PDBsum entry: 1n6d

Hydrolase

PDB id: 1n6d

Contents

Protein chains

(+ 0 more) 1023 a.a. *

Ligands

ARG-VAL-ARG-LYS ×6

D10 ×6

CHM ×6

Waters ×534

* Residue conservation analysis

PDB id:

1n6d

Name:

Hydrolase

Title:

Tricorn protease in complex with tetrapeptide chloromethyl ketone derivative

Structure:

Tricorn protease. Chain: a, b, c, d, e, f. Engineered: yes. Rvrk. Chain: g, h, i, j, k, l. Engineered: yes

Source:

Thermoplasma acidophilum. Organism_taxid: 2303. Expressed in: escherichia coli bl21. Expression_system_taxid: 511693. Synthetic: yes. Other_details: this sequence occurs naturally in thermoplasma acidophilum

Biol. unit:

Dodecamer (from PQS)

Resolution:

2.80Å R-factor: 0.285 R-free: 0.315

Authors:

J.-S.Kim,M.Groll,R.Huber,H.Brandstetter

Key ref:

J.S.Kim et al. (2002). Navigation inside a protease: substrate selection and product exit in the tricorn protease from Thermoplasma acidophilum. J Mol Biol, 324, 1041-1050. PubMed id: 12470958 DOI: 10.1016/S0022-2836(02)01153-1

Date:

10-Nov-02 Release date: 30-Dec-02

Protein chains

P96086  (TRI_THEAC) -  Tricorn protease

Seq: 1071 a.a.

Struc: 1023 a.a.

 Gene Ontology (GO) functional annotation 

• Cellular component: cytoplasm (1 term)
• Biological process: proteolysis (1 term)
• Biochemical function: protein binding (6 terms)

DOI no: 10.1016/S0022-2836(02)01153-1

J Mol Biol 324:1041-1050 (2002)

PubMed id: 12470958

Navigation inside a protease: substrate selection and product exit in the tricorn protease from Thermoplasma acidophilum.

J.S.Kim, M.Groll, H.J.Musiol, R.Behrendt, M.Kaiser, L.Moroder, R.Huber, H.Brandstetter.

ABSTRACT

The proposed pathway and mechanism of substrate entry and product egress in the hexameric D3 symmetric tricorn protease from Thermoplasma acidophilum were explored by crystallographic studies of ligand complexes and by structure-based mutagenesis. Obstruction of the pore within the 7-bladed beta-propeller (beta7) domain by alkylation or oxidation of an engineered double cysteine mutant strongly decreased enzymatic activities. In line herewith, the crystal structure of the tricorn protease in complex with a trideca-peptide inhibitor modifying the catalytic Ser965 revealed part of the peptide trapped inside the channel of the beta7 domain. The cysteine mutation widening the lumen of the 6-bladed beta-propeller (beta6) domain enhanced catalytic activity, which was restored to normal values after its alkylation. A charge reversal mutant at the putative anchor site of the substrate C terminus, R131E-R132E, drastically reduced the proteolytic activity. The complex crystal structure of a peptide inhibitor with a diketo group at the cleavage site mapped the substrate recognition site and confirmed the role of Arg131-Arg132 as an anchor site. Our results strongly suggest the wider beta7 domain to serve as a selective filter and guide of the substrate to the sequestered active site, while the narrower beta6 domain routes the product to the surface. Moreover, we identified the role of Arg131-Arg132 in anchoring the substrate C terminus.

Selected figure(s)

Figure 1.
Figure 1. Ribbon representation of the overall hexameric structure of the tricorn protease viewed along the molecular 3-fold axis. Individual subunits are drawn in alternating colors. One subunit is colored in yellow, blue, purple, red, and dark-green for the b6, b7, C1, PDZ, and C2 subdomain from the N terminus. Figure 1, Figure 2, Figure 4 and Figure 5 were prepared using Molscript, [26.] GRASP, [27.] Povscript [28.] and rendered with Raster3D. [29.]

Figure 4.
Figure 4. Cut-open surface representation with the averaged electron density map of the bound inh3 between the active site and the Arg131-Arg132 anchoring segment at 2.7 Å. Tricorn segments are drawn as a ribbon diagram, side-chains of key residues at the active site (Ser965 and His746), the anchor segment (Arg131-Arg132), and the inhibitor as colored stick models. The conformation of the Arg132 side-chain as seen in the chloromethyl ketone-based inhibitor and inhibitor-free structure was drawn in red sticks and labeled with same color. The inhibitor is bound via the first carbonyl carbon of the diketo group to the Og of Ser965 as a tetrahedral adduct.

The above figures are reprinted by permission from Elsevier: J Mol Biol (2002, 324, 1041-1050) copyright 2002.

Figures were selected by an automated process.