Fig. 4

Systematic interaction analysis reveals binding mechanism of HJURP on Mis18^core. a–f Amylose-resin pull-down assays were performed using MBP-tagged M18BP1^1–60, M18BP1^61–140, HJURP^394–540, and HJURP^541–748 with different constructs of Mis18^core. Binding buffer B containing 15 mM HEPES pH 7.5, 150 mM NaCl, 1 mM TCEP, and 0.01% Tween-20 was used for the pull-down assays presented in panel b and f. Binding buffer A was used for other pull-down assays. g Amino-acid sequence alignment showing the N-terminal region of mammalian Mis18α. The sequence similarity among highly conserved regions in HJURP R1, R2 and the N-terminus of Mis18α is shown. Regions predicted to form α-helices are indicated by green bars. h–k Amylose-resin pull-down assays were performed to identify the residues of Mis18α that modulate the binding of HJURP to Mis18^core. l A table summarizing the pull-down results presented here and in Supplementary Fig. 4c. single plus and double plus indicate weak and strong interactions, respectively