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.
Highlights
An Escherichia coli (E. coli) high temperature requirement A (HtrA) plays a role as a molecular chaperone at temperatures below 30oC, and its proteolytic activity rapidly increases at tem peratures above 30oC (Lipinska et al, 1989)
Rapid purification of human high temperature requrement 2 (HtrA2) recombinant proteins expressed in E. coli as GST-fusion
To characterize the structure-activity relationship of HtrA2, we used to GST-HtrA2 133 WT, S306A, and F149D constructs (Figure 1A)
Summary
An Escherichia coli (E. coli) HtrA (high temperature requirement A, known as DegP) plays a role as a molecular chaperone at temperatures below 30oC, and its proteolytic activity rapidly increases at tem peratures above 30oC (Lipinska et al, 1989). A monomeric structure of mature HtrA2 (amino acid residues 134-458) consists of functionally defined structures, a trimerization motif (aa 146-151), a C-terminal PDZ domain (aa 364-445), and a central serine protease domain (aa 150-343) that contains the His-198, Asp 228, and Ser-306 catalytic triad in its conserved active site (Faccio et al, 2000; Gray et al, 2000; Suzuki et al, 2001; Li et al, 2002).
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