Fig. 1: The ARM domain of HSF2BP binds with a nanomolar affinity to a 52 aa BRCA2 peptide.
a Schematic depiction of the truncation and substitution variants of HSF2BP used in this study. Substitutions tested previously are mapped above the bar, whereas those tested in this study are indicated below the bar. Truncation variants are colored based on their ability to bind BRCA2 peptides. b Schematic depiction of BRCA2 fragments and variants used in the study. Full-length BRCA2 is shown at the top with key domains indicated. Location of the fragment F9 identified previously and its truncations tested here are shown, with colors indicating the ability to bind HSF2BP. Fragments produced as recombinant proteins are indicated with blue labels. c Coimmunoprecipitation of GFP-HSF2BP (full-length wild-type (WT) or R200C variant, and fragments H3 (ARM) and H4) and indicated Flag-tagged BRCA2 variants. Proteins were transiently produced in HEK293T cells. d NMR characterization of BRCA2 residues involved in binding to the Armadillo domain of HSF2BP (ARM). 2D 1H-15N HSQC spectra were recorded at 950 MHz and 283 K on the 15N-labeled BRCA2 fragment F0 (S2213-Q2342), either free (100 μM; dark blue) or in the presence of the unlabeled ARM domain (1:1 ratio; cyan). Ratios of peak intensities in the two conditions revealed that a set of peaks, corresponding to BRCA2 fragment FNMR (S2252-Q2342), decreased by more than 60% after the addition of ARM. The points and curve fragments in purple, green, and brown correspond to residues encoded by exon 12, 13, and 14 of BRCA2, respectively. e ITC curves that reveal how either HSF2BP or its ARM domain (in the instrument cell) interacts with the BRCA2 fragment F0, FNMR, and F15X (N2291-Q2342) (in the instrument syringe). The dissociation constants (Kd) are indicated. All experiments were duplicated, and the dissociation constants, stoichiometry, and thermodynamics parameters of each experiment are recapitulated in Table 1.
