Structure And Function Of HtrA PDZ Domain From Streptococcs Pneumoniae And SAMPs From Haloferax Volcanii

This dissertation reports on the solution structure and function of HtrA PDZ domain from Streptococcs pneumpniae and SAMPs from Haloferax volcanii:(Ⅰ)High-temperature requirement A (HtrA), a highly conserved family of serine protease, plays crucial roles in protein quality control in prokaryotes and eukaryotes. The HtrA protein contains a C-terminal PDZ domain that mediates the proteolytic activity. Here we reported the solution structure of the HtrA PDZ domain from Streptococcus pneumoniae by NMR spectroscopy. Our results showed that the structure of HtrA PDZ domain, which contains threeα-helices and fiveβ-strands, illustrates conservation within the canonical PDZ domains. In addition, we demonstrated the interactions between S. pneumoniae HtrA PDZ domain and peptides with the motif XXX-YYF-COOH by surface plasmon resonance. Besides, we identified the ligand binding surface and the critical residues responsible for ligand binding of HtrA PDZ domain by chemical shift perturbation and site-directed mutagenesis. S. pneumoniae HtrA PDZ domain bound to the peptides via the groove formed byβ1 andα3. The residues located at the binding clefts interact with ligands mainly by hydrophobic; while the hydrogen bond might be also crucial for the ligand binding. The conserved structure and similar ligand recognition of S. pneumoniae HtrA PDZ domain in comparison to other HtrA PDZ domains might imply a similar activation mechanism for S. pneumoniae HtrA: ligands bind to the PDZ domain and induce conformational changes, which are then transmitted to the protease domain to degrade the substrate.(Ⅱ) Proteins can be modified through attachment to various small molecules and proteins. One such modification is conjugation to ubiquitin and ubiquitin-like proteins (Ubl). In eukaryotic cells, the conjugation of ubiquitin (Ub) and ubiquitin-like (Ubl) proteins to protein targets plays an integral role in a wide variety of processes, including proteasome-mediated proteolysis, heterochromatin remodelling and protein trafficking. In prokaryotes, the first example of a protein covalently attached to target proteins is Pup, which is distinct from ubiquitination in its use of deamidase and glutamine synthetase-like ligase reactions for conjugation and its disordered structure. Archaea, one of three major evolutionary lineages of life, encode proteasomes highly related to those of eukaryotes. In contrast, the presence of Ub-like protein conjugation systems in archaea is less clear. Recently, two ubiquitin-like proteins (SAMP1 and SAMP2) from Haloferax volcanii were reported. Here, we solved the solution structure of SAMPs (SAMP1 and SAMP2). In low salt concentrations, SAMP1 and SAMP2 each adopts two distinct conformations in solution. One is an ordered conformation which is similar to theβ-grasp fold conserved in ubiquitin-like proteins from eukaryotes. The other is a disordered conformation which resembles that of Pup. However, the disordered conformation is gradually disappearing with the increase of salt concentration; in contrast, the ordered conformation remained in high salt concentrations. By NMR chemical shift perturbation, SAMP1 was found to bind two proteasome-activating nucleotidase proteins, PanA and PanB. Thus, SAMP1 might associate with PanA/B to play a central role in the protein degradation pathway by proteasome.