Molecular and structural analysis of a mechanical transition of helices in the L. donovani coronin coiled-coil domain.

Protein-protein interactions of cellular importance are mediated by coiled coils (CCs), the ubiquitous structural motif formed by the association of two or more α-helices in a knobs into holes manner. Coronins, actin-associated multi-functional proteins that possess distinct cytoskeleton-dependent and independent functions, oligomerize through their C-terminal CC domain. The structure of the L. donovani coronin CC domain (LdCoroCC; PDB ID 5CX2) revealed, in addition to a novel topology and architecture, an inherent asymmetry, with one of the helices of the 4-helix bundle axially shifted (~2 turns). The structural analysis identified that steric hindrance by Ile 486, Leu 493 and Met 500 as the cause for this asymmetry. To experimentally validate this hypothesis and to better understand the sequence-structure relationship in CCs, these amino acids have been mutated (I486A, L493A, M500V and the double mutant I486A-L493A) and characterized. Thermal CD studies suggest that the I486A and M500V mutants have comparable Tm values to LdCoroCC, while the other mutants have lower melting temperatures. The mutant crystal structures (I486A, M500V and the double mutant) retain the 'ade' core packing as LdcoroCC. While the M500V structure is similar to LdCoroCC, the I486A and the I486A-L493A structures show an asymmetry to symmetry transition. This study reveals crucial role of residues at position 'a' in coiled-coil domain play an important role in stabilizing the asymmetry in LdCoroCC, which might be necessary pursue specific biological function(s) inside the Leishmania.

Structure of Leishmania donovani coronin coiled coil domain reveals an antiparallel 4 helix bundle with inherent asymmetry.

Coiled coils are ubiquitous structural motifs that serve as a platform for protein-protein interactions and play a central role in myriad physiological processes. Though the formation of a coiled coil requires only the presence of suitably spaced hydrophobic residues, sequence specificities have also been associated with specific oligomeric states. RhXXhE is one such sequence motif, associated with parallel trimers, found in coronins and other proteins. Coronin, present in all eukaryotes, is an actin-associated protein involved in regulating actin turnover. Most eukaryotic coronins possess the RhXXhE trimerization motif. However, a unique feature of parasitic kinetoplastid coronin is that the positions of R and E are swapped within their coiled coil domain, but were still expected to form trimers. To understand the role of swapped motif in oligomeric specificity, we determined the X-ray crystal structure of Leishmania donovani coronin coiled coil domain (LdCoroCC) at 2.2Å, which surprisingly, reveals an anti-parallel tetramer assembly. Small angle X-ray scattering studies and chemical crosslinking confirm the tetramer in solution and is consistent with the oligomerization observed in the full length protein. Structural analyses reveal that LdCoroCC possesses an inherent asymmetry, in that one of the helices of the bundle is axially shifted with respect to the other three. The analysis also identifies steric reasons that cause this asymmetry. The bundle adapts an extended a-d-e core packing, the e residue being polar (with an exception) which results in a thermostable bundle with polar and apolar interfaces, unlike the existing a-d-e core antiparallel homotetramers with apolar core. Functional implications of the anti-parallel association in kinetoplastids are discussed.