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

Abstract

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.

Three‐dimensional structure of the complexes of ribonuclease A with 2′,5′‐CpA and 3′,5′‐d(CpA) in aqueous solution, as obtained by NMR and restrained molecular dynamics

“…These results suggest that crystal packing may affect the conformation of the deoxyriboses in the RNase A/3',5'-d(CpA) complex. Similar conclusions have been obtained in other related complexes, such as the 3'-GMP/barnase complex (Meiering et al, 1993) and several mononucleotide/ RNase T1 complexes (Inagaki et al, 1985).…”

Structural Characterization of Protein Kinase A as a Function of Nucleotide Binding

“…In recent years, the approach of H-D exchange coupled with NMR analysis has revealed detailed site-specific information on bond formation and changes in bond energetics as a function of folding (41-51), conformational change (52), or ligand binding (53)(54)(55)(56)(57)(58)(59)(60). However, these methods are confined to a small (yet growing) subset of proteins that are amenable to NMR or crystallization and neutron diffraction analysis (61,62).…”

The role of glu73 of barnase in catalysis and the binding of barstar

“…This interaction was con®rmed in the structure of barnase complexed with 3 H -GMP (Meiering et al, 1993). The question is raised whether the difference in activity between the 73(bn) mutants is a result of a different af®nity for the substrate, or a result of differences in the general base activity (which would be seen as a difference in k cat ).…”

“…Instead, we chose to measure binding of 3 H -GMP to barnase using isothermal titration calorimetry (ITC; Figure 7). These measurements are signi®cant, since the end product of RNA degradation is 3 H -GMP, which retains many of the substrate contacts intact (Meiering et al, 1993). Both the enthalpy and the dissociation constant of binding are measured directly using ITC.…”