title: Long-range effects and functional consequences of stabilizing mutations in the ankyrin repeat domain of IκBα.
abstract: Protein domains containing three or more ankyrin repeats (ARs) are ubiquitous in all phyla. Sequence alignments previously identified certain conserved positions, which have been shown to stabilize AR domains and promote their folding. Consensus mutations [Y254L/T257A (YLTA) and C186P/A220P (CPAP)] stabilize the naturally occuring AR domain of human IκBα to denaturation; however, only the YLTA mutations stabilize the protein to proteasomal degradation. We present results from NMR experiments designed to probe the roles of these consensus mutations in IκBα. According to residual dipolar coupling analysis, the gross structures of the AR domains of both mutants appear to be similar to the wild type (WT). Comparison of chemical shifts of mutant and WT proteins reveals that the YLTA and CPAP consensus mutations cause unexpected long-range effects throughout the AR domains. Backbone dynamics experiments reveal that the YLTA mutations in the sixth AR order the C-terminal PEST sequence on the picosecond-to-nanosecond timescale, compared to either the WT or the CPAP mutant IκBαs. This property is likely the mechanism by which the half-life of YLTA IκBα is extended in vivo.
authors.affiliation: Department of Chemistry and Biochemistry, University of California San Diego, 9500 Gilman Drive, La Jolla, CA 92037-0378, USA.
authors.forename: Carla F
authors.initials: CF
authors.lastname: Cervantes
authors.forename: Lindsey D
authors.initials: LD
authors.lastname: Handley
authors.forename: Shih-Che
authors.initials: SC
authors.lastname: Sue
authors.forename: H Jane
authors.initials: HJ
authors.lastname: Dyson
authors.forename: Elizabeth A
authors.initials: EA
authors.lastname: Komives
mesh_terms: D017089:Ankyrin Repeat; D006207:Half-Life; D006801:Humans; D020672:I-kappa B Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008279:Magnetic Resonance Imaging; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics*; D000072000:NF-KappaB Inhibitor alpha; D011485:Protein Binding; D017510:Protein Folding; D017434:Protein Structure, Tertiary
chemical_list: D020672:I-kappa B Proteins; C000606204:NFKBIA protein, human; D000072000:NF-KappaB Inhibitor alpha