Crystal structures of human glutaminyl cyclase, an enzyme responsible for protein N-terminal pyroglutamate formation

Abstract

N-terminal pyroglutamate (pGlu) formation from its glutaminyl (or glutamyl) precursor is required in the maturation of numerous bioactive peptides. The aberrant formation of pGlu may be related to several pathological processes, such as osteoporosis and amyloidotic diseases. This N-terminal cyclization reaction, once thought to proceed spontaneously, is greatly facilitated by the enzyme glutaminyl cyclase (QC). To probe this important but poorly understood modification, we present here the structure of human QC in free form and bound to a substrate and three imidazole-derived inhibitors. The structure reveals an ␣͞␤ scaffold akin to that of two-zinc exopeptidases but with several insertions and deletions, particularly in the active-site region. The relatively closed active site displays alternate conformations due to the different indole orientations of Trp-207, resulting in two substrate (glutamine t-butyl ester)-binding modes. The single zinc ion in the active site is coordinated to three conserved residues and one water molecule, which is replaced by an imidazole nitrogen upon binding of the inhibitors. Together with structural and kinetic analyses of several active-site-mutant enzymes, a catalysis mechanism of the formation of protein N-terminal pGlu is proposed. Our results provide a structural basis for the rational design of inhibitors against QC-associated disorders.crystallography ͉ intramolecular cyclization ͉ posttranslational modification ͉ Alzheimer's disease ͉ aminopeptidase N -terminal pyroglutamate (pGlu) formation from its glutaminyl precursor is an important posttranslational or cotranslational event in the processing of numerous bioactive neuropeptides, hormones, and cytokines during their maturation in the secretory pathway. These regulatory peptides require the N-terminal pGlu to develop the proper conformation for binding to their receptors and͞or to protect the N termini of the peptides from exopeptidase degradation (1, 2). Previously, this Nterminal cyclization reaction was thought to proceed spontaneously, until the glutaminyl cyclases (QCs) were identified as catalysts that are responsible for this posttranslational modification (3, 4).QCs (EC 2.3.2.5) are acyltransferases that have been identified in both animal and plant sources (3-5). They are abundant in mammalian neuroendocrine tissues, such as hypothalamus and pituitary (4, 6), and are highly conserved from yeast to human. Animal QCs were shown to have distinct structure and protein stability from plant QCs despite their similar molecular masses [i.e., 33-40 kDa (5, 7)]. No bacterial QCs have been reported thus far; however, the mammalian QCs were predicted to exhibit remarkable homology to the bacterial double-zinc aminopeptidases (8, 9).In humans, several genetic diseases, such as osteoporosis, a multifactorial hormonal disease that is characterized by reduced bone mass and microarchitectural deterioration of bone tissue (10), appear to result from mutations of the QC gene. The gene encoding QC (QPCT) lies on chromosome 2p22.3. ...

Structures of Human Golgi-resident Glutaminyl Cyclase and Its Complexes with Inhibitors Reveal a Large Loop Movement upon Inhibitor Binding

“…We also determined the structures of gQC-PBD150 and sQC-PBD150, revealing a large loop movement in the active site of gQC upon inhibitor binding. To further compare the inhibitor binding modes between gQC and sQC, we also solved the high-resolution structures of gQC in complex with the inhibitors N--acetylhistamine and 1-benzylimidazole because the structures of sQC in complex with these two inhibitors are available (4). Lastly, we have analyzed the inhibition effects of these inhibitors on gQC and sQC to confirm our observations on the inhibitorbound QC structures.…”

“…For the structure of the sQC-PBD150 complex, 2 l of wild-type sQC (ϳ10 mg/ml) was mixed with 0.5 l of PBD150 (5 mM) and 2 l crystallization buffer (the same as for W207F) and then crystallized as described above. Subsequent cryoprotectant preparation and x-ray data collection were referenced from a previous report (4).…”

Microheterogeneity of Recombinant Antibodies: Analytics and Functional Impact

“…[64,65] Additionally, both reactions can also occur in an enzymatically driven fashion, catalyzed by the glutaminyl cyclase. [66] Non-enzymatic conversion of glutamate to pyroglutamate has also been observed on antibodies in vivo, without affecting the pharmacological properties of the molecules, such as its safety, pharmacodynamics, and pharmacokinetics. [25,60] The formation of PyroGlu from Gln is associated with the loss of the positively charged primary amine, which creates variants that have a more acidic isoelectric point.…”

Structural Biology of Glycoproteins

“…The structure of human QC had already been solved using protein produced in E. coli (PDB entry 2AFO) [29]. However, the glycosylated structure was shown to be more stable and to contain a disulphide bond not present in the non-glycosylated structure.…”