Structure and dynamics of barnase complexed with 3'-GMP studied by NMR spectroscopy.

The binding of 3'-GMP to the ribonuclease, barnase, has been studied using heteronuclear 2D and 3D NMR spectroscopy. The 1H and 15N NMR spectra of barnase complexed with 3'-GMP have been assigned. 2D and 3D NOESY spectra have been used to identify inter- and intramolecular NOEs, and a solution structure for the barnase-3'-GMP complex has been calculated. The position of the guanine ring of the ligand is reasonably well defined in the structures. The guanine ring forms hydrogen bonds with the NH protons of Ser57 and Arg59. These residues are located in a loop that is conserved among the microbial guanine-specific ribonucleases. The 2'-hydroxyl of 3'-GMP is close to His102 and Glu73, which have been shown to be involved in catalysis. The phosphate group of 3'-GMP is close to a number of positively charged residues that have also been shown to be important for activity. The position of the sugar moiety of 3'-GMP is less well defined in the structures. Structures calculated for the complex could not simultaneously satisfy all the observed intermolecular NOEs for the sugar protons, suggesting that the sugar samples several conformations when bound to barnase. The binding of 3'-GMP to barnase in solution is similar to that observed in the crystal structures of nucleotides bound to related ribonucleases. 3'-GMP binding causes no major conformational change in barnase. In contrast to the small structural changes that occur, there is a significant decrease in the rates of hydrogen/deuterium exchange and aromatic ring rotation in the active site of barnase upon ligand binding.