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Abstract

Naturally occurring coiled coils often deviate from the canonical model, which is characterized by a regular sequence pattern built from heptad repeats (abcdefgh) with hydrophobic residues in the core positions a and d. The occupation of single core positions of one or more consecutive heptads by polar residues is a rather common deviation from the standard. Such polar core residues locally destabilize the coiled coil structure, which in turn is often crucial for functionality, as has been shown for many examples. We have looked at the coiled-coil domains from various phage proteins and from trimeric autotransporter adhesins of Gram-negative bacteria. In these coiled coils, polar residues are rather frequent and found in up to 50 % of the core positions, in rare cases they even occupy

both core positions of a single repeat. Using bioinformatic analyses, we identified polar core motifs, such as the anion-binding motif N@d (hxxNxx) and a Ca2+ -binding motif with N@a, Q@d and D@e (NxxQDx), and characterized them biophysically and structurally. Analysis of both motifs in the GCN4 background showed that the coordination of ions in the core of trimeric coiled coils provides structural specificity. Furthermore, we designed a trimeric coiled

coil comprising a central segment of identical, consecutive heptads with exclusively polar core residues flanked by GCN4-pII adaptors. Starting from this idealized construct of high sequence symmetry, we created a second design more similar to naturally occurring sequences by increasing the sequence length, replacing individual residues, inserting specific salt bridges, and introducing a trigger motif. Finally, we shortened the flanking GCN4 adaptors to assess the impact of flanking canonical repeats on the folding propensity and stability of coiled-coil segments with polar cores. The structural and biophysical data obtained in this work provide a clear picture of the strategies used by trimeric coiled coils to accommodate polar side chains in their cores.