The homotrimeric structure of HtrA2 is indispensable for executing its serine protease activity.
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.
Nam
Min-Kyung
MK
Research Institute of Molecular Genetics, Catholic University, Seoul 137-701, Korea.
Seong
Young-Mo
YM
Park
Hyo-Jin
HJ
Choi
Ju-Youn
JY
Kang
Seongman
S
Rhim
Hyangshuk
H
D000409:Alanine / Q000378:metabolism; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D002460:Cell Line; D002850:Chromatography, Gel; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000235:genetics; D005982:Glutathione Transferase / Q000378:metabolism; D000074923:High-Temperature Requirement A Serine Peptidase 2; D006868:Hydrolysis; D024101:Mitochondrial Proteins; D008969:Molecular Sequence Data; D010649:Phenylalanine / Q000378:metabolism; D017354:Point Mutation; D011233:Precipitin Tests; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D012697:Serine Endopeptidases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D013329:Structure-Activity Relationship; D014162:Transfection
D024101:Mitochondrial Proteins; D011993:Recombinant Fusion Proteins; D010649:Phenylalanine; D005982:Glutathione Transferase; D012697:Serine Endopeptidases; D000074923:High-Temperature Requirement A Serine Peptidase 2; D000409:Alanine