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-␤ 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 gQCinhibitor 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 inhibitorbound 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.

Dipeptidyl peptidase-IV inhibitors used in type-2 diabetes inhibit a phospholipase C: a case of promiscuous scaffolds in proteins

“…Our findings rationalize the elevated levels of serum lipase found in patients undergoing DPP4 inhibitor based therapies 28, 29 , although these reports are in disagreement with other findings 30, 31 . While it is logical and expected to find scaffolds that are congruent to trypsin and DPP4 active sites in lipases based on the current results and our previous findings 22 , we also show the presence of the serine catalytic triad in close proximity to the active site residues of proteins which have a completely different enzymatic mechanism (for example, in glutaminyl cyclase which is a transferase 32 ). This corroborates the current belief that convergent evolution occurs more frequently than previously believed 33 .…”

“…However, our methodology also detects other proteins, often with a completely different enzymatic mechanism from hydrolases. A glutaminyl cyclase (PDBid:3PB4 32 ), a transferase, has a significantly congruent domain with Motif1 (lesser congruence with Motif2, as indicated by the RMSD) ( Table 1). Figure 3 shows the proximity of the promiscuous scaffold to the active site of the cyclase, and also the congruence of the scaffold to Motif1.…”

Soluble Variants of Human Recombinant Glutaminyl Cyclase

“…The 15 N HSQC performed on the 70 µM 15 N, 13 C 6xmut hQPCT is reported in Figure 3C. NMR was also used to demonstrate the binding of the ligand PBD150, a known inhibitor of wild type hQPCT [13] that we have found to inhibit also the activity of the 2xmut (see above). A titration of the 2xmut 15 N labeled protein with increasing amounts of this inhibitor was performed.…”

“…The mapping is possible whenever the following conditions are met: i ) a structure of the protein and ii ) the assignment of the 1 H- 15 N HSQC spectrum are available [8], [9], [10], [11]. While the first condition has been achieved by X-ray crystal structure determinations (PDB id: 2ZED, 3SI1, 2AFM, 3PBB), [4], [5], [12], [13] no NMR assignment exists yet for hQPCT, nor 1 H- 15 N HSQC spectra have been reported. Given the size of the protein, this might be accomplished via triple resonance NMR experiments that require 15 N, 13 C and partial 2 H enrichment on protein forms with a solubility of hundreds of µM [14].…”

Inhibition of glutaminyl cyclase attenuates cell migration modulated by monocyte chemoattractant proteins

“…These two QCs share ∼ 45 % sequence identity and similar catalytic characteristics [7]. Previous studies indicated that the enzymatic activity of isoQC is ∼ 3-fold weaker on several synthetic substrates than the activity of sQC [8]. A tissue distribution analysis revealed that the expression of isoQC is ubiquitous [7], whereas sQC expression is higher in neuronal tissues and in several peripheral blood lymphocytes [1].…”