One of the CBI Molecules being studied in the University of Massachusetts Amherst Chemistry-Biology Interface Program at UMass Amherst and on display at the Molecular Playground
PcrH
Introduction
Pseudomonas aeruginosa is a bacterium widely found in the environment, nonetheless is capable of infecting patients with a weak immune system.
P. aeruginosa has been linked to more than 65% of cases of human infections that exhibit great resistance to antibiotics; moreover, is the predominant cause of mortality in cystic fibrosis patients. As many Gram-negative pathogens,
P. aeruginosa is armed with a sophisticated Type III Secretion System (T3SS) used to inject toxic proteins into human host cells. T3SS has been shown to have an important role in disease and pathogenicity of this bacterium. The T3SS is structured like a syringe that protrudes from the bacterial membrane towards the target cell membrane.
Model of
P. aeruginosa Type III secretion system. PcrH remains in the bacterial cytoplasm bound to PopB and PopD to keep them soluble and competent for secretion.
Within this system, two secreted proteins called PopB and PopD are found to insert into the target cell membrane and form a proteinaceous structure called the translocon that will connect with the syringe to serve as a gateway for the toxic proteins into the target cell cytoplasm.
[1] Substantial genetic and biochemical data indicate the translocon is composed by two T3S proteins, in the case of
Pseudomonas aeruginosa, the translocators protein are PopB and PopD
[1]. While they are in the bacterial cytoplasm they need to be maintained in a state competent for secretion and also not toxic for the bacterial cell, the system achieves this by employing a Type II chaperone called PcrH.
PrcH is a small (18.4 kDa) non-secreted bacterial co-chaperone that binds uniquely to these translocator proteins.
PcrH Structure
PcrH's characteristic fold is composed of Tetratricopeptide Repeats (TPR repeats). TPR repeats consist of a 34 amino-acid sequence motif that folds into two antiparallel alpha-helices that serve as interaction modules and multi-protein complex mediators.[2] In this case, PcrH from Pseudomonas aeruginosa consist of three . The first Tpr motif is represented in red, the second in green and the third in cyan color, also an additional alpha-helix is represented in blue. This fold is also present in Eukaryotic co-chaperons such as HOP(Heat shock protein Organizing Protein).
PcrH dimerization has been reported to be required for stability of PcrH in the absence of the cargo but not their function. But their dimerization interface is controversial. In the solved structure of PcrH, it showed a dimer with (each monomer is labeled in green and blue) [3]. The interaction only seems to happen at Y95, L98 and M99 (labeled in yellow) between helix 4 in the neighboring monomers. However, Tomalka et al [2]showed by mutagenesis that the dimer should be head-to-head where monomers interact via two N terminal helices (TPR1). This result resembles the solved structure of [4]which is the chaperone of translocators in Yersinia and shares 59.3% identity with PcrH. (each monomer is labeled in green and blue, mutation in residue A61 and L65 labeled in yellow has been shown to disrupt dimerization)
PcrH bound to PopB/D
PcrH crystal structure has also been solved carrying a homologous sequence that is present in these two translocators.[5] [3] PcrH has a (Histidine and Arginine residues shown in yellow) where it binds to a (VXLXXP) present in both PopB and PopD (9 residue peptide (rainbow) shown bound to Chain A). The VXLXXP sequence motif binding is displayed as three hydrophobic pockets in the concave side of the TPR domain receive three hydrophobic residues within the CBM present in the synthetic peptide. This interaction keeps them in a metastable non oligomeric state in the cytosol, thus protecting them from degradation and aggregation. It is also believed that PcrH has an active role in delivering PopB and PopD to the basal body of the T3S system, where an ATPse unfolds and secretes the translocators PopB and PopD.
Given its key role in molecular pathogenesis PcrH is a potential target for drug design.
