Coiled-coil fusions are a useful approach to enforce dimerization in protein engineering. However, the final structures of coiled-coil fusion proteins have received relatively little attention. Here, we determine the structural outcome of adjacent parallel and antiparallel coiled coils. The targets are coiled coils that stabilize myosin-10 in single-molecule biophysical studies. We reveal the solution structure of a short, antiparallel, myosin-10 coiled-coil fused to the parallel GCN4-p1 coiled coil. Surprisingly, this structure is a continuous, antiparallel coiled coil where GCN4-p1 pairs with myosin-10 rather than itself. We also show that longer myosin-10 segments in these parallel/antiparallel fusions are dynamic and do not fold cooperatively. Our data resolve conflicting results on myosin-10 selection of actin filament bundles, demonstrating the importance of understanding coiled-coil orientation and stability.
Bibliographical noteFunding Information:
SAXS data collection was aided by Xiaobing Zuo and the staff at beamline 12-ID-B at the Advanced Photon Source, Argonne National Laboratory (GUP38797), supported by the U.S. Department of Energy under contract No. DE-AC02-06CH11357 . This work was supported by NIH R01s GM078450 and GM109863 (to R.S.R.) and NIH T32 GM007183 (to K.C.V.). Funding for NMR instrumentation was provided by the Office of the Vice President for Research , the Medical School , the College of Biological Science , NIH , NSF , and the Minnesota Medical Foundation . We acknowledge the University of Chicago Research Computing Center for support of this work.