PDBsum entry 1orf

Hydrolase/hydrolase inhibitor

PDB id: 1orf

Contents

Protein chain

232 a.a. *

Ligands

0G6

SO4

Waters ×128

* Residue conservation analysis

PDB id:

1orf

Name:

Hydrolase/hydrolase inhibitor

Title:

The oligomeric structure of human granzyme a reveals the molecular determinants of substrate specificity

Structure:

Granzyme a. Chain: a. Synonym: cytotoxic t-lymphocyte proteinase 1, hanukkah factor, h factor, hf, granzyme 1, ctl tryptase, fragmentin 1. Engineered: yes. Mutation: yes

Source:

Homo sapiens. Human. Organism_taxid: 9606. Gene: gzma. Expressed in: pichia pastoris. Expression_system_taxid: 4922.

Biol. unit:

Tetramer (from PDB file)

Resolution:

2.40Å R-factor: 0.191 R-free: 0.232

Authors:

J.K.Bell,D.H.Goetz,S.Mahrus,J.L.Harris,R.J.Fletterick,C.S.Craik

Key ref:

J.K.Bell et al. (2003). The oligomeric structure of human granzyme A is a determinant of its extended substrate specificity. Nat Struct Biol, 10, 527-534. PubMed id: 12819769 DOI: 10.1038/nsb944

Date:

12-Mar-03 Release date: 01-Jul-03

Protein chain

P12544  (GRAA_HUMAN) -  Granzyme A from Homo sapiens

Seq: 262 a.a.

Struc: 232 a.a.*

* PDB and UniProt seqs differ at 2 residue positions (black crosses)

Enzyme reactions

• Enzyme class: E.C.3.4.21.78 - granzyme A.
• Reaction: Hydrolysis of proteins, including fibronectin, type IV collagen and nucleolin. Preferential cleavage: Arg-|-Xaa, Lys-|-Xaa >> Phe-|-Xaa in small molecule substrates.

DOI no: 10.1038/nsb944

Nat Struct Biol 10:527-534 (2003)

PubMed id: 12819769

The oligomeric structure of human granzyme A is a determinant of its extended substrate specificity.

J.K.Bell, D.H.Goetz, S.Mahrus, J.L.Harris, R.J.Fletterick, C.S.Craik.

ABSTRACT

The cell death-inducing serine protease granzyme A (GzmA) has a unique disulfide-linked quaternary structure. The structure of human GzmA bound to a tripeptide CMK inhibitor, determined at a resolution of 2.4 A, reveals that the oligomeric state contributes to substrate selection by limiting access to the active site for potential macromolecular substrates and inhibitors. Unlike other serine proteases, tetrapeptide substrate preferences do not correlate well with natural substrate cleavage sequences. This suggests that the context of the cleavage sequence within a macromolecular substrate imposes another level of selection not observed with the peptide substrates. Modeling of inhibitors bound to the GzmA active site shows that the dimer also contributes to substrate specificity in a unique manner by extending the active-site cleft. The crystal structure, along with substrate library profiling and mutagenesis, has allowed us to identify and rationally manipulate key components involved in GzmA substrate specificity.

Selected figure(s)

Figure 1.
Figure 1. Overall structure of dimeric human granzyme A. (a) Ribbon diagram of dimeric human GzmA. The secondary structural elements are colored in a gradient from N to C terminus. The Ser195-His57-Asp102 catalytic triad and P1 coordinating Asp189 are shown in ball and stick (carbons, light green) as is the D-Phe-Pro-Arg-chloromethylketone (carbons, cyan). The disulfide linkage is depicted as space-filling model for residue 93 and its symmetry mate. A sulfate ion contributed from the crystallization solution is bound at the base of loop 184-B -197 by Arg186 and Arg188. (b) Surface representation mapped with potentials shows the overall positive charge of the molecule reflected by its pI > 9 and the distinct negative charge of Asp189 emanating from the S1 pocket.

Figure 3.
Figure 3. The active site of human granzyme A. (a) Ball-and-stick representation of the bound inhibitor, D-Phe-Pro-Arg-CMK (carbons, cyan) and residues that frame the substrate binding pocket depicted in the context of the molecular surface. The molecular surfaces of the proposed S1' and S2' subsites are colored in magenta; S1 subsite, orange; S2, blue; S3, red; S4, green. (b) Ligplot representation showing direct interactions between GzmA and the bound inhibitor. D-Phe-Pro-Arg-CMK bonds and carbons are cyan. Bonds between the irreversible inhibitor and GzmA are magenta. (c) Stereo view of the refined (2F[o] - F[c]) electron density for the CMK inhibitor (carbons, yellow) bound to the GzmA active site (carbons, gray).

The above figures are reprinted by permission from Macmillan Publishers Ltd: Nat Struct Biol (2003, 10, 527-534) copyright 2003.

Figures were selected by the author.