PDBsum entry 3bhx

Hydrolase

PDB id: 3bhx

Contents

Protein chain

694 a.a. *

Ligands

NAG-NAG ×3

NAG-NAG-BMA-MAN

NAG ×3

BHX

Metals

_ZN ×2

_CA

_CL

Waters ×665

* Residue conservation analysis

PDB id:

3bhx

Name:

Hydrolase

Title:

X-ray structure of human glutamate carboxypeptidase ii (gcpii) in complex with a transition state analog of asp-glu

Structure:

Glutamate carboxypeptidase 2. Chain: a. Fragment: extracellular domain residues 44-750. Synonym: glutamate carboxypeptidase ii, membrane glutamate carboxypeptidase, mgcp, n- acetylated-alpha-linked acidic dipeptidase i, naaladase i, pteroylpoly-gamma-glutamate carboxypeptidase, folylpoly-gamma- glutamate carboxypeptidase, fgcp, folate hydrolase 1, prostate- specific membrane antigen, psma, psm. Engineered: yes

Source:

Homo sapiens. Human. Organism_taxid: 9606. Gene: folh1, folh, naalad1, psm, psma. Expressed in: drosophila melanogaster. Expression_system_taxid: 7227.

Resolution:

1.60Å R-factor: 0.181 R-free: 0.202

Authors:

J.Lubkowski,C.Barinka

Key ref:

C.Barinka et al. (2008). Structural basis of interactions between human glutamate carboxypeptidase II and its substrate analogs. J Mol Biol, 376, 1438-1450. PubMed id: 18234225 DOI: 10.1016/j.jmb.2007.12.066

Date:

29-Nov-07 Release date: 08-Jan-08

Protein chain

Q04609  (FOLH1_HUMAN) -  Glutamate carboxypeptidase 2 from Homo sapiens

Seq: 750 a.a.

Struc: 694 a.a.

Enzyme reactions

• Enzyme class: E.C.3.4.17.21 - glutamate carboxypeptidase Ii.
• Reaction: Release of an unsubstituted, C-terminal glutamyl residue, typically from Ac-Asp-Glu or folylpoly-gamma-glutamates.
• Cofactor: Zn(2+)

DOI no: 10.1016/j.jmb.2007.12.066

J Mol Biol 376:1438-1450 (2008)

PubMed id: 18234225

Structural basis of interactions between human glutamate carboxypeptidase II and its substrate analogs.

C.Barinka, K.Hlouchova, M.Rovenska, P.Majer, M.Dauter, N.Hin, Y.S.Ko, T.Tsukamoto, B.S.Slusher, J.Konvalinka, J.Lubkowski.

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.

Selected figure(s)

Figure 2.
Fig. 2. The substrate-binding cavity of GCPII can be closed by the ‘entrance lid.’ (a) Superposition of the ‘entrance lids’ from rhGCPII/MPE and rhGCPII/SPE complexes. The model of rhGCPII/MPE is shown in cartoon representation and colored gray. The ‘entrance lid,’ formed by the amino acids Trp541–Gly548, is painted red and blue for the open (observed in the rhGCPII/MPE complex) and closed (taken from the superimposed structure of the rhGCPII/SPE complex) conformations, respectively. The active-site-bound SPE inhibitor is represented by sticks, and the active-site Zn^2+ ions are represented by magenta spheres. (b and c) A close-up view of the ‘entrance lid’ in open/closed conformation. The protein is represented by its molecular surface, with the ‘entrance lid’ colored red or blue for the open (rhGCPII/MPE, b) and closed (rhGCPII/SPE, c) conformations, respectively. The molecule of MPE, visible in the current projection, is represented by sticks (b), while a molecule of SPE is buried by the ‘entrance lid’ (c). A surface defined by the active-site zinc ions is colored magenta.

Figure 3.
Fig. 3. The electron density maps of the substrate-binding cavity of human GCPII in complex with SPE (a) and EPE (b). The protein residues are shown in ball-and-stick representation, while Zn^2+ and Cl^− ions are depicted as blue and yellow spheres, respectively. The F[o] − F[c] electron density omit maps around inhibitor molecules are contoured at the 3 σ level (green), and the 2F[o] − F[c] electron density maps are contoured at the 1σ level (blue). The picture was generated using MolScript^28 and BobScript^29 and rendered with PovRay.

The above figures are reprinted by permission from Elsevier: J Mol Biol (2008, 376, 1438-1450) copyright 2008.

Figures were selected by an automated process.