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Dynamic Allostery of the Catabolite Activator Protein Revealed by Interatomic Forces.

Louet M, Seifert C, Hensen U, Gräter F - PLoS Comput. Biol. (2015)

Bottom Line: We recover the experimentally observed entropic penalty for the second cAMP binding event with our recently developed force covariance entropy estimator and reveal allosteric communication pathways with Force Distribution Analyses (FDA).We identified crucial relays in the mostly symmetric allosteric activation network, and suggest point mutants to test this mechanism.Our study suggests inter-residue forces, as opposed to coordinates, as a highly sensitive measure for structural adaptations that, even though minute, can very effectively propagate allosteric signals.

View Article: PubMed Central - PubMed

Affiliation: Heidelberg Institutes for Theoretical Studies gGmbH, Heidelberg, Germany.

ABSTRACT
The Catabolite Activator Protein (CAP) is a showcase example for entropic allostery. For full activation and DNA binding, the homodimeric protein requires the binding of two cyclic AMP (cAMP) molecules in an anti-cooperative manner, the source of which appears to be largely of entropic nature according to previous experimental studies. We here study at atomic detail the allosteric regulation of CAP with Molecular dynamics (MD) simulations. We recover the experimentally observed entropic penalty for the second cAMP binding event with our recently developed force covariance entropy estimator and reveal allosteric communication pathways with Force Distribution Analyses (FDA). Our observations show that CAP binding results in characteristic changes in the interaction pathways connecting the two cAMP allosteric binding sites with each other, as well as with the DNA binding domains. We identified crucial relays in the mostly symmetric allosteric activation network, and suggest point mutants to test this mechanism. Our study suggests inter-residue forces, as opposed to coordinates, as a highly sensitive measure for structural adaptations that, even though minute, can very effectively propagate allosteric signals.

No MeSH data available.


Related in: MedlinePlus

Global motions of CAP.(A) Estimated entropic contributions-TΔS to the binding free energy of the first (red) or the second (blue) cAMP binding event, and the overall entropy change for the binding of both cAMP (purple). Estimates from force covariance (FC) and quasi-harmonic (QH) analyses of either protein main chain (“MC”) or the full protein including DBD (“full”). For comparison, NMR-based estimates (“NMR”) are given for the entropy change of the first and second binding event of a truncated CAP construct (“CBD”) without DBD [8] and, respectively, for the binding of both cAMP to the full protein (“full”) [23]. (B) Functional motion of CAP for DNA binding as sampled in MD simulations. Projection of CAP X-ray structures and all simulation data from apo (black), cap1 (blue) and cap2 (orange) states on the first eigenvector obtained from a PCA of available 2 cAMP-bound X-ray structures, either solved in absence of DNA (1–6: 1GN6 [9], 1HW5 [27], and 1I6X, 3RDI, 3ROU, 1I5Z –all unpublished) or in presence of DNA (9–11: 1RUO [28], 1RUN [28] and 1CGP [26]). The two intermediate structures (PDB ids: 3QOP (unpublished) and 3KCC [29]) are not bound to DNA but to two cAMP molecules, localized in between the DBD and β-strand 5, triggering a rotation of the DBD.
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pcbi.1004358.g002: Global motions of CAP.(A) Estimated entropic contributions-TΔS to the binding free energy of the first (red) or the second (blue) cAMP binding event, and the overall entropy change for the binding of both cAMP (purple). Estimates from force covariance (FC) and quasi-harmonic (QH) analyses of either protein main chain (“MC”) or the full protein including DBD (“full”). For comparison, NMR-based estimates (“NMR”) are given for the entropy change of the first and second binding event of a truncated CAP construct (“CBD”) without DBD [8] and, respectively, for the binding of both cAMP to the full protein (“full”) [23]. (B) Functional motion of CAP for DNA binding as sampled in MD simulations. Projection of CAP X-ray structures and all simulation data from apo (black), cap1 (blue) and cap2 (orange) states on the first eigenvector obtained from a PCA of available 2 cAMP-bound X-ray structures, either solved in absence of DNA (1–6: 1GN6 [9], 1HW5 [27], and 1I6X, 3RDI, 3ROU, 1I5Z –all unpublished) or in presence of DNA (9–11: 1RUO [28], 1RUN [28] and 1CGP [26]). The two intermediate structures (PDB ids: 3QOP (unpublished) and 3KCC [29]) are not bound to DNA but to two cAMP molecules, localized in between the DBD and β-strand 5, triggering a rotation of the DBD.

Mentions: As a final sanity check of our models, we also compared the experimentally and computationally observed motions of the DBD involved in DNA binding. To this end, we projected our simulations on the first eigenvector derived from 11 X-ray structures, all of which possessed two cAMP, but for which the DNA could be absent or present. This eigenvector corresponds to the previously described large-scale DNA binding motion [9,26], and involves an almost rigid rotation of the DBD by ~25 degrees. X-ray structures with bound DNA clearly separated from structures not coupled to DNA along this eigenvector, with only two intermediate conformations (which show non-canonical cAMP binding). Remarkably, in spite of the limited time scale of our MD simulations, projections of conformations explored during the MD simulations and experimentally resolved structures [9,26–29], on this particular eigenvector showed that the cap2 state is able to follow this motion further than the apo and cap1 states and even overlaps with DNA-bound X-ray structures (Fig 2B). This suggests that even though we started from the same active cap2 structure, the sampled conformational space partially diverged during the unbiased MD simulations along directions in agreement with experiments. We note that even, the cap2 ensemble preferentially populates a region to the left of the experimental structures, a diversion which however, is minor, and could be due to crystal packing effects.


Dynamic Allostery of the Catabolite Activator Protein Revealed by Interatomic Forces.

Louet M, Seifert C, Hensen U, Gräter F - PLoS Comput. Biol. (2015)

Global motions of CAP.(A) Estimated entropic contributions-TΔS to the binding free energy of the first (red) or the second (blue) cAMP binding event, and the overall entropy change for the binding of both cAMP (purple). Estimates from force covariance (FC) and quasi-harmonic (QH) analyses of either protein main chain (“MC”) or the full protein including DBD (“full”). For comparison, NMR-based estimates (“NMR”) are given for the entropy change of the first and second binding event of a truncated CAP construct (“CBD”) without DBD [8] and, respectively, for the binding of both cAMP to the full protein (“full”) [23]. (B) Functional motion of CAP for DNA binding as sampled in MD simulations. Projection of CAP X-ray structures and all simulation data from apo (black), cap1 (blue) and cap2 (orange) states on the first eigenvector obtained from a PCA of available 2 cAMP-bound X-ray structures, either solved in absence of DNA (1–6: 1GN6 [9], 1HW5 [27], and 1I6X, 3RDI, 3ROU, 1I5Z –all unpublished) or in presence of DNA (9–11: 1RUO [28], 1RUN [28] and 1CGP [26]). The two intermediate structures (PDB ids: 3QOP (unpublished) and 3KCC [29]) are not bound to DNA but to two cAMP molecules, localized in between the DBD and β-strand 5, triggering a rotation of the DBD.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4526232&req=5

pcbi.1004358.g002: Global motions of CAP.(A) Estimated entropic contributions-TΔS to the binding free energy of the first (red) or the second (blue) cAMP binding event, and the overall entropy change for the binding of both cAMP (purple). Estimates from force covariance (FC) and quasi-harmonic (QH) analyses of either protein main chain (“MC”) or the full protein including DBD (“full”). For comparison, NMR-based estimates (“NMR”) are given for the entropy change of the first and second binding event of a truncated CAP construct (“CBD”) without DBD [8] and, respectively, for the binding of both cAMP to the full protein (“full”) [23]. (B) Functional motion of CAP for DNA binding as sampled in MD simulations. Projection of CAP X-ray structures and all simulation data from apo (black), cap1 (blue) and cap2 (orange) states on the first eigenvector obtained from a PCA of available 2 cAMP-bound X-ray structures, either solved in absence of DNA (1–6: 1GN6 [9], 1HW5 [27], and 1I6X, 3RDI, 3ROU, 1I5Z –all unpublished) or in presence of DNA (9–11: 1RUO [28], 1RUN [28] and 1CGP [26]). The two intermediate structures (PDB ids: 3QOP (unpublished) and 3KCC [29]) are not bound to DNA but to two cAMP molecules, localized in between the DBD and β-strand 5, triggering a rotation of the DBD.
Mentions: As a final sanity check of our models, we also compared the experimentally and computationally observed motions of the DBD involved in DNA binding. To this end, we projected our simulations on the first eigenvector derived from 11 X-ray structures, all of which possessed two cAMP, but for which the DNA could be absent or present. This eigenvector corresponds to the previously described large-scale DNA binding motion [9,26], and involves an almost rigid rotation of the DBD by ~25 degrees. X-ray structures with bound DNA clearly separated from structures not coupled to DNA along this eigenvector, with only two intermediate conformations (which show non-canonical cAMP binding). Remarkably, in spite of the limited time scale of our MD simulations, projections of conformations explored during the MD simulations and experimentally resolved structures [9,26–29], on this particular eigenvector showed that the cap2 state is able to follow this motion further than the apo and cap1 states and even overlaps with DNA-bound X-ray structures (Fig 2B). This suggests that even though we started from the same active cap2 structure, the sampled conformational space partially diverged during the unbiased MD simulations along directions in agreement with experiments. We note that even, the cap2 ensemble preferentially populates a region to the left of the experimental structures, a diversion which however, is minor, and could be due to crystal packing effects.

Bottom Line: We recover the experimentally observed entropic penalty for the second cAMP binding event with our recently developed force covariance entropy estimator and reveal allosteric communication pathways with Force Distribution Analyses (FDA).We identified crucial relays in the mostly symmetric allosteric activation network, and suggest point mutants to test this mechanism.Our study suggests inter-residue forces, as opposed to coordinates, as a highly sensitive measure for structural adaptations that, even though minute, can very effectively propagate allosteric signals.

View Article: PubMed Central - PubMed

Affiliation: Heidelberg Institutes for Theoretical Studies gGmbH, Heidelberg, Germany.

ABSTRACT
The Catabolite Activator Protein (CAP) is a showcase example for entropic allostery. For full activation and DNA binding, the homodimeric protein requires the binding of two cyclic AMP (cAMP) molecules in an anti-cooperative manner, the source of which appears to be largely of entropic nature according to previous experimental studies. We here study at atomic detail the allosteric regulation of CAP with Molecular dynamics (MD) simulations. We recover the experimentally observed entropic penalty for the second cAMP binding event with our recently developed force covariance entropy estimator and reveal allosteric communication pathways with Force Distribution Analyses (FDA). Our observations show that CAP binding results in characteristic changes in the interaction pathways connecting the two cAMP allosteric binding sites with each other, as well as with the DNA binding domains. We identified crucial relays in the mostly symmetric allosteric activation network, and suggest point mutants to test this mechanism. Our study suggests inter-residue forces, as opposed to coordinates, as a highly sensitive measure for structural adaptations that, even though minute, can very effectively propagate allosteric signals.

No MeSH data available.


Related in: MedlinePlus