De-novo modeling and ESR validation of a cyanobacterial FoF1–ATP synthase subunit bb′ left-handed coiled coil

The structure and functional role of the dimeric external stalk of FoF1–ATP synthases have been very actively researched over the last years. To understand the function, detailed knowledge of the structure and protein packing interactions in the dimer is required. In this paper we describe the application of structural prediction and molecular modeling approaches to elucidate the structural packing interaction of the cyanobacterial ATP synthase external stalk. In addition we present biophysical evidence derived from ESR spectroscopy and site directed spin labeling of stalk proteins that supports the proposed structural model. The use of the heterodimeric bb′ dimer from a cyanobacterial ATP synthase (Synechocystis sp. PCC 6803) allowed, by specific introduction of spin labels along each individual subunit, the evaluation of the overall tertiary structure of the subunits by calculating inter-spin distances. At defined positions in both b and b′ subunits, reporter groups were inserted to determine and confirm inter-subunit packing. The experiments showed that an approximately 100 residue long section of the cytoplasmic part of the bb′-dimer exists mostly as an elongated α-helix. The distant C-terminal end of the dimer, which is thought to interact with the δ-subunit, seemed to be disordered in experiments using soluble bb′ proteins. A left-handed coiled coil packing of the dimer suggested from structure prediction studies and shown to be feasible in molecular modeling experiments was used together with the measured inter-spin distances of the inserted reporter groups determined in ESR experiments to support the hypothesis that a significant portion of the bb′ structure exists as a left-handed coiled coil.