A smallest 6 kda metalloprotease, mini-matrilysin, in living world: a revolutionary conserved zinc-dependent proteolytic domain- helix-loop-helix catalytic zinc binding domain (ZBD)

Journal of Biomedical Science

Research

Shuan-Su C Yu¹  Wei-Hsuan Yu¹  Chen Lin¹  Po-Tsang Huang²  Kuo-Long Lou³ 

[1] Institute of Biochemistry and Molecular Biology, College of Medicine, National Taiwan University, Ren-Ai Road, Taipei, Taiwan;Institute of Biochemistry and Molecular Biology, College of Medicine, National Taiwan University, Ren-Ai Road, Taipei, Taiwan;Graduate Institute of Oral Biology, College of Medicine, National Taiwan University, Ren-Ai Road, Taipei, Taiwan;Institute of Biochemistry and Molecular Biology, College of Medicine, National Taiwan University, Ren-Ai Road, Taipei, Taiwan;Graduate Institute of Oral Biology, College of Medicine, National Taiwan University, Ren-Ai Road, Taipei, Taiwan;NTU-DRCP Lectures and Core for Membrane Proteins, Center for Biotechnology, National Taiwan University, Chang Sing Street, Taipei, Taiwan;Institute of Biotechnology, National Taiwan University, Chang Sing Street, Taipei, Taiwan;

关键词: Matrilysin;    Zinc-dependent proteolytic domain;    Catalytic zinc binding domain;    Helix-loop-helix;    SC44463;   

DOI  :  10.1186/1423-0127-19-54

 received in 2012-03-14, accepted in 2012-05-29,  发布年份 2012

来源: Springer

【 摘 要 】

BackgroundThe Aim of this study is to study the minimum zinc dependent metalloprotease catalytic folding motif, helix B Met loop-helix C, with proteolytic catalytic activities in metzincin super family. The metzincin super family share a catalytic domain consisting of a twisted five-stranded β sheet and three long α helices (A, B and C). The catalytic zinc is at the bottom of the cleft and is ligated by three His residues in the consensus sequence motif, HEXXHXXGXXH, which is located in helix B and part of the adjacent Met turn region. An interesting question is - what is the minimum portion of the enzyme that still possesses catalytic and inhibitor recognition?”MethodsWe have expressed a 60-residue truncated form of matrilysin which retains only the helix B-Met turn-helix C region and deletes helix A and the five-stranded β sheet which form the upper portion of the active cleft. This is only 1/4 of the full catalytic domain. The E. coli derived 6 kDa MMP-7 ZBD fragments were purified and refolded. The proteolytic activities were analyzed by Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2 peptide assay and CM-transferrin zymography analysis. SC44463, BB94 and Phosphoramidon were computationally docked into the 3day structure of the human MMP7 ZBD and TAD and thermolysin using the docking program GOLD.ResultsThis minimal 6 kDa matrilysin has been refolded and shown to have proteolytic activity in the Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2 peptide assay. Triton X-100 and heparin are important factors in the refolding environment for this mini-enzyme matrilysin. This minienzyme has the proteolytic activity towards peptide substrate, but the hexamer and octamer of the mini MMP-7 complex demonstrates the CM-transferrin proteolytic activities in zymographic analysis. Peptide digestion is inhibited by SC44463, specific MMP7 inhibitors, but not phosphorimadon. Interestingly, the mini MMP-7 can be processed by autolysis and producing ~ 6 ~ 7 kDa fragments. Thus, many of the functions of the enzyme are retained indicating that the helix B-Met loop-helix C is the minimal functional “domain” found to date for the matrixin family.ConclusionsThe helix B-Met loop-helix C folding conserved in metalloprotease metzincin super family is able to facilitate proteolytic catalysis for specific substrate and inhibitor recognition. The autolysis processing and producing 6 kDa mini MMP-7 is the smallest metalloprotease in living world.

【 授权许可】

CC BY   
© Yu et al.; licensee BioMed Central Ltd. 2012