PDBsum entry 2ddf

Hydrolase

PDB id: 2ddf

Contents

Protein chains

253 a.a. *

Ligands

INN ×2

IPA ×4

IMD

CIT

Metals

_ZN ×2

_CA

Waters ×333

* Residue conservation analysis

PDB id:

2ddf

Name:

Hydrolase

Title:

Crystal structure of tace in complex with tapi-2

Structure:

Adam 17. Chain: a, b. Synonym: a disintegrin and metalloproteinase domain 17, tnf-alpha- converting enzyme, tnf-alpha convertase, snake venom-like protease, cd156b antigen. Engineered: yes. Mutation: yes

Source:

Homo sapiens. Human. Organism_taxid: 9606. Gene: adam17, csvp, tace. Expressed in: trichoplusia ni. Expression_system_taxid: 7111

Resolution:

1.70Å R-factor: 0.204 R-free: 0.230

Authors:

P.Orth

Key ref:

R.N.Ingram et al. (2006). Stabilization of the autoproteolysis of TNF-alpha converting enzyme (TACE) results in a novel crystal form suitable for structure-based drug design studies. Protein Eng Des Sel, 19, 155-161. PubMed id: 16459338

Date:

28-Jan-06 Release date: 14-Mar-06

Protein chains

P78536  (ADA17_HUMAN) -  Disintegrin and metalloproteinase domain-containing protein 17 from Homo sapiens

Seq: 824 a.a.

Struc: 253 a.a.*

* PDB and UniProt seqs differ at 3 residue positions (black crosses)

Enzyme reactions

• Enzyme class: E.C.3.4.24.86 - Adam 17 endopeptidase.
• Cofactor: Zn(2+)

Protein Eng Des Sel 19:155-161 (2006)

PubMed id: 16459338

Stabilization of the autoproteolysis of TNF-alpha converting enzyme (TACE) results in a novel crystal form suitable for structure-based drug design studies.

R.N.Ingram, P.Orth, C.L.Strickland, H.V.Le, V.Madison, B.M.Beyer.

ABSTRACT

The crystallization of TNF-alpha converting enzyme (TACE) has been useful in understanding the structure-activity relationships of new chemical entities. However, the propensity of TACE to undergo autoproteolysis has made enzyme handling difficult and impeded the identification of inhibitor soakable crystal forms. The autoproteolysis of TACE was found to be specific (Y352-V353) and occurred within a flexible loop that is in close proximity to the P-side of the active site. The rate of autoproteolysis was found to be proportional to the concentration of TACE, suggesting a bimolecular reaction mechanism. A limited specificity study of the S(1)' subsite was conducted using surrogate peptides and suggested substitutions that would stabilize the proteolysis of the loop at positions Y352-V353. Two mutant proteases (V353G and V353S) were generated and proved to be highly resistant to autoproteolysis. The kinetics of the more resistant mutant (V353G) and wild-type TACE were compared and demonstrated virtually identical IC(50) values for a panel of competitive inhibitors. However, the k(cat)/K(m) of the mutant for a larger substrate (P6 - P(6)') was approximately 5-fold lower than that for the wild-type enzyme. Comparison of the complexed wild-type and mutant structures indicated a subtle shift in a peripheral P-side loop (comprising the mutation site) that may be involved in substrate binding/turnover and might explain the mild kinetic difference. The characterization of this stabilized form of TACE has yielded an enzyme with similar native kinetic properties and identified a novel crystal form that is suitable for inhibitor soaking and structure determination.