A computational investigation of allostery in the catabolite activator protein

The catabolite activator protein is a dimer that consists of two cAMP-binding subunits, each containing a C-terminus DNA-binding module and a N-terminus ligand binding domain. The system is well-known to exhibit negative cooperativity, whereby the binding of one CAMP molecule reduces the binding affinity of the other CAMP molecule by 2 orders of magnitude, despite the large separation between the CAMP binding pockets. Here we use extensive explicit-solvent molecular dynamics simulations (135 ns) to investigate the allosteric, mechanism of CAP. Six trajectories were carried out for apo, singly liganded, and doubly liganded CAP, both in the presence and absence of DNA. Thorough analyses of the dynamics through the construction of dynamical cross-correlated maps, as well as essential dynamics analyses, indicated that the system experienced a switch in motion as a result of CAMP binding, in accordance with recent NMR experiments carried out on a truncated form of the protein. Analyses of conformer structures collected from the simulations revealed a remarkable event: the DNA-binding module was found to dissociate from the N-terminus ligand binding domain. An interesting aspect of this structural change is that it only occurred in unoccupied subunits, suggesting that the binding of CAMP provides additional stability to the system, consistent with the increase in entropy that was observed in our calculations and from isothermal titration calorimetry. Analysis of the distribution of intrinsic disorder propensities in CAP amino acid sequence using PONDR VLXT and VSL1 predictors revealed that the region connecting ligand-binding and DNA-binding domains of CAP have the potential to exhibit increased flexibility. We complemented these trajectories with free energy calculations following the MM-PBSA approach on more than 2000 snapshots that included 880 normal mode analysis. The resulting free energy differences between the singly liganded and doubly liganded states were in excellent agreement with isothermal titration calorimetry data. When the free energy calculations were carried out in the presence of DNA, we discovered that a switch in cooperativity occurred, so that the binding of the first CAMP promoted the binding of the other CAMP. The components of the free energy reveal that this effect is mainly entropic in nature, whereby the DNA reduces the degree of tightening that is observed in its absence, thereby promoting binding of the second CAMP. This finding prompted us to propose a new mechanism by which CAP triggers the transcription activation that is based on an order to disorder transition mediated by CAMP binding as well as DNA.