Three-dimensional solution structure of the calcium-signaling protein Apo-S100Al as determined by NMR

S100A1, a member of the S100 protein family, is an EF-hand containing Ca2+-binding protein (93 residues per subunit) with noncovalent interactions at its dimer interface. Each subunit of S100A1 has four alpha-helices and a small antiparallel beta-sheet consistent with two helix-loop-helix calcium-binding domains [Baldiserri et at. (1999) J. Biomol. NMR 14, 87-88]. In this study, the three-dimensional structure of reduced apo-S100A1 was determined by NMR spectroscopy using a total of 2220 NOE distance constraints, 258 dihedral angle constraints, and 168 backbone hydrogen bond constraints derived from a series of 2D, 3D, and 4D NMR experiments. The final structure was found to be globular and compact with the four helices in each subunit aligning to form a unicornate-type four-helix bundle, Intermolecular NOE correlations were observed between residues in helices 1 and 4 from one Subunit to residues in helices 1' and 4' of the other subunit, respectively, consistent with the antiparallel alignment of the two subunits to form a symmetric X-type four-helix bundle as found for other members of the S 100 protein family. Because of the similarity of the S100A1 dimer interface to that round for S 10013, it was possible to calculate a model of the S100A1/B heterodimer. This model is consistent with a number of NMR chemical shift changes observed when S100A1 is titrated into a sample of N-15-labeled S100B. Helix 3 (and 3') of S100A1 was found to have an interhelical angle of -150degrees with helix 4 (and 4') in the apo state. This crossing angle is quite different (>50degrees) from that typically found in other EF-hand containing proteins such as apocalmodulin and apotroponin C but more similar to apo-S100B, which has an interhelical angle of -166degrees. As with S100B, it is likely that the second EF-hand of apo-S100A1 reorients dramatically upon the addition of Ca2+, which can explain the Ca2+ dependence that S100A1 has for binding several of its biological targets.