UNUSUAL HELIX-CONTAINING GREEK KEYS IN DEVELOPMENT-SPECIFIC CA2+-BINDING PROTEIN-S - H-1, N-15, AND C-13 ASSIGNMENTS AND SECONDARY STRUCTURE DETERMINED WITH THE USE OF MULTIDIMENSIONAL DOUBLE AND TRIPLE-RESONANCE HETERONUCLEAR NMR-SPECTROSCOPY

Multidimensional heteronuclear NMR spectroscopy has been used to determine almost complete backbone and side-chain H-1, N-15, and C-13 resonance assignments of calcium loaded Myxococcus xanthus protein S (173 residues). Of the range of constant-time triple resonance experiments recorded, HNCACB and CBCA(CO)NH, which correlate C alpha and C beta with backbone amide resonances of the same and the succeeding residue respectively, proved particularly useful in resolving assignment ambiguities created by the 4-fold internal homology of the protein S amino acid sequence. Extensive side-chain H-1 and C-13 assignments have been obtained by analysis of HCCH-TOCSY and N-15-edited TOCSY-HMQC spectra. A combination of NOE, backbone amide proton exchange, (3)J(NH alpha), coupling constant, and chemical shift data has been used to show that each of the protein S repeat units consists of four beta-strands in a creek key arrangement. Two of the Greek keys contain a regular alpha-helix between the third and fourth strands, resulting in an unusual and possibly unique variation on this common folding motif. Despite similarity between two nine-residue stretches in the first and third domains of protein S and one of the Ca2+-binding sequences in bovine brain calmodulin [Inouye, S., Franceschini, T., and Inouye, M. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 6829-6833], the protein S topology in these regions is incompatible with an EF-hand calmodulin-type Ca2+-binding site.