PDBsum entry 1klt

Serine protease

PDB id: 1klt

Contents

Protein chain

226 a.a.

Ligands

PMS

Waters ×663

References listed in PDB file

Key reference

Title

Crystal structure of phenylmethanesulfonyl fluoride-Treated human chymase at 1.9 a.

Authors

M.E.Mcgrath, T.Mirzadegan, B.F.Schmidt.

Ref.

Biochemistry, 1997, 36, 14318-14324. [DOI no: 10.1021/bi971403n]

PubMed id

9400368

Abstract

The X-ray crystal structure of human chymase has been determined to 1.9 A resolution using molecular replacement methods. This first structure of human chymase is present as the Ser 195 ester of alpha-toluenesulfonic acid. The refined structure (Rcryst = 0.183) shows that the inhibitor phenyl moiety lies at the top of the major specificity pocket, S1, while the sulfur is covalently linked to Ser 195-O gamma. The sulfonyl oxygens interact with the oxyanion hole and with His 57-N delta 1. The presence of the inhibitor disturbs the usual gauche position of His 57 and forces it to the trans conformer. Though the primary binding pockets are similarly specific in chymase and chymotrypsin, examination of the extended substrate binding sites reveals the structural basis for chymase's greater discrimination in choosing substrates. The larger 30s loop and its proximity to the active site indicates that it contacts substrate residues C-terminal to the scissile bond. Modeling of substrate at the chymase active site suggests that binding energy may be gained by three main-chain hydrogen bonds provided by substrate residues P2' and P4' and that discriminating interactions with substrate side chains are also likely. The presence of Lys 40 in S1' of human chymase explains its preference for Asp/Glu at P1'. Moreover, the cationic nature of S1' provides a structural basis for human chymase's poor catalytic efficiency when angiotensin II is the substrate.

Secondary reference #1

Title

Production of crystallizable human chymase from a bacillus subtilis system.

Authors

M.E.Mcgrath, A.E.Osawa, M.G.Barnes, J.M.Clark, K.D.Mortara, B.F.Schmidt.

Ref.

Febs Lett, 1997, 413, 486-488. [DOI no: 10.1016/S0014-5793(97)00962-9]

PubMed id

9303560

Figure 1.
Fig. 1. A schematic representation of the expression cassette used to produce human mast cell chymase C7S in B. subtilis. Expression is driven by the B. subtilis aprE promoter [28] shown as the thick black arrow. The chymase gene is represented by the white box and downstream is the transcription terminator from the B. amyloliquefaciens aprE gene [29] shown as a thick black line. The DNA and protein sequence of the aprE signal sequence (underlined) fused to the mature chymase gene is shown below the schematic figure. Amino acids 9–30 of the signal sequence are derived from the B. amyloliquefaciens aprE gene.

Figure 2.
Fig. 2. A silver-stained 4–20% SDS-PAGE gel showing chymase at various purification steps. Chymase is the major protein band seen in the lane containing the pool from the S-100 chromatography step.

The above figures are reproduced from the cited reference with permission from the Federation of European Biochemical Societies