Structure and ubiquitin interactions of the conserved zinc finger domain of Npl4 - PubMed (original) (raw)

A, surface plasmon resonance biosensor analysis of the Ub/Npl4 NZF interaction. Ubiquitin was injected in triplicate at concentrations of 0, 2.29, 4.38, 8.75, 17.5, 35.0, 70.0, 140, and 280 µ

m

over GST-Npl4 NZF captured on an anti-GST surface. The inset depicts the response obtained for 280 µ

m

ubiquitin injected over recombinant GST captured on an anti-GST surface (negative control). B, isotherms for Ub binding to the NZF domains from Npl4 and Vps36p. Fitting the data to simple 1:1 binding models yielded KD = 122 ± 2 µ

m

for Npl4 NZF (filled squares) and KD = 199 ± 17 µ

m

for Vps36p NZF (open squares). C, isotherms for Ub binding to wild type Npl4 NZF (filled squares, KD = 108 ± 13 µ

m

) and a series of single and double substitution mutants at Npl4 positions 13 and 14. F14V (open squares, KD = 2830 ± 30 µ

m

), T13L (filled diamonds, KD = 6940 ± 30 µ

m

), T13N (open diamonds, KD = 8450 ± 30 µ

m

), F14E (filled triangles, KD = 10 m

m

), T13L/F14V (open triangles, KD = 10 m

m

), and T13N/F14E (filled circles, KD = 10 m

m

).