Crystal structure of a designed, thermostable; heterotrimeric coiled coil

Electrostatic interactions are often critical for determining the specifici ty of protein-protein complexes. To study the role of electrostatic interac tions for assembly of helical bundles, we previously designed a thermostabl e, heterotrimeric coiled coil, ABC, in which charged residues were employed to drive preferential association of three distinct, 34-residue helices. T o investigate the basis for heterotrimer specificity, we have used multiwav elength anomalous diffraction (MAD) analysis to determine the 1.8 Angstrom resolution crystal structure of ABC. The structure shows that ABC forms a h eterotrimeric coiled coil with the intended arrangement of parallel chains. Over half of the ion pairs engineered to restrict helix associations were apparent in the experimental electron density map. As seen in other trimeri c coiled coils, ABC displays acute knobs-into-holes packing and a buried an ion coordinated by core polar amino acids. These interactions validate the design strategy and illustrate how packing and polar contacts determine str uctural uniqueness.