Modulation of global low-frequency motions underlies allosteric regulation: demonstration in CRP/FNR family transcription factors.

PLoS Biol. 2013 Sep; 11(9): e1001651. Published online 2013 Sep 10. doi: 10.1371/journal.pbio.1001651 PMCID: PMC3769225 PMID: 24058293 Modulation of Global Low-Frequency Motions Underlies Allosteric Regulation: Demonstration in CRP/FNR Family Transcription Factors Thomas L. Rodgers,# 1 , 2 Philip D. Townsend,# 1 , 3 David Burnell, 1 , 2 Matthew L. Jones, 4 Shane A. Richards, 3 Tom C. B. McLeish, 1 , 2 , 4 Ehmke Pohl, 1 , 2 , 3 Mark R. Wilson, 1 , 2 and Martin J. Cann 1 , 3 , * Thomas L. Rodgers 1Biophysical Sciences Institute, Durham University, Durham, United Kingdom 2Department of Chemistry, Durham University, Durham, United Kingdom Find articles by Thomas L. Rodgers Philip D. Townsend 1Biophysical Sciences Institute, Durham University, Durham, United Kingdom 3School of Biological and Biomedical Sciences, Durham University, Durham, United Kingdom Find articles by Philip D. Townsend David Burnell 1Biophysical Sciences Institute, Durham University, Durham, United Kingdom 2Department of Chemistry, Durham University, Durham, United Kingdom Find articles by David Burnell Matthew L. Jones 4Department of Physics, Durham University, Durham, United Kingdom Find articles by Matthew L. Jones Shane A. Richards 3School of Biological and Biomedical Sciences, Durham University, Durham, United Kingdom Find articles by Shane A. Richards Tom C. B. McLeish 1Biophysical Sciences Institute, Durham University, Durham, United Kingdom 2Department of Chemistry, Durham University, Durham, United Kingdom 4Department of Physics, Durham University, Durham, United Kingdom Find articles by Tom C. B. McLeish Ehmke Pohl 1Biophysical Sciences Institute, Durham University, Durham, United Kingdom 2Department of Chemistry, Durham University, Durham, United Kingdom 3School of Biological and Biomedical Sciences, Durham University, Durham, United Kingdom Find articles by Ehmke Pohl Mark R. Wilson 1Biophysical Sciences Institute, Durham University, Durham, United Kingdom 2Department of Chemistry, Durham University, Durham, United Kingdom Find articles by Mark R. Wilson Martin J. Cann 1Biophysical Sciences Institute, Durham University, Durham, United Kingdom 3School of Biological and Biomedical Sciences, Durham University, Durham, United Kingdom Find articles by Martin J. Cann Daniel Herschlag, Academic Editor Author information Article notes Copyright and License information Disclaimer 1Biophysical Sciences Institute, Durham University, Durham, United Kingdom 2Department of Chemistry, Durham University, Durham, United Kingdom 3School of Biological and Biomedical Sciences, Durham University, Durham, United Kingdom 4Department of Physics, Durham University, Durham, United Kingdom Stanford University, United States of America #Contributed equally. * E-mail: [email protected] The authors have declared that no competing interests exist. The author(s) have made the following declarations about their contributions: Conceived and designed the experiments: TCBM EP MRW MJC. Performed the experiments: TLR PDT DB MLJ. Analyzed the data: TLR PDT SAR EP. Wrote the paper: TCBM MRW MJC. Received 2013 Jan 21; Accepted 2013 Jul 31. Copyright © 2013 Rodgers et al This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited. This article has been cited by other articles in PMC. Associated DataSupplementary Materials Figure S1: ENM representation of CAP. Alpha helices are represented in magenta and beta sheets in yellow. Blue spheres show the positions of the Cα atoms, and the black lines display the connectivity of the Hookean springs with a cutoff of 8 Å. Apo and singly bound ENMs were constructed by manually removing cAMP from the holoenzyme. (TIF) pbio.1001651.s001.tif (422K) GUID: B22D29BB-3A83-4C34-8A8A-16E198EFA9B8 Figure S2: Validation of ENM methodology. (A) CAP B-factors are independent of coarse-grained methodology. The chart represents the B-factor plotted against amino acid number for the crystal structure, ENM, and molecular dynamics. (B) Mode frequencies are independent of methodology. The chart represents the mode frequency plotted against mode number for ENM and molecular dynamics. (TIF) pbio.1001651.s002.tif (775K) GUID: 457A0D02-F2F2-4504-942C-0B089F23274F Figure S3: ENM predicted residue interactions that impact on cooperativity. (A) The change in cooperativity that occurs when k R/k is varied at the indicated residue (legend) against every amino acid within the same monomer (within an 8 Å cutoff). (B) The change in cooperativity that occurs when k R/k is varied at the indicated residue (legend) against every amino acid within the opposing monomer (within an 8 Å cutoff). (TIF) pbio.1001651.s003.tif (764K) GUID: 241EEFDA-95E6-4432-8C36-FD6EB98A40BC Figure S4: Least-squares superposition of one representative chain of each of the seven doubly cAMP-bound crystal structures treating the two domains (dimerization/cAMP-binding domain and DNA-binding domain) as rigid bodies with a flexible linker (wild-type, green; V132A, cyan; V132L, dark cyan; V140A, magenta; V140L, orange; H160L, red). The transformation matrices were obtained using RAPIDO [76]. (TIF) pbio.1001651.s004.tif (164K) GUID: 28742CAF-9FF5-4207-A6C6-BFE42F79C7CA Figure S5: Fitting of ITC data. Binding isotherm for a representative data set for the calorimetric titration of cAMP to wild-type CAP protein showing experimental data and fitted curves for two and three molecules of ligand cAMP. The inset shows the structure of CAP (green) with three bound molecules of cAMP (blue). (TIF) pbio.1001651.s005.tif (112K) GUID: 0784091A-4690-4CA4-8566-3774404E476F Figure S6: Calculated and observed values for cooperativity in CAP. (A) The ratio of the second to first dissociation constants for cAMP (K 2/K 1) for wild-type and mutant CAP proteins were calculated from the ENMs (calculated) or obtained by ITC (observed). The coloured lines correspond to the value for K 2/K 1 in the wild-type to enable comparison of the direction of change. (B) Values for K 2/K 1 obtained by ITC plotted against values for K 2/K 1 predicted by the ENM demonstrating the correlation between the extents of experimentally observed and predicted values for K 2/K 1. Dotted line represents the 95% confidence interval for the linear regression (R2 = 0.85). (TIF) pbio.1001651.s006.tif (654K) GUID: 5A742F08-0DF0-4F4E-9A8C-1E3AD24FD020 Figure S7: Mapping local dynamics in CAP. (A) The effect of mutation of V140 and H160 on local dynamics over the CAP monomer. The chart represents the percentage variation in B-factor from the wild-type ENM plotted against amino acid number. Inset shows the same chart with an expansion of the y-axis. (B) The chart is identical to that show (...truncated)