What is the average conformation of bacteriophage T4 lysozyme in solution?A domain orientation study using dipolar couplings measured by solution NMR

Lysozyme from T4 bacteriophage is comprised of two domains that are both in volved in binding substrate. Although wild-type lysozyme has been exclusive ly crystallized in a closed form that is similar to the pepti-doglycan-boun d conformation, a more open structure is thought to be required for ligand binding. To determine the relative arrangement of domains within T4 lysozym e in the solution state, dipolar couplings were measured in several differe nt dilute Liquid crystalline media by solution NMR methods. The dipolar cou pling data were analyzed with a domain orientation procedure described prev iously that utilizes high-resolution X-ray structures. The cleft between th e domains is significantly larger in the average solution structure than wh at is observed in the X-ray structure of the ligand-free form of the protei n (similar to 17 degrees closure from solution to X-ray structures). A comp arison of the solution domain orientation with X-ray-derived structures in the protein data base shows that the solution structure resembles a crystal structure obtained for the M6I mutant. Dipolar couplings were also measure d on the lysozyme mutant T21C/T142C, which was oxidized to form an inter-do main disulfide bond (T4SS). Ln this case, the inter-domain solution structu re was found to be more closed than was observed in the crystal (similar to 11 degrees). Direct refinement of lysozyme crystal structures with the mea sured dipolar couplings using the program CNS, establishes that this degree of closure can be accommodated whilst maintaining the inter-domain cystine bond. The differences between the average solution conformations obtained using dipolar couplings and the crystal conformations for both forms of lys ozyme investigated in this study illustrate the impact that crystal packing interactions can have on the arrangement of domains within proteins and th e importance of alternative methods to X-ray crystallography for evaluating inter-domain structure. (C) 2001 Academic Press.