| EPR Spectroscopy Shows a Microtubule-Dependent Conformational Change in the Kinesin Switch 1 Domain Biophysical Journal, Volume 84, Issue 5, 1 May 2003, Pages 3190-3196 Nariman Naber, Sarah Rice, Marija Matuska, Ronald D. Vale, Roger Cooke and Edward Pate Abstract We have used site-directed spin-labeling and electron paramagnetic resonance spectroscopy to monitor a conformational change at the nucleotide site of kinesin. Cys-lite kinesin (K349 monomer) with the mutation S188C was spin labeled with MSL or MTSL. This residue is at the junction between the switch 1 region (which is a structure known to be sensitive to bound nucleotide in the G-proteins) and the 3-helix, adjacent to the nucleotide site. The spectra showed two or more components of mobility, which were independent of nucleotide in the absence of microtubules (MTs). The spectra of both labels showed a change of mobility upon binding to MTs. A more mobile spectral component became enhanced for all triphosphate analogs examined, AMPPNP, ADP•AlFx, or ADP•BeFx, in the presence of MTs, although the magnitude of the new component and the degree of mobility varied with nucleotide analog. The ADP state showed a much-reduced spectral change with a small shift to the more immobilized component in the presence of MTs. For kinesin•ADP•MT, a van’t Hoff plot gave Δ°=−96kJ/mol implying that the conformational change was extensive. We conclude there is a conformational change in the switch 1-3-helix domain when kinesin binds to MTs. Abstract | Full Text | PDF (232 kb) |
| Molecular Dynamics Study of the Energetic, Mechanistic, and Structural Implications of a Closed Phosphate Tube in ncd Biophysical Journal, Volume 80, Issue 3, 1 March 2001, Pages 1151-1168 Todd J. Minehardt, Roger Cooke, Edward Pate and Peter A. Kollman Abstract The switch 1 region of myosin forms a lid over the nucleotide phosphates as part of a structure known as the phosphate-tube. The homologous region in kinesin-family motors is more open, not interacting with the nucleotide. We used molecular dynamics (MD) simulations to examine a possible displacement of switch 1 of the microtubule motor, ncd, from the open conformation to the closed conformation seen in myosin. MD simulations were done of both the open and the closed conformations, with either MgADP or MgATP at the active site. All MD structures were stable at 300K for 500ps, implying that the open and closed conformers all represented local minima on a global free energy surface. Free energy calculations indicated that the open structure was energetically favored with MgADP at the active site, suggesting why only the open structure has been captured in crystallographic work. With MgATP, the closed and open structures had roughly equal energies. Simulated annealing MD showed the transformation from the closed phosphate-tube ncd structure to an open configuration. The MD simulations also showed that the coordination of switch 1 to the nucleotide dramatically affected the position of both the bound nucleotide and switch 2 and that a closed phosphate-tube may be necessary for catalysis. Abstract | Full Text | PDF (1671 kb) |
| The Structural Mechanism of Translocation and Helicase Activity in T7 RNA Polymerase Cell, Volume 116, Issue 3, 6 February 2004, Pages 393-404 Y.Whitney Yin and Thomas A Steitz Summary RNA polymerase functions like a molecular motor that can convert chemical energy into the work of strand separation and translocation along the DNA during transcription. The structures of phage T7 RNA polymerase in an elongation phase substrate complex that includes the incoming nucleoside triphosphate and a pretranslocation product complex that includes the product pyrophosphate (PP) are described here. These structures and the previously determined posttranslocation elongation complex demonstrate that two enzyme conformations exist during a cycle of single nucleotide addition. One orientation of a five-helix subdomain is stabilized by the phosphates of either the incoming NTP or by the product PP A second orientation of this subdomain is stable in their absence and is associated with translocation of the heteroduplex product as well as strand separation of the downstream DNA. We propose that the dissociation of the product PP after nucleotide addition produces the protein conformational change resulting in translocation and strand separation. Summary | Full Text | PDF (838 kb) |
Copyright © 1996 The Biophysical Society. All rights reserved.
Biophysical Journal, Volume 70, Issue 4, 1590-1602, 1 April 1996
doi:10.1016/S0006-3495(96)79745-X
Research Article
C.A. Smith and I. Rayment
Institute for Enzyme Research, University of Wisconsin, Madison 53705, USA.
The phosphate binding loop (P-loop) is a common feature of a large number of enzymes that bind nucleotide whose consensus sequence is often used as a fingerprint for identifying new members of this group. We review here the binding sites of nine purine nucleotide binding proteins, with a focus on their relationship to the active site of myosin. This demonstrates that there is considerable conversation in the distribution and nature of the ligands that coordinate the triphosphate moiety. This comparison further suggests that at least myosin and the G-proteins utilize a similar mechanism for nucleotide hydrolysis.