Fig. 3: NMR titrations of Sla1 SH3 domains 1 and 2 with Las17 peptides.
A Schematic diagram of Las17. The PPR from residues 300-535 is subdivided into PPR-N (300-422) and PPR-C (423-535). The amino acid sequence is shown for PPR-N with three polyproline (PP) motifs indicated. Lines above the sequence are SH3 domain binding consensus sequences with green indicating type-I SH3 domain binding motifs, and blue type-II SH3 domain binding motifs. Underscored arginine residues in red text motifs have previously been identified as important in G-actin binding. Pink text indicates a predicted new ABS region. B A region of the HSQC spectrum from the titration of 15N-labelled Sla1 SH3 domains 1 and 2 with peptide 3 is shown. For clarity only the titrations with 0, 1, 2, 3, 5 and 8 peptide equivalents are shown (red, orange, yellow, green, blue, purple respectively). C Chemical shift changes in protein signals were fitted to standard equations (Williamson 2013) to estimate binding affinity. The estimated affinities clearly fell into two groups: signals from SH3#1 (top panel) fitted to a common affinity of 24 μM, while signals from SH3#2 (bottom panel) fitted to a common affinity of 190 μM. The signals shown are (top) 15N shifts for I38 (blue), W42 (red) and W41 (purple) (bottom) 1H shifts for W108 (blue), G124 (red) and N85 (purple). D Chemical shift changes on addition of each peptide (PP1, PP2, PP3) are shown as the weighted chemical shift changes for 1H and 15N, [ΔδH2 + (0.14ΔδN2)]1/2. The three peptides bind at similar locations, although peptide 1 binds approximately three times more weakly. The approximate affinities for PP1, PP2 and PP3 obtained from these data are respectively 70, 22 and 24 μM for SH3#1 and 550, 160 and 190 μM for SH3#2. E The binding site on the protein for peptide 3. Chemical shift changes on addition of peptide 3 were used to calculate the mean and standard deviation weighted shift change for each protein residue. Residues with shift changes larger than (mean  + sd) are indicated in orange for domain 1 and red for domain 2, and comprise V11, Y12, Y14, S36, I38, D39, W41 and W42 (domain 1), and D105, A106 and W108 (domain 2). The figure shows two views rotated by 180°.
