Structural insight into the XTACC3/XMAP215 interaction from CD and NMR studies on model peptides

Abstract

TACC3 is a centrosomal adaptor protein that plays important roles during mitotic spindle assembly. It interacts with chTOG/XMAP215, which catalyzes the addition of tubulin dimers during microtubule growth. A 3D coiled-coil model for this interaction is available but the structural details are not well described. To characterize this interaction at atomic resolution, we have designed a simplified version of the system based on small peptides. Four different peptides have been studied by circular dichroism and nuclear magnetic resonance both singly and in all possible combinations; namely, five peptide pairs and two trios. In cosolvents, all single peptides tend to adopt helical conformations resembling those of the full-length protein. However, neither the single peptides nor pairs of peptides form coiled coils. We show that the simultaneous presence of all preformed helices is a prerequisite for binding. The simplest 3D model for the interaction, based on the NMR results, is proposed. Interestingly, the peptide's structure remains unaffected by mutations at essential positions for TACC3 activity. This suggests that the lack of interaction of this TACC3 mutant with XMAP does not correlate with changes in the protein structure and that specific interactions are likely responsible for the interaction and stability of the complex.

Switching the N-Capping Region from all-L to all-D Amino Acids in a VEGF Mimetic Helical Peptide

“…To estimate the helix population, the observed Hα chemical shift values were compared with the random coil values (ΔδHα = δHα observed − δHα random coil ). Then, the average ΔδHα values for all helical residues were divided by the ΔδHα value, corresponding to 100% helix (−0.39 ppm for the Hα [ 53 ] and multiplied by 100% [ 54 ].…”

Probing the helical stability in a VEGF-mimetic peptide

“…ΔδHα values from all the residues were averaged and the resulting mean divided by ΔδHα value corrisponding to 100% helix. [26] Values of H random-coil chemical shifts were taken from Wuthrich. [27]…”