HIGH-RESOLUTION SOLUTION NMR STRUCTURE OF THE Z-DOMAIN OF STAPHYLOCOCCAL PROTEIN-A

Staphylococcal protein A (SpA) is a cell-wall-bound pathogenicity fact or from the bacterium Staphylococcus aureus. Because of their small si ze and immunoglobulin (IgG)-binding activities, domains of protein A a re 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 (a n analog of the B domain of SpA), has been determined by simulated ann ealing with restrained molecular dynamics on the basis of 671 conforma tional constraints. The Z domain contains three well-defined alpha-hel ices corresponding to polypeptide segments Lys7 to Leu17 (helix 1), Gl u24 to Asp36 (helix 2), and Ser41 to Ala54 (helix 3). A family of ten conformers representing the solution structure of the Z domain was com puted by simulated annealing of restrained molecular dynamics using th e program CONGEN. The average of the root-mean-square deviations (r.m. s.d.) of the individual NMR conformers, relative to the mean coordinat es, for the backbone atoms N, C-alpha and C' of residues Phe5 through Ala56 is 0.69 Angstrom; the corresponding backbone r.m.s.d. for the th ree-helical core is 0.44 Angstrom. Helices 1, 2 and 3 are antiparallel in orientation (Ohm(12) = -170(+/-4)degrees, Ohm(13) = +16(+/-3)degre es, Ohm(23) = +173(+/-7)degrees). A comparison of backbone amide hydro gen/deuterium exchange rates in free and IgG-bound Z domains demonstra tes that the amide protons of helices 1, 2 and 3 are protected from ra pid exchange in both states, indicating that all three helices are als o intact in the IgG-bound state. These solution NMR results differ fro m the previously determined X-ray structure of the similar SpA B domai n in complex with the F-c fragment of a human IgG antibody, where heli x 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 i s tilted by similar to-30 degrees with respect to helices 2 and 3. Hyd rogen-bonded N-cap and C-cap formation is observed for all three helic es of the Z domain; these capping interactions appear to be highly con served in the five homologous domains of SpA. (C) 1997 Academic Press Limited.