Unique Residues Involved in Activation of the Multitasking Protease/Chaperone HtrA from Chlamydia trachomatis

Abstract

DegP, a member of the HtrA family of proteins, conducts critical bacterial protein quality control by both chaperone and proteolysis activities. The regulatory mechanisms controlling these two distinct activities, however, are unknown. DegP activation is known to involve a unique mechanism of allosteric binding, conformational changes and oligomer formation. We have uncovered a novel role for the residues at the PDZ1:protease interface in oligomer formation specifically for chaperone substrates of Chlamydia trachomatis HtrA (DegP homolog). We have demonstrated that CtHtrA proteolysis could be activated by allosteric binding and oligomer formation. The PDZ1 activator cleft was required for the activation and oligomer formation. However, unique to CtHtrA was the critical role for residues at the PDZ1:protease interface in oligomer formation when the activator was an in vitro chaperone substrate. Furthermore, a potential in vivo chaperone substrate, the major outer membrane protein (MOMP) from Chlamydia, was able to activate CtHtrA and induce oligomer formation. Therefore, we have revealed novel residues involved in the activation of CtHtrA which are likely to have important in vivo implications for outer membrane protein assembly.

Proteolytic activation of Chlamydia trachomatis HTRA is mediated by PDZ1 domain interactions with protease domain loops L3 and LC and beta strand β5

“…None of the activators could induce proteolytic activity in either the R362L or R224A mutant (Fig. 5A, B), while wild-type CtHtrA proteolysis was activated 2.7-fold in the presence of MOMP, 1.9-fold in the presence of Act1, 1.2-fold in the presence of Act2, 1.1-fold in the presence of OmpA, and 1.2-fold in the presence of PmpC, as previously observed [26]. Given the proteolytic inactivity of these mutants, we then tested whether this correlated with an oligomeric preference for inactive hexamers, but oligomerization assays showed that both mutants readily oligomerized to 24-mer in the presence of each activator (Fig.…”

“…The active site residues (His143, Asp173 and Ser247) are bordered with a red box, while the residues investigated in this study via mutagenesis are bordered with a black box. CtHtrA residue numbers are according to the full-length protein sequence as deposited at the National Centre for Biotechnology Information (NCBI; accession number: YP_001653297.1) [26]. EcHtrA residue numbers are according the standard system used for all published EcHtrA crystal structures [19], which begin at residue position 27 (underlined) in the full-length EcHtrA protein sequence (NCBI accession number: BAL37467.1).…”

“…Each mutation was confirmed by DNA sequencing prior to transformation into BL21 (DE3) cells for protein expression. Protein expression, purification and characterization Proteins were heterologously expressed in E. coli BL21 (DE3) cells, transformed with pET-22b-CtHtrA or SDM plasmids, and purified using affinity chromatography as previously reported [26]. Briefly, cells were grown at 37ºC in LB, and induced at A 600 of 0.6-0.8 with 0.1 mM isopropyl-β-D-1-thiogalactopyranoside (IPTG) for 5 h, before being harvested by centrifugation at 4,000 g for 20 min.…”

“…Assays were performed as previously described [26]. Briefly, 0.5-1.0 mg/ml CtHtrA samples in 0.1 M sodium phosphate buffer (pH 7.0) were incubated at 37ºC for 30 min.…”

“…Unlike DegP, DegS and MtHtrA2, the specific structural mechanism of activation for Chlamydia trachomatis HtrA (CtHtrA) has not yet been defined. We previously showed that CtHtrA can be allosterically activated by PDZ1-activating peptides (activators) and that this mechanism is potentially mediated by a distinct interaction between the protease and PDZ1 domains [26]. Here, we used a combination of homology modeling and in vitro analyses to identify specific residues and structural elements that contribute to this allosteric mechanism, furthering our understanding of the CtHtrA mode of activation.…”