PDBsum entry 5fht

Hydrolase

PDB id: 5fht

Contents

Protein chain

300 a.a.

Ligands

MES

Metals

_NA

_CL

__K

Waters ×200

PDB id:

5fht

Name:

Hydrolase

Title:

Htra2 protease mutant v226k

Structure:

Serine protease htra2, mitochondrial. Chain: a. Synonym: high temperature requirement protein a2,htra2,omi stress- regulated endoprotease,serine protease 25,serine proteinase omi. Engineered: yes

Source:

Homo sapiens. Human. Organism_taxid: 9606. Gene: htra2, omi, prss25. Expressed in: escherichia coli bl21(de3). Expression_system_taxid: 469008

Resolution:

1.95Å R-factor: 0.177 R-free: 0.228

Authors:

P.Golik,G.Dubin,D.Zurawa-Janicka,B.Lipinska,M.Jarzab,T.Wenta, A.Gieldon,A.Ciarkowski

Key ref:

A.Gieldon et al. (2016). Distinct 3D Architecture and Dynamics of the Human HtrA2(Omi) Protease and Its Mutated Variants. PLoS One, 11, e0161526. PubMed id: 27571206

Date:

22-Dec-15 Release date: 07-Sep-16

Protein chain

O43464  (HTRA2_HUMAN) -  Serine protease HTRA2, mitochondrial from Homo sapiens

Seq: 458 a.a.

Struc: 300 a.a.*

* PDB and UniProt seqs differ at 2 residue positions (black crosses)

Enzyme reactions

• Enzyme class: E.C.3.4.21.108 - HtrA2 peptidase.

PLoS One 11:e0161526 (2016)

PubMed id: 27571206

Distinct 3D Architecture and Dynamics of the Human HtrA2(Omi) Protease and Its Mutated Variants.

A.Gieldon, D.Zurawa-Janicka, M.Jarzab, T.Wenta, P.Golik, G.Dubin, B.Lipinska, J.Ciarkowski.

ABSTRACT

HtrA2(Omi) protease controls protein quality in mitochondria and plays a major role in apoptosis. Its HtrA2S306A mutant (with the catalytic serine routinely disabled for an X-ray study to avoid self-degradation) is a homotrimer whose subunits contain the serine protease domain (PD) and the regulatory PDZ domain. In the inactive state, a tight interdomain interface limits penetration of both PDZ-activating ligands and PD substrates into their respective target sites. We successfully crystalized HtrA2V226K/S306A, whose active counterpart HtrA2V226K has had higher proteolytic activity, suggesting higher propensity to opening the PD-PDZ interface than that of the wild type HtrA2. Yet, the crystal structure revealed the HtrA2V226K/S306A architecture typical of the inactive protein. To get a consistent interpretation of crystallographic data in the light of kinetic results, we employed molecular dynamics (MD). V325D inactivating mutant was used as a reference. Our simulations demonstrated that upon binding of a specific peptide ligand NH2-GWTMFWV-COOH, the PDZ domains open more dynamically in the wild type protease compared to the V226K mutant, whereas the movement is not observed in the V325D mutant. The movement relies on a PDZ vs. PD rotation which opens the PD-PDZ interface in a lid-like (budding flower-like in trimer) fashion. The noncovalent hinges A and B are provided by two clusters of interfacing residues, harboring V325D and V226K in the C- and N-terminal PD barrels, respectively. The opening of the subunit interfaces progresses in a sequential manner during the 50 ns MD simulation. In the systems without the ligand only minor PDZ shifts relative to PD are observed, but the interface does not open. Further activation-associated events, e.g. PDZ-L3 positional swap seen in any active HtrA protein (vs. HtrA2), were not observed. In summary, this study provides hints on the mechanism of activation of wtHtrA2, the dynamics of the inactive HtrA2V325D, but does not allow to explain an increased activity of HtrA2V226K.