2FV9

Crystal structure of TACE in complex with JMV 390-1

Summary for 2FV9

• Entry DOI: 10.2210/pdb2fv9/pdb
• Related: 1bkc 2ddf 2fv5
• Related PRD ID: PRD_000919
• Descriptor: ADAM 17, ZINC ION, N-{(2R)-2-[2-(hydroxyamino)-2-oxoethyl]-4-methylpentanoyl}-3-methyl-L-valyl-N-(2-aminoethyl)-L-alaninamide, ... (5 entities in total)
• Functional Keywords: tace adam33 zn-endopeptidase, hydrolase
• Biological source: Homo sapiens (human)
• Cellular location: Membrane; Single-pass type I membrane protein: P78536
• Total number of polymer chains: 2
• Total formula weight: 59243.07

Authors

Orth, P. (deposition date: 2006-01-30, release date: 2006-03-14, Last modification date: 2023-08-30)

Primary citation

Ingram, R.N.,Orth, P.,Strickland, C.L.,Le, H.V.,Madison, V.,Beyer, B.M.
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, 2006

PubMed 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.

PubMed: 16459338
DOI: 10.1093/protein/gzj014

Experimental method

X-RAY DIFFRACTION (2.02 Å)