title: Structural basis of interactions between human glutamate carboxypeptidase II and its substrate analogs.
abstract: Human glutamate carboxypeptidase II (GCPII) is involved in neuronal signal transduction and intestinal folate absorption by means of the hydrolysis of its two natural substrates, N-acetyl-aspartyl-glutamate and folyl-poly-gamma-glutamates, respectively. During the past years, tremendous efforts have been made toward the structural analysis of GCPII. Crystal structures of GCPII in complex with various ligands have provided insight into the binding of these ligands, particularly to the S1' site of the enzyme. In this article, we have extended structural characterization of GCPII to its S1 site by using dipeptide-based inhibitors that interact with both S1 and S1' sites of the enzyme. To this end, we have determined crystal structures of human GCPII in complex with phosphapeptide analogs of folyl-gamma-glutamate, aspartyl-glutamate, and gamma-glutamyl-glutamate, refined at 1.50, 1.60, and 1.67 A resolution, respectively. The S1 pocket of GCPII could be accurately defined and analyzed for the first time, and the data indicate the importance of Asn519, Arg463, Arg534, and Arg536 for recognition of the penultimate (i.e., P1) substrate residues. Direct interactions between the positively charged guanidinium groups of Arg534 and Arg536 and a P1 moiety of a substrate/inhibitor provide mechanistic explanation of GCPII preference for acidic dipeptides. Additionally, observed conformational flexibility of the Arg463 and Arg536 side chains likely regulates GCPII affinity toward different inhibitors and modulates GCPII substrate specificity. The biochemical experiments assessing the hydrolysis of several GCPII substrate derivatives modified at the P1 position, also included in this report, further complement and extend conclusions derived from the structural analysis. The data described here form an a solid foundation for the structurally aided design of novel low-molecular-weight GCPII inhibitors and imaging agents.
authors.affiliation: Center for Cancer Research, National Cancer Institute, Frederick, MD 21702, USA. cyril@ncifcrf.gov
authors.forename: Cyril
authors.initials: C
authors.lastname: Barinka
authors.forename: Klara
authors.initials: K
authors.lastname: Hlouchova
authors.forename: Miroslava
authors.initials: M
authors.lastname: Rovenska
authors.forename: Pavel
authors.initials: P
authors.lastname: Majer
authors.forename: Miroslawa
authors.initials: M
authors.lastname: Dauter
authors.forename: Niyada
authors.initials: N
authors.lastname: Hin
authors.forename: Yao-Sen
authors.initials: YS
authors.lastname: Ko
authors.forename: Takashi
authors.initials: T
authors.lastname: Tsukamoto
authors.forename: Barbara S
authors.initials: BS
authors.lastname: Slusher
authors.forename: Jan
authors.initials: J
authors.lastname: Konvalinka
authors.forename: Jacek
authors.initials: J
authors.lastname: Lubkowski
mesh_terms: D000954:Antigens, Surface / Q000737:chemistry* / Q000378:metabolism; D001120:Arginine / Q000378:metabolism; D001665:Binding Sites; D018360:Crystallography, X-Ray; D043425:Glutamate Carboxypeptidase II / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D008958:Models, Molecular; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity; D015032:Zinc / Q000737:chemistry
chemical_list: D000954:Antigens, Surface; D011994:Recombinant Proteins; D001120:Arginine; C473848:FOLH1 protein, human; D043425:Glutamate Carboxypeptidase II; D015032:Zinc