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[Preprint]. 2023 May 22:2023.05.22.541727. [Version 1] doi: 10.1101/2023.05.22.541727

Figure 2:

Figure 2:

Structure-guided mutagenesis reveals MSN residues involved in CD44 binding. (A) Schematic representation of the in vitro TR-FRET assay for MSN–CD44 interaction using purified components. Signal generated from Tb-linked anti-6HIS antibody bound to MSN is transferred to the FITC-conjugated CD44 peptide. (B) Inhibition of the TR-FRET signal using unlabelled CD44 peptide as a competitor (IC50 = 66 nM). (C) Sidechains of mutated MSN residues (L281 and H288) within the MSN–CD44 binding pocket are shown (green). (D) Comparison of MSN–CD44 TR-FRET response using varying levels of 6His-tagged WT or MSN mutant protein (0.01–0.29 nM). CD44 peptide concentration was kept constant (8 nM) (E) Competition of mutant MSN protein in MSN–CD44 TR-FRET assay. Dose response of untagged WT or mutant MSN protein in TR-FRET assay containing wild-type 6His-MSN (2 nM) and CD44 peptide (8 nM). IC50 values of competitor proteins are shown on the right (n=3). (F) Thermal shift assay, showing melting temperature (Tm) curves of WT and mutant proteins (left panel). The ΔTm, compared to WT protein, is shown in the right panel (n=3; ±SD).