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. 2017 Jul 5;25(7):1011–1024.e4. doi: 10.1016/j.str.2017.05.005

Figure 2.

Figure 2

Mapping the HD-PTP Binding Interactions with SARA and Endofin

(A) Y2H assays between endofin(1–84) constructs or endofin(85–1,539) and HD-PTPFL or HD-PTPBro1-CC shows that binding is restricted to the endofin N terminus and that endofin L15 is important for binding. Data are from a representative experiment performed in triplicate.

(B) In vitro translated endofin1–84-strep, but not endofin(1–84/L15P)-strep, is co-immunoprecipitated with bacterially expressed His6-HD-PTPBro1-CC after an incubation with anti-His6 antibody; (i) is an example experiment, (ii) is quantitation from three independent experiments (mean ± SD).

(C) Biosensor binding isotherms for the HD-PTPCC and HD-PTPBro1-CC binding to endofin and SARA 22-mer peptides, used to determine the binding affinities (HD-PTPBro1 and HD-PTPBro1-CC binding to the endofin peptide have KD 3.1 ± 0.1 μM and 2.7 ± 0.1 μM, respectively, and HD-PTPBro1 has KD 11.3 ± 0.3 μM to SARA).

(D) Biosensor sensogram showing the lack of binding of the endofin peptide to AlixBro1.

(E and F) Sensograms showing that mutation of endofin L15 to proline abolishes binding to HD-PTPBro1 (E) and HD-PTPBro1-CC (F) compared with the control experiment using the wild-type endofin peptide.