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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.


Allosteric network upon binding the second cAMP obtained from FDA.Residue pairwise force differences between the cap1 and cap2 states are shown as blue sticks at (A) 50 pN and (B) 40 pN cut-off. (C) Zoom of B highlighting the allosteric connection pathway between the two protomers, which resembles pathway B in Fig 3D. (D) Zoom of B, highlighting the force changes in the Glu58-Arg87 and Glu58-Q174 pairs which connects the NBD with the DBD and which are symmetrically present in both protomers.
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pcbi.1004358.g005: Allosteric network upon binding the second cAMP obtained from FDA.Residue pairwise force differences between the cap1 and cap2 states are shown as blue sticks at (A) 50 pN and (B) 40 pN cut-off. (C) Zoom of B highlighting the allosteric connection pathway between the two protomers, which resembles pathway B in Fig 3D. (D) Zoom of B, highlighting the force changes in the Glu58-Arg87 and Glu58-Q174 pairs which connects the NBD with the DBD and which are symmetrically present in both protomers.

Mentions: Binding of the second cAMP entails local force changes within host protomer resembling those already seen for the first binding event (Fig 5A, also compare Fig 3A). Again, large residue-residue force differences were observed in the protomer hosting the additionally bound nucleotide, including the P-loop region and residues nearby (His31’ to Ala36’), the loop from Leu73’ to Gln80’ and also again the upper β4/β5-sheet (see Fig 1B). Similar interaction changes, upon the binding of the first and second cAMP, at least in the proximity of the binding pockets, are expected, given the high similarity of ligand-protein interactions. FDA encouragingly recovered this expected behaviour. However, at the same cut-off of 50 pN, the network after the second binding event now extends over ~15 Å from the rather local network around the cAMP binding cleft detected upon the first binding event. It now reaches the β-strands 2 and 7 to finally join the P-loop of both protomers, and even residues in close contact with and within the DBDs (Fig 5A and 5B). This new distant signal propagation towards the DBD is likely connected to the DBD activation motion observed in our MD simulations (Fig 2) and in available experimental structures [9,25]. The long-range nature of the cap1-cap2 force network is also in agreement with the global stiffening of CAP after the binding of the second cAMP observed experimentally [8]. An even larger, but slightly weaker (40 pN) force network now reaches all the way to the C-terminal region of the H3-helix (see Fig 1B). We would like to emphasize that this network was absent before binding of the second cAMP. This region is crucial for regulation and is known to undergo large conformational changes after CAP activation. Experimental structures showed that cAMP binding promotes addition of two helix turns at this C-terminal region [25,34].


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

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

Allosteric network upon binding the second cAMP obtained from FDA.Residue pairwise force differences between the cap1 and cap2 states are shown as blue sticks at (A) 50 pN and (B) 40 pN cut-off. (C) Zoom of B highlighting the allosteric connection pathway between the two protomers, which resembles pathway B in Fig 3D. (D) Zoom of B, highlighting the force changes in the Glu58-Arg87 and Glu58-Q174 pairs which connects the NBD with the DBD and which are symmetrically present in both protomers.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4526232&req=5

pcbi.1004358.g005: Allosteric network upon binding the second cAMP obtained from FDA.Residue pairwise force differences between the cap1 and cap2 states are shown as blue sticks at (A) 50 pN and (B) 40 pN cut-off. (C) Zoom of B highlighting the allosteric connection pathway between the two protomers, which resembles pathway B in Fig 3D. (D) Zoom of B, highlighting the force changes in the Glu58-Arg87 and Glu58-Q174 pairs which connects the NBD with the DBD and which are symmetrically present in both protomers.
Mentions: Binding of the second cAMP entails local force changes within host protomer resembling those already seen for the first binding event (Fig 5A, also compare Fig 3A). Again, large residue-residue force differences were observed in the protomer hosting the additionally bound nucleotide, including the P-loop region and residues nearby (His31’ to Ala36’), the loop from Leu73’ to Gln80’ and also again the upper β4/β5-sheet (see Fig 1B). Similar interaction changes, upon the binding of the first and second cAMP, at least in the proximity of the binding pockets, are expected, given the high similarity of ligand-protein interactions. FDA encouragingly recovered this expected behaviour. However, at the same cut-off of 50 pN, the network after the second binding event now extends over ~15 Å from the rather local network around the cAMP binding cleft detected upon the first binding event. It now reaches the β-strands 2 and 7 to finally join the P-loop of both protomers, and even residues in close contact with and within the DBDs (Fig 5A and 5B). This new distant signal propagation towards the DBD is likely connected to the DBD activation motion observed in our MD simulations (Fig 2) and in available experimental structures [9,25]. The long-range nature of the cap1-cap2 force network is also in agreement with the global stiffening of CAP after the binding of the second cAMP observed experimentally [8]. An even larger, but slightly weaker (40 pN) force network now reaches all the way to the C-terminal region of the H3-helix (see Fig 1B). We would like to emphasize that this network was absent before binding of the second cAMP. This region is crucial for regulation and is known to undergo large conformational changes after CAP activation. Experimental structures showed that cAMP binding promotes addition of two helix turns at this C-terminal region [25,34].

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.