. The zinc-coordinated amino acid residues (Asp¹⁸⁶, Glu²²⁶, and His³⁵¹) and water molecule are drawn with the yellow stick models and a red ball, respectively. The single-letter code sequence with green background (Ser⁵³–Pro⁷¹) represents the N-terminal flexible region of our structure. The remaining sequence with white background depicts the transmembrane (TM) (Leu³⁵–Trp⁵²) and cytosolic (Met¹–Arg³⁴) domains of an entire gQC molecule, as predicted with the program HMMTOP (45).

FIGURE 2.

Sequence alignment of human gQC with several representative gQCs and sQCs. The secondary structural elements, e.g. α-helices, β-strands, and 3₁₀-helices, according to our refined structures of gQC, are illustrated. The residues that are identical in five out of the six sequences are shaded in gray, and the completely conserved residues are depicted in black. The zinc-coordinated residues are marked with orange balls under the alignment. The residues at the loops that have different conformations between gQC and sQC, as described under “Results,” are boxed in orange. The GenBank accession numbers of these sequences are as follows: NP_060129 (human gQC), NP_001069408 (bovine gQC), NP_080387 (mouse gQC), NP_036545 (human sQC), NP_803472 (bovine sQC), and AAI51028 (mouse sQC).

FIGURE 3.

Comparison of the gQC and sQC structures. A, superimposition of the structures of gQC (red), open form sQC (blue), and closed form sQC (green) in stereo view. The loops with different conformations between gQC and sQC are marked with black stars. B, surface charge potential of the gQC and sQC structures. The charge potentials were calculated by using the software PyMOL (46) with the values ranging from −67.258 to 67.258, colored from red to blue. Note that gQC has a more negatively charged surface around its active site. The notable residue substitutions that contribute to the distinct charge potentials between these two QCs are labeled. The tryptophan residue with a large positional change is also labeled. C, superimposition of the active site structures of gQC (red), open form sQC (blue), and closed form sQC (green) in stereo view. Note that the tryptophan residue, i.e. Trp²³¹ in gQC and Trp²⁰⁷ in sQC, moves ∼15 Å in between these two QCs. By contrast, the catalytic glutamate residue (Glu²²⁵ in gQC) and the catalytically essential hydrogen-bond network (Glu²²⁵···Asp³²⁶···Asp²⁶⁹ in gQC) in these structures are superimposed very well.

FIGURE 4.

The active site structures of gQC. A, a close-up and stereo view of the active site structure of gQC at pH 6.0. The model representations are the same as in Fig. 1. Wat, water molecule. B, a close-up view of the tetrahedral zinc coordination environment of gQC at pH 6.0. The 7.0σ F_o − F_c stimulated annealing omit densities for the zinc ion (gold) and the zinc-coordinated residues (gold) and water molecule (green) are overlaid with the refined models. The coordination bonds are drawn with distances indicated in angstroms. C, a close-up view of the zinc coordination environment of gQC at pH 6.5. Note that the zinc-coordinated water is replaced by a cacodylate molecule. The chemical structure of cacodylate is shown on the left. The F_o − F_c omit densities are also shown, with that for the cacodylate molecule being colored in green.

FIGURE 5.

The QC inhibitors used in the present study. A, chemical structure of the QC inhibitors. B, the F_o − F_c stimulated annealing omit densities for the inhibitors bound in various QC structures, as indicated, are overlaid with the final refined models. The contour levels of the densities are also indicated.

FIGURE 6.

Comparison of the binding modes of QC inhibitors in gQC and sQC. A, the free-form gQC. B, the N-ω-acetylhistamine-bound gQC (AH-gQC). To enhance the clarity of the hydrogen bondings, the side chain of Glu³²⁵ is sliced out of view. C, the 1-benzylimidazole-bound gQC (BI-gQC). Note that the loop Lys²²⁹–Lys²³⁴ shows two distinct conformations, with the one identical to that of free-form enzyme being colored in orange. D, the PBD150-bound gQC. Note that the loop Lys²²⁹–Lys²³⁴ shows a large conformational change when compared with that of free-form enzyme. E, the free-form sQC (open form). F, the N-ω-acetylhistamine-bound sQC (open form). Note that the inhibitor forms three hydrogen bonds with sQC, in contrast to the two with gQC as observed in panel B. For clarity, the side chain of Gln³⁰⁴ is sliced out of view. G, the 1-benzylimidazole-bound sQC (open form). The inhibitor shows fewer hydrophobic contacts with sQC when compared with that with gQC as observed in panel C. H, the PBD150-bound sQC (open form). Note that the indole ring of Trp²⁰⁷ rotates to an opposite orientation when compared with that of free-form enzyme. I, the free-form sQC (closed form). J, the N-ω-acetylhistamine-bound sQC (closed form). The inhibitor exhibits a binding mode as observed in panel F. For clarity, the side chain of Gln³⁰⁴ is also sliced out of view. K, the 1-benzylimidazole-bound sQC (closed form). The inhibitor shows a binding mode as observed in panel G, except for the additional contact with Trp²⁰⁷. L, the PBD150-bound sQC (closed form). Except in the free-form QCs, only the residues that bind to the inhibitors or show a large positional change are labeled. The structures for free-form, N-ω-acetylhistamine-bound, and 1-benzylimidazole-bound sQCs are from the previous report (4).

FIGURE 7.

A close-up view of the interactions of 1-benzylimidazole and PBD150 with gQC and sQC. A, comparison of 1-benzylimidazole (BI) bound to gQC and sQC. The possible hydrophobic contacts are drawn with black dashed lines, with distances being indicated in angstroms. B, comparison of PBD150 bound to gQC and sQC. The possible hydrophobic contacts and hydrogen bonds are drawn with dashed lines colored in black and red, respectively, with distances being indicated in angstroms.

FIGURE 8.

Surface representation showing the large loop movement in the active site of gQC upon binding to PBD150. The residues that undergo a large positional change upon inhibitor binding are labeled and painted with various colors.