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Title: Structural and functional analysis of human HtrA3 protease and its subdomains

Human HtrA3 protease, which induces mitochondria-mediated apoptosis, can be a tumor suppressor and a potential therapeutic target in the treatment of cancer. However, there is little information about its structure and biochemical properties. HtrA3 is composed of an N-terminal domain not required for proteolytic activity, a central serine protease domain and a C-terminal PDZ domain. HtrA3S, its short natural isoform, lacks the PDZ domain which is substituted by a stretch of 7 C-terminal amino acid residues, unique for this isoform. This paper presents the crystal structure of the HtrA3 protease domain together with the PDZ domain (ΔN-HtrA3), showing that the protein forms a trimer whose protease domains are similar to those of human HtrA1 and HtrA2. The ΔN-HtrA3 PDZ domains are placed in a position intermediate between that in the flat saucer-like HtrA1 SAXS structure and the compact pyramidal HtrA2 X-ray structure. The PDZ domain interacts closely with the LB loop of the protease domain in a way not found in other human HtrAs. ΔN-HtrA3 with the PDZ removed (ΔN-HtrA3-ΔPDZ) and an N-terminally truncated HtrA3S (ΔN-HtrA3S) were fully active at a wide range of temperatures and their substrate affinity was not impaired. This indicates that the PDZ domain is dispensablemore » for HtrA3 activity. As determined by size exclusion chromatography, ΔN-HtrA3 formed stable trimers while both ΔN-HtrA3-ΔPDZ and ΔN-HtrA3S were monomeric. This suggests that the presence of the PDZ domain, unlike in HtrA1 and HtrA2, influences HtrA3 trimer formation. The unique C-terminal sequence of ΔN-HtrA3S appeared to have little effect on activity and oligomerization. Additionally, we examined the cleavage specificity of ΔN-HtrA3. Results reported in this paper provide new insights into the structure and function of ΔN-HtrA3, which seems to have a unique combination of features among human HtrA proteases.« less
Authors:
 [1] ;  [2] ;  [1] ;  [1] ;  [1] ;  [1] ;  [3] ;  [1] ;  [2] ;  [1] ;  [4]
  1. Univ. of Gdansk (Poland). Dept. of Biochemistry.
  2. Argonne National Lab., Argonne, IL (United States). Midwest Center for Structural Genomics and Structural Biology Center.
  3. Univ. of Gdansk (Poland). Dept. of Molecular and Cellular Biology; Medical Univ. of Gdansk (Poland)
  4. Centro Nacional de Biotecnologia - CSIC (Spain)
Publication Date:
OSTI Identifier:
1212403
Grant/Contract Number:
AC02-06CH11357
Type:
Accepted Manuscript
Journal Name:
PLoS ONE
Additional Journal Information:
Journal Volume: 10; Journal Issue: 6; Journal ID: ISSN 1932-6203
Publisher:
Public Library of Science
Research Org:
Argonne National Laboratory (ANL), Argonne, IL (United States)
Sponsoring Org:
USDOE Office of Science (SC), Biological and Environmental Research (BER) (SC-23); National Institute of Health; National Science Center (Poland)
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES proteases; crystal structure; protein structure; peptides; oligomers; serine proteases; size-exclusion chromatography; X-ray crystallography