Exp Mol Med.  2006 Feb;38(1):36-43.

The homotrimeric structure of HtrA2 is indispensable for executing its serine protease activity

• Nam MK
• Seong YM
• Park HJ
• Choi JY
• Kang S
• Rhim H

Abstract

Serine protease activity of high temperature requrement 2 (HtrA2) is essential for promoting cell death, as well as for protecting against cellular stresses. An X-ray crystallographic study described the formation of a pyramid shaped homotrimer that is a proteolytically competent form of HtrA2; however, little is known about effects of the trimeric structure of HtrA2 on the natural substrates. In this study, we generated the HtrA2 protein that has a single point mutation at the homotrimerization motif to assess relationship between structure and the proteolytic activity of HtrA2 on its substrates. Using gel filtration, a native gel electrophoresis system, and a co-precipitation assay, we confirm that phenylalanine 149 in HtrA2 is a crucial determinant for the formation of the HtrA2 homotrimeric structure. Moreover, we described that the HtrA2 monomeric form abolished not only autoproteolytic activity, but also the proteolytic activity against XIAP (X-linked inhibitor of apoptosis protein) known as the HtrA2 substrate. Taken together, the results indicate that the homotrimeric structure of HtrA2 is required for executing its serine protease activity.

Keyword

Omi serine protease; protein structure; tertiary; serine endopeptidases; structure-activity relationships; X-linked inhibitor of apoptosis protein

MeSH Terms

Alanine/metabolism
Amino Acid Motifs
Amino Acid Sequence
Amino Acid Substitution
Cell Line
Chromatography, Gel
Crystallography, X-Ray
Escherichia coli/genetics
Glutathione Transferase/metabolism
Hydrolysis
Molecular Sequence Data
Phenylalanine/metabolism
Point Mutation
Precipitin Tests
Protein Structure, Tertiary
Recombinant Fusion Proteins/metabolism
Research Support, Non-U.S. Gov't
Sequence Homology, Amino Acid
Serine Endopeptidases/*chemistry/genetics/isolation & purification/*metabolism
Structure-Activity Relationship
Transfection