Structural Basis Of Mycobacterium Tuberculosis Small Heat Shock Protein Hsp16.3 In Modulating Its Chaperone-like Activities

Molecular chaperones are predominantly involved in assisting newly synthesized protein folding and in preventing denaturing proteins from misfolding and aggregation. Small heat shock proteins (sHsps), as one sub-class of molecular chaperones, are known for the existence of a highly conserved 80-100 amino acids α-crystallin domain. In present dissertation, I am attempting to reveal the mechanism of Hsp16.3, one sHsps from the human pathogen Mycobacterium tuberculosis, in modulating its chaperone-like activities to suppress the aggregation of denaturing proteins, as shown by the following three sections.The oligomeric dissociation was previously revealed to be correlated with chaperone-like activities of sHsps. Here our further studies by chemical cross-linking showed that the oligomeric dissociation of Hsp16.3 is a prerequisite for its chaperone-like activities. Furthermore, we demonstrated that Hsp16.3 is able to modulate its chaperone-like activities by adjusting the rate of oligomeric dissociation while maintaining its static oligomeric size unchangeable. As a result, a kinetic model to describe the dynamic oligomeric dissociation and chaperone activities is proposed to explain some contradictory observations. In addition, Hsp16.3 was found to modulate its chaperone-like activities in a range of physiological temperatures (25°C-37.5°C), thus indicating that the protein is capable of binding non-native proteins in vivo. In an attempt to reveal the relation of oligomeric structure of Hsp16.3 with its primary structure, we found that the N-terminal region is a substrate-binding site and essential for chaperone-like activities. Remarkably, these regions in Hsp16.3 nine subunits, being essential as a whole for the nonameric assembly and important for stabilization of trimers, of which in at least three subunits are unessential for such an assembly. The C-terminal extension, especially the conserved IXI/V motif in this region, is also critical for the nonameric assembly of Hsp16.3, however, the dissociated oligomers with this motif truncated exhibits greatly enhanced chaperone activities. Gly59 in the conserved motif LPGV was found to be located in the subunit interface of Hsp16.3, of which the replacement by Trp results in the disappearance of nonamer and enhanced chaperone-like activities. In light of these observations, we propose that the nonamric structure of Hsp16.3 is not a prerequisite for its chaperone-like activities and the dissociated oligomers are active. The structural feature of molecular chaperone family was summarized as highly conformational flexibility and spontaneously folding/assembly, however, the mechanism is far from clear. The frequency of Cys and Trp in molecular chaperones is significantly less than that in other protein families. Further more, we for the first time presented evidence to show that disulfide bonds will convert Hsp16.3 from a chaperone to a non-chaperone, thus partially explaining the above intriguing evolution observation and implying that the conformational flexibility is important for molecular chaperones.