Glycosylation Promotes the Random Coil to Helix Transition in a Region of a Protist Skp1 Associated with F-Box Binding

作者：Xu Xianzhong; Eletsky Alexander; Sheikh M. Osman; Prestegard James H.; West Christopher M.^*

来源：Biochemistry, 2018, 57(5): 511-515.

DOI：10.1021/acs.biochem.7b01033

Cullin-ring-ligases mediate protein polyubiquitination, a signal for degradation in the 26S proteasome. The CRL1 class consists of Skp1/cullin-1/F-box protein/Rbx1 (SCF) complexes that cyclically associate with ubiquitin-E2 to build the polyubiquitin chain. Within the SCF complex, the 162-amino acid DdSkp1 from Dictyostelium bridges culling with an F-box protein (FBP), the specificity factor for substrate selection. The hydroxylation-dependent glycosylation of Pro143 of DdSkp1 by a pentasaccharide forms the basis of a novel O-2-sensing mechanism in the social amoeba Dictyostelium and other protists. Previous evidence indicated that glycosylation promotes increased alpha-helical content correlating with enhanced interaction with three F-box proteins. To localize these differences, we used nuclear magnetic resonance (NMR) methods to compare nonglycosylated DdSkp1 and a glycoform with a single GlcNAc sugar (Gn-DdSkp1). We report NMR assignments of backbone H-1(N), N-15, C-13(alpha) and (CO)-C-13 nuclei as well as side-chain C-13(beta) and methyl C-13/H-1 nuclei of Ile(delta 1), Leu, and Val in both unmodified DdSkp1 and Gn-DdSkp1. The random coil index and N-15{H-1} HNOE indicate that the C-terminal region, which forms a helix-loop-helix motif centered on Pro143 at the crystallographically defined binding interface with F-box domains, remains dynamic in both DdSkp1 and Gn-DdSkp1. Chemical shifts indicate that the variation of conformation in Gn-DdSkp1, relative to DdSkp1, is limited to this region and characterized by increased helical fold. Extension of the glycan chain results in further changes, also limited to this region. Thus, glycosylation may control F-box protein interactions via a local effect on DdSkp1 conformation, by a mechanism that may be general to many unicellular eukaryotes.

• 出版日期2018-2-6