TY - JOUR

T1 - What is the average conformation of bacteriophage T4 lysozyme in solution? A domain orientation study using dipolar couplings measured by solution NMR

AU - Goto, NK

AU - Skrynnikov, NR

AU - Dahlquist, FW

AU - Kay, LE

PY - 2001/5/11

Y1 - 2001/5/11

N2 - Lysozyme from T4 bacteriophage is comprised of two domains that are both involved in binding substrate. Although wild-type lysozyme has been exclusively crystallized in a closed form that is similar to the pepti-doglycan-bound conformation, a more open structure is thought to be required for ligand binding. To determine the relative arrangement of domains within T4 lysozyme in the solution state, dipolar couplings were measured in several different dilute Liquid crystalline media by solution NMR methods. The dipolar coupling data were analyzed with a domain orientation procedure described previously that utilizes high-resolution X-ray structures. The cleft between the domains is significantly larger in the average solution structure than what is observed in the X-ray structure of the ligand-free form of the protein (similar to 17 degrees closure from solution to X-ray structures). A comparison 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 measured on the lysozyme mutant T21C/T142C, which was oxidized to form an inter-domain disulfide bond (T4SS). Ln this case, the inter-domain solution structure was found to be more closed than was observed in the crystal (similar to 11 degrees). Direct refinement of lysozyme crystal structures with the measured 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 lysozyme investigated in this study illustrate the impact that crystal packing interactions can have on the arrangement of domains within proteins and the importance of alternative methods to X-ray crystallography for evaluating inter-domain structure. (C) 2001 Academic Press.

AB - Lysozyme from T4 bacteriophage is comprised of two domains that are both involved in binding substrate. Although wild-type lysozyme has been exclusively crystallized in a closed form that is similar to the pepti-doglycan-bound conformation, a more open structure is thought to be required for ligand binding. To determine the relative arrangement of domains within T4 lysozyme in the solution state, dipolar couplings were measured in several different dilute Liquid crystalline media by solution NMR methods. The dipolar coupling data were analyzed with a domain orientation procedure described previously that utilizes high-resolution X-ray structures. The cleft between the domains is significantly larger in the average solution structure than what is observed in the X-ray structure of the ligand-free form of the protein (similar to 17 degrees closure from solution to X-ray structures). A comparison 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 measured on the lysozyme mutant T21C/T142C, which was oxidized to form an inter-domain disulfide bond (T4SS). Ln this case, the inter-domain solution structure was found to be more closed than was observed in the crystal (similar to 11 degrees). Direct refinement of lysozyme crystal structures with the measured 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 lysozyme investigated in this study illustrate the impact that crystal packing interactions can have on the arrangement of domains within proteins and the importance of alternative methods to X-ray crystallography for evaluating inter-domain structure. (C) 2001 Academic Press.

KW - T4 lysozyme

KW - domain orientation

KW - dipolar couplings

KW - NMR

KW - MALTODEXTRIN-BINDING-PROTEIN

KW - LIQUID-CRYSTALLINE MEDIUM

KW - LABELED SIDE-CHAINS

KW - HIGH-RESOLUTION NMR

KW - MOLECULAR-DYNAMICS

KW - INDUCED ALIGNMENT

KW - BIOLOGICAL MACROMOLECULES

KW - IMPROVED SENSITIVITY

KW - PHAGE-T4 LYSOZYME

KW - STRUCTURAL BASIS

U2 - 10.1006/jmbi.2001.4614

DO - 10.1006/jmbi.2001.4614

M3 - статья

VL - 308

SP - 745

EP - 764

JO - Journal of Molecular Biology

JF - Journal of Molecular Biology

SN - 0022-2836

IS - 4

ER -