Fig. 3.
Evolutionarily conserved features in Hip1R subfamily of ANTH domains. a Native MS was used to investigate PIP2 binding to several members of the ANTH family: ANTHSla2, ANTHSla2,mut (four residues to Ala in the canonical PIP2-binding site, see Constructs) and CALM from indicated species. Signal intensities from mass spectra of at least three independent measurements were summed over all charge states, normalized to the corrected signal of the unbound protein and the average of the relative signal intensities and their standard deviations were plotted (back). After correction for unspecific clustering, relative signals of unbound and PIP2-bound ANTH domains are obtained (front). Signal for proteins with three attached PIP2 molecules observed in raw spectra disappear after correction. b Composite model of the ENTH and ANTHSla2 complex based on the low-resolution EM structure14 and the high-resolution X-ray crystal structures presented here (ENTH2/PIP2, yellow, and superimposed ANTH domains of Sla2 (violet), AP180 (cyan), and CALM (pale cyan). Several elements contributing to the interface are highlighted, including the inserted α helix in the ANTHSla2 domain, the proximate Arg 37 of ANTHSla2 and Thr 104 from ENTH, and the location of PIP2 from the secondary site of the ENTH2/PIP2 crystal structure. c Growth defects of Sla2(1–360) strains mutated in Sla2/Hip1R ANTH-specific features. Tenfold serial dilutions of sla2Δ strains expressing Sla2(1–360) wt, Sla2(1–360) NHL with α8–α9 loop replaced by residues NHL as occurring in Yap1802, Sla2(1–360) dYL deletion of conserved Tyr 252 and Leu 253 and Sla2(1–360) R29A were incubated on SC-Ura plates at 30, 34, and 37 °C for 2 days