title: Changes in the quaternary structure and function of MjHSP16.5 attributable to deletion of the IXI motif and introduction of the substitution, R107G, in the α-crystallin domain.
abstract: The archael small heat-shock protein (sHSP), MjHSP16.5, forms a 24-subunit oligomer with octahedral symmetry. Here, we demonstrate that the IXI motif present in the C-terminal domain is necessary for the oligomerization of MjHSP16.5. Removal increased the in vitro chaperone activity with citrate synthase as the client protein. Less predictable were the effects of the R107G substitution in MjHSP16.5 because of the differences in the oligomerization of metazoan and non-metazoan sHSPs. We present the crystal structure for MjHSP16.5 R107G and compare this with an improved (2.5 Å) crystal structure for wild-type (WT) MjHSP16.5. Although no significant structural differences were found in the crystal, using cryo-electron microscopy, we identified two 24mer species with octahedral symmetry for the WT MjHSP16.5 both at room temperature and at 60°C, all showing two major species with the same diameter of 12.4 nm. Similarly, at room temperature, there are also two kinds of 12.4 nm oligomers for R107G MjHSP16.5, but in the 60°C sample, a larger 24mer species with a diameter of 13.6 nm was observed with significant changes in the fourfold symmetry axis and dimer-dimer interface. This highly conserved arginine, therefore, contributes to the quaternary organization of non-metazoan sHSP oligomers. Potentially, the R107G substitution has functional consequences as R107G MjHSP16.5 was far superior to the WT protein in protecting βL-crystallin against heat-induced aggregation.
authors.affiliation: Biophysical Sciences Institute, University of Durham, , South Road, Durham DH1 LE, UK.
authors.forename: Roy A
authors.initials: RA
authors.lastname: Quinlan
authors.forename: Yan
authors.initials: Y
authors.lastname: Zhang
authors.forename: Andrew
authors.initials: A
authors.lastname: Lansbury
authors.forename: Ian
authors.initials: I
authors.lastname: Williamson
authors.forename: Ehmke
authors.initials: E
authors.lastname: Pohl
authors.forename: Fei
authors.initials: F
authors.lastname: Sun
mesh_terms: D020816:Amino Acid Motifs; D001105:Archaea / Q000235:genetics / Q000378:metabolism*; D019843:Archaeal Proteins / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D017353:Gene Deletion; D019848:Gene Expression Regulation, Archaeal / Q000502:physiology; D006360:Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D038201:alpha-Crystallins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D019843:Archaeal Proteins; D006360:Heat-Shock Proteins; D038201:alpha-Crystallins