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Structural and Functional Analysis of Human HtrA3 Protease and Its Subdomains.

Glaza P, Osipiuk J, Wenta T, Zurawa-Janicka D, Jarzab M, Lesner A, Banecki B, Skorko-Glonek J, Joachimiak A, Lipinska B - PLoS ONE (2015)

Bottom Line: The PDZ domain interacts closely with the LB loop of the protease domain in a way not found in other human HtrAs.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Faculty of Biology, University of Gdansk, 80-308 Gdansk, Poland.

ABSTRACT
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 dispensable 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.

No MeSH data available.


Related in: MedlinePlus

The key residues involved in HtrA3 trimer formation.(A) The trimer is stabilized by three phenylalanines in the PD of each monomer. (B) A “lock201D structure formed by F140, F142 and F255. The HtrA1 protein PD has an analogous trimer arrangement with F140 replaced by tyrosine. The chains A, B and C of the ΔN-HtrA3 trimer are shown in green, magenta and blue, respectively.
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pone.0131142.g004: The key residues involved in HtrA3 trimer formation.(A) The trimer is stabilized by three phenylalanines in the PD of each monomer. (B) A “lock201D structure formed by F140, F142 and F255. The HtrA1 protein PD has an analogous trimer arrangement with F140 replaced by tyrosine. The chains A, B and C of the ΔN-HtrA3 trimer are shown in green, magenta and blue, respectively.

Mentions: The structure of the ΔN-HtrA3S305A protein (Figs 1, 2 and 3) closely resembles the structures of two other human HtrA proteins: HtrA1 and HtrA2 [9] [11] [12]. The asymmetric unit of the ΔN-HtrA3 crystals contains three molecules of the monomeric protein which represents the trimeric HtrA3 biological assembly. The trimer is stabilized by the interaction of three phenylalanines (F140, F142, and F255) from each monomer, forming a “lock” between monomers similar to other HtrA proteins [9] (Fig 4). The only difference in the “lock” structure is the presence of HtrA3 F140 in place of a tyrosine in both HtrA1 and HtrA2. Chain A is the only molecule visible in full length in the ΔN-HtrA3 structure. It encompasses residues 135–459 with the exclusion of residues 163–168 and 277–289 of the loops LA and L3, respectively. The loops are named according to the chymotrypsin nomenclature (Fig 3) [54]. Two other chains encompass only PDs, residues 130–344 for chain B and 134–344 for chain C. Owing to the lack of electron density, the PDZ domains in the B and C chains are missing in the final structure, probably as a result of multiple domain positions inside the crystal. The PD-PDZ domain conformation observed in chain A could be one of several conformations for the chain B and must be excluded in the case of the chain C molecule due to the crystal packing. The chain C PDZ domain must have a different position than the one defined in chain A to avoid a collision with neighboring molecules inside the crystal and has to be moved to a vacant space (the ΔN-HtrA3S305A crystal solvent content is 55%). A similar situation was observed for the HtrA1S328A protein structure where only the PDs were modeled and all PDZ domains were undefined (PDB entry 3NUM) [9].


Structural and Functional Analysis of Human HtrA3 Protease and Its Subdomains.

Glaza P, Osipiuk J, Wenta T, Zurawa-Janicka D, Jarzab M, Lesner A, Banecki B, Skorko-Glonek J, Joachimiak A, Lipinska B - PLoS ONE (2015)

The key residues involved in HtrA3 trimer formation.(A) The trimer is stabilized by three phenylalanines in the PD of each monomer. (B) A “lock201D structure formed by F140, F142 and F255. The HtrA1 protein PD has an analogous trimer arrangement with F140 replaced by tyrosine. The chains A, B and C of the ΔN-HtrA3 trimer are shown in green, magenta and blue, respectively.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4481513&req=5

pone.0131142.g004: The key residues involved in HtrA3 trimer formation.(A) The trimer is stabilized by three phenylalanines in the PD of each monomer. (B) A “lock201D structure formed by F140, F142 and F255. The HtrA1 protein PD has an analogous trimer arrangement with F140 replaced by tyrosine. The chains A, B and C of the ΔN-HtrA3 trimer are shown in green, magenta and blue, respectively.
Mentions: The structure of the ΔN-HtrA3S305A protein (Figs 1, 2 and 3) closely resembles the structures of two other human HtrA proteins: HtrA1 and HtrA2 [9] [11] [12]. The asymmetric unit of the ΔN-HtrA3 crystals contains three molecules of the monomeric protein which represents the trimeric HtrA3 biological assembly. The trimer is stabilized by the interaction of three phenylalanines (F140, F142, and F255) from each monomer, forming a “lock” between monomers similar to other HtrA proteins [9] (Fig 4). The only difference in the “lock” structure is the presence of HtrA3 F140 in place of a tyrosine in both HtrA1 and HtrA2. Chain A is the only molecule visible in full length in the ΔN-HtrA3 structure. It encompasses residues 135–459 with the exclusion of residues 163–168 and 277–289 of the loops LA and L3, respectively. The loops are named according to the chymotrypsin nomenclature (Fig 3) [54]. Two other chains encompass only PDs, residues 130–344 for chain B and 134–344 for chain C. Owing to the lack of electron density, the PDZ domains in the B and C chains are missing in the final structure, probably as a result of multiple domain positions inside the crystal. The PD-PDZ domain conformation observed in chain A could be one of several conformations for the chain B and must be excluded in the case of the chain C molecule due to the crystal packing. The chain C PDZ domain must have a different position than the one defined in chain A to avoid a collision with neighboring molecules inside the crystal and has to be moved to a vacant space (the ΔN-HtrA3S305A crystal solvent content is 55%). A similar situation was observed for the HtrA1S328A protein structure where only the PDs were modeled and all PDZ domains were undefined (PDB entry 3NUM) [9].

Bottom Line: The PDZ domain interacts closely with the LB loop of the protease domain in a way not found in other human HtrAs.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Faculty of Biology, University of Gdansk, 80-308 Gdansk, Poland.

ABSTRACT
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 dispensable 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.

No MeSH data available.


Related in: MedlinePlus