Squire, CYoung, PBaker, ENYosaatmadja, Yuliana2018-08-062018http://hdl.handle.net/2292/37613In the last decade, two new intramolecular covalent bonds were discovered in elongated, single molecule wide surface proteins that markedly changed our view of protein stability. Intramolecular isopeptide bonds were first discovered in 2007 in a surface adhesin from the Gram-positive bacterium Streptococcus pyogenes, and form spontaneously between lysine and asparagine (or aspartate) side chains. Another twist on spontaneously-forming intramolecular crosslinks was revealed in 2014 and featured an ester bond formed between the side chains of threonine and glutamine. This was reported in the surface protein Cpe0147 of another Gram-positive bacterium, Clostridium perfringens, and was the focus of this PhD project. Just like the isopeptide bond, the ester crosslink forms spontaneously, in this case utilising a serine protease-like mechanism and a recognisable catalytic triad. However, unlike the serine protease mechanism where a water molecule attacks and hydrolyses the acyl intermediate to regenerate the active site, the ester bond in Cpe0147 is stable and does not react further. This earlier work by Kwon et al. using a single Cpe0147 domain, also showed that variants lacking the crosslink have little or no tertiary structure, suggesting an inherent instability without an ester bond in place. However, if the protein is so unstable without the ester bond, how do the reactive and accessory residues come together forming an intricate enzyme-like reactive site? To elucidate the mechanism by which this ester bond is formed in the Cpe0147 protein, and to further define the structural and chemical factors involved in this spontaneous reaction, we created a range of single- and three-domain Cpe0147 constructs, including split constructs enabling the bond formation and folding events to be captured. Using a combination of biophysical and biochemical techniques we explored the steric, chemical, and thermodynamic determinants of bond formation in Cpe0147. Site-directed mutagenesis experiments confirm a serine protease-like mechanism, whereby a histidine abstracts a proton from the bond forming threonine, and a stabilising aspartic acid plays a role equivalent to an oxyanion hole. Structural analysis of the wild-type Cpe0147 protein along with other ester domains from Mobiluncus mulieris, show that the accessory histidine is sequestered following ester bond formation, preventing ester bond hydrolysis. A conservative substitution of the bond forming threonine to a serine produces a hydrolysable variant at high pH, effectively completing the second half of the canonical serine protease mechanism. Wild-type protein at high pH and even in the presence of 6M urea, shows negligible hydrolysis. The stability of the wild-type protein, along with the presence of conserved water molecules near the ester bond in our structures, suggest that the hydrolysis reaction requires a subtle rearrangement of the solvent structure in the reactive site, and that the reaction uses an hydroxide ion rather than histidine as nucleophile. The rate of ester bond formation was followed via 2D NMR (HSQC) using a single-domain Cpe0147439-587 T450S variant. NMR analysis shows ester bond formation is tightly coupled to folding, where the protein transiently samples many conformations, with the correct conformation subsequently trapped by rapid ester bond formation. Biophysical methods including SEC-MALLS, SAXS and NMR, probed folding further and showed that all singledomain un-crosslinked Cpe0147 proteins (truncated and mutated) are unfolded or at least highly dynamic. However, when the same domain is attached to two intact domains, all three Cpe0147 domains appear folded, suggesting a cooperative folding mechanism with cross-talk between adjacent domains. Such cooperativity would explain folding and ester bond formation in the native secreted protein.Items in ResearchSpace are protected by copyright, with all rights reserved, unless otherwise indicated. Previously published items are made available in accordance with the copyright policy of the publisher.https://researchspace.auckland.ac.nz/docs/uoa-docs/rights.htmThesisCopyright: The authorhttp://purl.org/eprint/accessRights/OpenAccessQ111964069