Fig. 2
From: Evolution of AF6-RAS association and its implications in mixed-lineage leukemia
The αN helix is present in AF6 and augments its binding to RAS. a CSI versus residue number for the AF6 RA1 domain extended 31 residues at the N terminus (6–137). Four positive CSI values indicate α-helix; four negative values indicate β-strand. Resulting secondary structure arrangement depicted at top reveals the N-terminal α-helix (yellow). b Ribbons diagram depicting our homology model of the AF6 RA1 domain and αN helix (blue) complexed with RAS-GMPPNP (red-yellow) based on structural alignment with RASSF5-RAS (PDB 3DDC). Leu27 and Phe28, bottom of αN helix, contact switch II residues Met67, Tyr64, and Ile36 of RAS (marked, sticks). c Amino acid alignment of residues upstream of the core RA1 domain in seven evolutionarily conserved AF6 orthologues. A Leu-Phe motif in the loop between αN and β1 is completely conserved (boxed). d ITC analyses of the interactions between AF6 and RAS-GMPPNP. Core RA1 domain binds RAS with a Kd of 17.8 μM (black), an order of magnitude weaker than most RAS effector interactions. The αN-extended domain exhibits a 4.5-fold increase in affinity (red; Kd of 4.1 μM), while a L27D/F28D double mutant showed no heats of association (blue). e Experimental validation of the model by two NMR-based approaches. Left axis, normalized chemical shift perturbations induced in1H/15N-HSQC spectra of the AF6 RA1 domain by a purified AF6 fragment encompassing the αN helix (6–36). Right axis, combined chemical shift differences (Δp.p.m.) of backbone 1H/15N resonances from the core RA1 domain (37–136) vs extended RA1 domain that incorporates the αN helix (6–136). The αN helix in either experiment affects the same three regions (yellow), indicated in domain diagram at top
