A high-resolution structure of ligand-free human glutamate carboxypeptidase II

Bařinka, Cyril; Starková, Jana; Konvalinka, Jan; Łubkowski, J.

doi:10.1107/s174430910700379x

Cited by 46 publications

(57 citation statements)

References 17 publications

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“…It is interesting to note that in the highresolution structures of GCPII complexes and ligand-free GCPII published previously, a portion of the 'entrance lid' was not modeled, due to the lack of corresponding electron density, implying high positional flexibility of this loop. 20,31 Our observations suggest that the closed conformation of the 'entrance lid' might be stabilized by ligand binding in the active site of GCPII (i.e., the occupancy of the S1 pocket by a glutamate/aspartate moiety of the substrate/inhibitor), despite the lack of direct contacts between a ligand and the 'entrance lid.' Additionally, when larger substrates such as folylpoly-γ-glutamates are processed by GCPII, the 'entrance lid' probably remains open during the course of hydrolysis.…”

Section: Discussionmentioning

confidence: 91%

“…It should be noted that in the rhGCPII/ SPE complex, Arg536 adopts both 'stacking' and 'binding' conformations, with the 'stacking' conformation being more populated (about 70%). The arrangement of the Arg536 side chain is similar to that in the ligand-free GCPII structure, 31 implying that the SPE binding is not associated with significant conformational changes within the S1 site. A similar arrangement of the S1 site and a comparable set of enzyme-ligand interactions are observed in the rhGCPII/EPE complex (Fig.…”

Section: Gcpii-phosphapeptide Interactions In the S1 Sitementioning

confidence: 83%

“…Since the crystals of the rhGCPII complexes were isomorphous with the crystals of ligand-free rhGCPII 31 [Protein Data Bank (PDB) code 2OOT], the latter was used as the starting model for the structure determination using difference Fourier methods. The initial structure determinations, carried out at a resolution of 2.5 Å, were followed by the refinement of individual atomic coordinates and Bfactors, with gradual extension of the resolution to the limits of the experimental data.…”

Section: Structure Determination and Refinementmentioning

confidence: 99%

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Structural Basis of Interactions between Human Glutamate Carboxypeptidase II and Its Substrate Analogs

Bařinka

Hlouchová

Rovenská

et al. 2008

Journal of Molecular Biology

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119

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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.

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Section: Discussionmentioning

confidence: 91%

Section: Gcpii-phosphapeptide Interactions In the S1 Sitementioning

confidence: 83%

Section: Structure Determination and Refinementmentioning

confidence: 99%

See 1 more Smart Citation

Structural Basis of Interactions between Human Glutamate Carboxypeptidase II and Its Substrate Analogs

Bařinka

Hlouchová

Rovenská

et al. 2008

Journal of Molecular Biology

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119

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show abstract

“…The distances are in . A) Active site of thermolysin (PDB ID: 1LNF [4] ); B) glutamate carboxypeptidase II (GCPII, PDB ID: 2OOT [5] ); C) bovine lens leucine aminopeptidase (blLAP, PDB ID: 1LAM [6] ).…”

Section: Introductionmentioning

confidence: 99%

Theoretical Aspects of Hydrolysis of Peptide Bonds by Zinc Metalloenzymes

Navrátil

Klusák

Rulı́s̆ek

2013

Chemistry A European J

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Activation and reaction energies for four model systems capturing the essential physicochemical features of the hydrolysis of the peptide bond have been calculated at various level of theory, including the presumably accurate CCSD(T) calculations. The models studied covered a part of the spectrum encountered in biological systems: the hydrolysis in the absence of metal ions (represented by formamide and Ala-Ala) and the hydrolysis in the presence of one and two zinc(II) ions, mimicking the active sites of mono- and dizinc metallopeptidases, respectively (by using thermolysin and glutamate carboxypeptidase II as the model catalytic systems and formamide as the model substrate). The results obtained using CCSD(T)/def2-TZVP and CCSD(T)/aug-cc-pVTZ calculations were used as the benchmark values to which the set of cheaper methods, such as (RI-)DFT, (RI-)MP2, and SCS-MP2, were referenced. It was shown that deviations of 3-5 kcal mol(-1) (translating to 2-3 orders in reaction constants) with respect to the reference CCSD(T) barriers are frequently encountered for many correlated methods and most of studied DFT functionals. It has been concluded that from the set of wave-function methods, both MP2 and SCS-MP2 methods can be recommended for smaller models (measured by the mean absolute deviation of the activation barriers over the four systems studied), whereas among the popular DFT functionals, B3LYP and especially M06-2X are likely to be reasonable choices for calculating the activation barriers of zinc metallopeptidases. Finally, with the model of glutamate carboxypeptidase II, issues related to the convergence of the calculated barriers with the size of the model system used as the representative of the enzyme active site were addressed. The intricacies related to system truncation are demonstrated, and suggest that the correlated wave-function methods may suffer from problems, such as intramolecular BSSE, which make their usage for the larger system questionable. Altogether, the presented data should contribute to efforts to understand enzymatic catalysis more deeply and to gain control of the accuracy and deficiencies of the available theoretical methods and computational approaches.

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“…For each complex, a complete dataset was collected from a single crystal and data were processed using the HKL2000 software package. 26 Difference Fourier methods were used to determine structures of GCPII/inhibitor complexes with the ligand-free GCPII structure (PDB code 2OOT) 27 used as a starting model. Calculations were performed using the program Refmac 5.5.…”

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confidence: 99%

Structural characterization of P1′-diversified urea-based inhibitors of glutamate carboxypeptidase II

Pavlíček

Ptacek

Černý

et al. 2014

Bioorganic & Medicinal Chemistry Letters

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Urea-based inhibitors of human glutamate carboxypeptidase II (GCPII) have advanced into clinical trials for imaging metastatic prostate cancer. In parallel efforts, agents with increased lipophilicity have been designed and evaluated for targeting GCPII residing within the neuraxis. Here we report the structural and computational characterization of six complexes between GCPII and P1′-diversified urea-based inhibitors that have the C-terminal glutamate replaced by more hydrophobic moieties. The X-ray structures are complemented by quantum mechanics calculations that provide a quantitative insight into the GCPII/inhibitor interactions. These data can be used for the rational design of novel glutamate-free GCPII inhibitors with tailored physicochemical properties.

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A high-resolution structure of ligand-free human glutamate carboxypeptidase II

Cited by 46 publications

References 17 publications

Structural Basis of Interactions between Human Glutamate Carboxypeptidase II and Its Substrate Analogs

Structural Basis of Interactions between Human Glutamate Carboxypeptidase II and Its Substrate Analogs

Theoretical Aspects of Hydrolysis of Peptide Bonds by Zinc Metalloenzymes

Structural characterization of P1′-diversified urea-based inhibitors of glutamate carboxypeptidase II

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