Abstract
The N-terminal regions, which are highly variable in small heat-shock proteins, were found to be structurally disordered in all the 24 subunits of Methanococcus jannaschii Hsp16.5 oligomer and half of the 12 subunits of wheat Hsp16.9 oligomer. The structural and functional roles of the corresponding region (potentially disordered) in Mycobacterium tuberculosis Hsp16.3, existing as nonamers, were investigated in this work. The data demonstrate that the mutant Hsp16.3 protein with 35 N-terminal residues removed (DeltaN35) existed as trimers/dimers rather than as nonamers, failing to bind the hydrophobic probe (1,1'-bi(4-anilino)naphthalene-5,5'-disulfonic acid) and exhibiting no chaperone-like activity. Nevertheless, another mutant protein with the C-terminal extension (of nine residues) removed, although existing predominantly as dimers, exhibited efficient chaperone-like activity even at room temperatures, indicating that pre-existence as nonamers is not a prerequisite for its chaperone-like activity. Meanwhile, the mutant protein with both the N- and C-terminal ends removed fully exists as a dimer lacking any chaperone-like activity. Furthermore, the N-terminal region alone, either as a synthesized peptide or in fusion protein with glutathione S-transferase, was capable of interacting with denaturing proteins. These observations strongly suggest that the N-terminal region of Hsp16.3 is not only involved in self-oligomerization but also contains the critical site for substrate binding. Such a dual role for the N-terminal region would provide an effective mechanism for the small heat-shock protein to modulate its chaperone-like activity through oligomeric dissociation/reassociation. In addition, this study demonstrated that the wild-type protein was able to form heterononamers with DeltaN35 via subunit exchange at a subunit ratio of 2:1. This implies that the 35 N-terminal residues in three of the nine subunits in the wild-type nonamer are not needed for the assembly of nonamers from trimers and are thus probably structurally disordered.
Highlights
Small heat-shock proteins,1 as one subclass of molecular chaperones, have been found to exist in all types of
The Removal of the Potentially Disordered N-terminal 35 Residues of Hsp16.3 Had Little Effect on the Protein Secondary Structure—Sequence alignment of the whole sequences of Hsp16.3, Hsp16.5, and Hsp16.9 (Fig. 1A) revealed that the 35 N-terminal residues of Hsp16.3 corresponded to the N-terminal regions of Hsp16.9 and Hsp16.5, which are structurally disordered in half of the 12 subunits of the former and all 24 subunits of the latter [12, 18]
Removing the 35 N-terminal residues led to the formation of trimers/dimers lacking chaperone-like activity, suggesting a critical role of this region in the assembly of nonamers from trimers as well as in the exhibition of chaperone-like activity. This finding attributes a role for the N-terminal region of Hsp16.3 somehow similar to that of the ␣-crystallin as proposed by de Jong et al [42]
Summary
Small heat-shock proteins (sHSPs), as one subclass of molecular chaperones, have been found to exist in all types of. Small heat-shock proteins usually exist as oligomers (with 9 – 40 subunits) that undergo dynamic dissociation/reassociation, a structural feature that is apparently required for their chaperone-like activities (9 –15). Does the N-terminal region in Hsp16.3 help the protein to form its nonameric structure or/ and to exhibit chaperone-like activity? Previous studies have demonstrated that Hsp16.3: 1) exists as nonamers, which are assembled by using trimers and hexamers as intermediates [27, 31]; 2) undergo dynamic oligomeric dissociation/reassociation [13, 14]; and 3) exhibit chaperone-like activities that are modulated by adjusting the equilibrium and/or rate of the oligomeric dissociation process [13,14,15]
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
