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

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.

Structural insights into the activation mechanisms of human HtrA serine proteases

“…The simulations demonstrated that upon binding of a specific peptide ligand (GWTMFWV) the PDZ domains rotate vs. PD by~30 -50 , opening the PD-PDZ interface in a lid-like (budding flower-like in trimer) fashion. Furthermore, the opening of the subunit interfaces progresses in a sequential manner [132]. These changes are consistent with the thermal induction model [123] though it is not possible to state if the extent of the PDZ rotation stimulated by the peptide binding is similar to the one caused by the temperature increase.…”

“…These changes are consistent with the thermal induction model [123] though it is not possible to state if the extent of the PDZ rotation stimulated by the peptide binding is similar to the one caused by the temperature increase. The MDS studies pointed also to the fact that the position of the L3 loop in the generated active HtrA2 is different from a canonical position present in any active HtrA protein, enabling L3 to make contact with the LD* loop [132]. This seems to be an important issue which needs clarification.…”

“…The latter included the peptide corresponding to the cytoplasmic C-terminal tail of presenilin-1 [125], the C-terminal The peptide-bound HtrA2 homotrimer was modeled as described previously [132]; it is based on the crystal structure of the PDZ domain in complex with a selectively bound peptide (i.e. GWTMFWV) (PDB ID: 2PDZ) [96] and is compatible with the HtrA2 crystal structure (PDB ID: 1LCY) [117].…”

Chiral Interface of Amyloid Beta (Aβ): Relevance to Protein Aging, Aggregation and Neurodegeneration

“…Most studied amyloid beta (Aβ) peptides includes Aβ (1-16), Aβ (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19), Aβ (20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33)(34), Aβ (20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33), Aβ , Aβ (23-34), Aβ (34)(35)(36)(37)…”

“…Coincidence of phosphorylation and racemization at Ser and Asp residues of Aβ (1-42). Beta (1-42) N- Terminal 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 C-Terminal Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val His His Gln Lys Leu Val Phe Phe Ala Glu Asp Val Gly Ser Asn Lys Gly Ala Ile Ile Gly Leu Met Val Gly Gly Val Val Ile Ala First, the pathological role of mitochondrial enzymes Ser proteases (SerPs) is attributed to neurodegenerative disorders such as AD and Parkinson's and disease [25][26][27]. The HtrA (high-temperature requirement) family represents a class of oligomeric SerPs [25].…”