NMR studies on the interaction of sugars with the C-terminal domain of an R-type lectin from the earthworm Lumbricus terrestris.
The R-type lectin EW29, isolated from the earthworm Lumbricus terrestris, consists of two homologous domains (14,500 Da) showing 27% identity with each other. The C-terminal domain (Ch; C-half) of EW29 (EW29Ch) has two sugar-binding sites in subdomains alpha and gamma, and the protein uses these sugar-binding sites for its function as a single-domain-type hemagglutinin. In order to determine the sugar-binding ability and specificity for each of the two sugar-binding sites in EW29Ch, ligand-induced chemical-shift changes in EW29Ch were monitored using (1)H-(15)N HSQC spectra as a function of increasing concentrations of lactose, melibiose, D-galactose, methyl alpha-D-galactopyranoside and methyl beta-D-galactopyranoside. Shift perturbation patterns for well-resolved resonances confirmed that all of these sugars associated independently with the two sugar-binding sites of EW29Ch. NMR titration experiments showed that the sugar-binding site in subdomain alpha had a slow or intermediate exchange regime on the chemical-shift timescale (K(d) = 10(-2) to 10(-1) mM), whereas that in subdomain gamma had a fast exchange regime for these sugars (K(d) = 2-6 mM). Thus, our results suggest that the two sugar-binding sites of EW29Ch in the same molecule retain its hemagglutinating activity, but this activity is 10-fold lower than that of the whole protein because EW29Ch has two sugar-binding sites in the same molecule, one of which has a weak binding mode.
Hemmi
Hikaru
H
National Food Research Institute, National Agriculture and Food Research Organization (NARO), Tsukuba, Ibaraki, Japan. hemmi@affrc.go.jp
Kuno
Atsushi
A
Ito
Shigeyasu
S
Suzuki
Ryuichiro
R
Hasegawa
Tsunemi
T
Hirabayashi
Jun
J
D000818:Animals; D001665:Binding Sites; D004187:Disaccharides / Q000737:chemistry / Q000378:metabolism*; D005690:Galactose / Q000737:chemistry / Q000378:metabolism; D007785:Lactose / Q000737:chemistry / Q000378:metabolism; D037102:Lectins / Q000737:chemistry* / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D008553:Melibiose / Q000737:chemistry / Q000378:metabolism; D009835:Oligochaeta / Q000378:metabolism*; D017434:Protein Structure, Tertiary
D004187:Disaccharides; D037102:Lectins; D008553:Melibiose; D007785:Lactose; D005690:Galactose