Extended Data Fig. 7: Biochemical and structural characterization of the IST2/OSH6 interaction.
From: Structural basis for lipid transport at membrane contact sites by the IST2–OSH6 complex
a, Left, SDS-PAGE gel of pulldowns of OSH6 with fusion-constructs of peptides carrying the indicated region of IST2 fused to GST by binding to GSH resin (n = 1). Neg. refers to GST not containing any attached peptide. Right, intensity ratio of bands corresponding to OSH6 and the respective GST-IST2 fusion constructs. Dashed line indicates value of negative control (neg.). b, Comparison of size exclusion chromatography profiles of purified OSH6 expressed in S. cerevisiae and E. coli showing the same elution volume of the main protein peak. Inset shows SDS-PAGE gel of OSH6 purified from E. coli (n = 1). c, d, Binding of a peptide encompassing residues 732-761 of IST2 (IST2732-761, showing an independent biological replicate from data displayed in Fig. 3a) (c) or residues 732-756 (IST2732-756) (d) to OSH6. Binding isotherms were obtained from titrations of either peptide to OSH6. The data show fits to a model assuming a single binding site with Kd valued of 714.0 ± 86.7 nM (IST2732-761) and 1.1 ± 0.17 μM (IST2732-756). The dashed line in b refers to the fit of IST2732-761. Errors represent fitting errors. The thermograms are shown as inset (right). e, Superposition of OSH6 structures obtained in absence (cyan, PDBID 4B2Z) and in complex with IST2732-761 (red). The displayed region illustrates conformational differences in the lid helix α1 covering the lipid binding site. f, 2Fo-Fc density (grey mesh) superimposed on different regions of the OSH6/IST2 interface. g, Structural details of IST2/OSH6 interactions. h, Sequence alignment of OSH6 binding region of IST2 of three Saccheromyces species (S. paradoxus, S. kudriavzeii, S. cerevisiae). Residues in contact with OSH6 are colored in green. Numbering refers to the S. cerevisiae IST2. i, Sequence alignment of IST2 interactions of OSH6 and OSH7 of the same strains. Residues in contact with IST2 are colored in yellow. Numbering refers to the S. cerevisiae OSH6. j, Intra- and intermolecular interactions of the Arg 750 sidechain of IST2. k, Pulldown of OSH6 by a peptide of the IST2732-761 region carrying the point mutation R750A in comparison to WT. Experiments were caried out as described for a. Bars show mean of three technical replicates, errors are s.d. l, Binding of a peptide encompassing residues 732-761 of IST2 carrying the mutation R750A to OSH6. Binding isotherms were obtained from isothermal titrations of the peptide to OSH6. Dashed line refers to the fit of IST2732-761. The thermogram is shown as inset (right).
