High-resolution solution NMR structure of the Z domain of staphylococcal protein A

Abstract

Staphylococcal protein A (SpA) is a cell-wall-bound pathogenicity factor from the bacterium Staphylococcus aureus. Because of their small size and immunoglobulin (IgG)-binding activities, domains of protein A are targets for protein engineering efforts and for the development of computational approaches for de novo protein folding. The NMR solution structure of an engineered IgG-binding domain of SpA, the Z domain (an analog of the B domain of SpA), has been determined by simulated annealing with restrained molecular dynamics on the basis of 671 conformational constraints. The Z domain contains three well-defined α-helices corresponding to polypeptide segments Lys7 to Leu17 (helix 1), Glu24 to Asp36 (helix 2), and Ser41 to Ala54 (helix 3). A family of ten conformers representing the solution structure of the Z domain was computed by simulated annealing of restrained molecular dynamics using the program CONGEN. The average of the root-mean-square deviations (r.m.s.d.) of the individual NMR conformers, relative to the mean coordinates, for the backbone atoms N, C α and C′ of residues Phe5 through Ala56 is 0.69 Å; the corresponding backbone r.m.s.d. for the three-helical core is 0.44 Å. Helices 1, 2 and 3 are antiparallel in orientation (Ω 12 = −170(±4)°, Ω 13 = +16(±3)°, Ω 23 = +173(±7)°). A comparison of backbone amide hydrogen/deuterium exchange rates in free and IgG-bound Z domains demonstrates that the amide protons of helices 1, 2 and 3 are protected from rapid exchange in both states, indicating that all three helices are also intact in the IgG-bound state. These solution NMR results differ from the previously determined X-ray structure of the similar SpA B domain in complex with the F c fragment of a human IgG antibody, where helix 3 is not observed in the electron density map and from the solution NMR structure of the B domain, where helix 3 is observed but helix 1 is tilted by ∼30° with respect to helices 2 and 3. Hydrogen-bonded N-cap and C-cap formation is observed for all three helices of the Z domain; these capping interactions appear to be highly conserved in the five homologous domains of SpA.