Nuclear Overhauser effect studies of the conformations and binding site environments of deoxynucleoside triphosphate substrates bound to DNA polymerase I and its large fragment

Abstract

The conformations and binding site environments of Mg2+TTP and Mg2+dATP bound to Escherichia coli DNA polymerase I and its large (Klenow) fragment have been investigated by proton NMR. The effect of the large fragment of Pol I on the NMR line widths of the protons of Mg2+TTP detected one binding site for this substrate with a dissociation constant of 300 +/- 100 microM and established simple competitive binding of deoxynucleoside triphosphates at this site in accord with previous equilibrium dialysis experiments with whole Pol I [Englund, P. T., Huberman, J.A., Jovin, T.M., & Kornberg, A. (1969) J. Biol. Chem. 244, 3038]. Primary negative nuclear Overhauser effects were used to calculate interproton distances on enzyme-bound Mg2+dATP and Mg2+TTP. These distances established that each substrate was bound with an anti-glycosidic torsional angle (chi) of 50 +/- 10 degrees for Mg2+dATP and 40 +/- 10 degrees for Mg2+TTP. The sugar pucker of both substrates was predominantly O1'-endo, with a C5'-C4'-C3'-O3' exocyclic torsional angle (delta) of 95 +/- 10 degrees for Mg2+dATP and 100 +/- 10 degrees for Mg2+TTP. The consistency of these conformations with those previously proposed, on the basis of distances from Mn2+ at the active site [Sloan, D. L., Loeb, L. A., Mildvan, A.S., & Feldman, R.J. (1975) J. Biol. Chem. 250, 8913], indicates a unique conformation for each bound nucleotide. The chi and delta values of the bound substrates are appropriate for nucleotide units of B DNA.(ABSTRACT TRUNCATED AT 250 WORDS)

CORCEMA evaluation of the potential role of intermolecular transferred NOESY in the characterization of ligand-receptor complexes

“…This work has been motivated because of reports of intermolecular transferred NOE contacts observed experimentally (e.g., Refs. [15][16][17][18][19][20][21][22][23], as well as the realization by us [11,14] and *To whom correspondence should be addressed. A copy of the CORCEMA program may be obtained from this author.…”

“…When the exchange rate is very fast on the relaxation rate scale, the intensities become independent of the exchange rates, and the analysis of the intraand inter-TrNOESY is simpler [14]. The experimental observation of inter-TrNOESY contacts between a ligand and a large macromolecule by a number of investigators [15][16][17][18][19][20][21][22][23]32] establishes that a quantitative measurement of these intensities is indeed possible for small to moderate-sized complexes. Anglister and co-workers [21] have successfully identified interTrNOESY contacts between an antibody/antigen complex using difference-NOESY methods as well as a perdeuterated antibody.…”

Poliovirus RNA-Dependent RNA Polymerase (3Dpol): Pre-Steady-State Kinetic Analysis of Ribonucleotide Incorporation in the Presence of Mg2+

“…In the first reports on KF, it was suggested that the first conformational change may reflect reorientation of the triphosphate moiety of the incoming nucleotide to interact with side chains of the enzyme (28). This hypothesis was put forward to explain the observation made by Mildvan and colleagues that the orientation of the triphosphate and its interaction with divalent cation change in a KF ternary complex (38,39). Recently, it has been suggested that the conformational change defined kinetically may be related to changes in the orientation of the fingers subdomain observed crystallographically (12,40,41).…”

Remote Site Control of an Active Site Fidelity Checkpoint in a ViralRNA-dependent RNAPolymerase

“…Further complicating matters is the absence of a clear structural explanation for the conformational change observed kinetically. Structural explanations range from inter-and intra-domain rearrangements (10 -12) to triphosphate reorientation (1,7,(13)(14)(15)(16)(17).…”