Water-mediated interactions in the CRP–cAMP–DNA complex: Does water mediate sequence-specific binding at the DNA primary-kink site?

The cyclic AMP receptor protein (CRP) of Escherichia coli binds preferentially to DNA sequences possessing a T:A base pair at position 6 (at which the DNA becomes kinked), but with which it does not form any direct interactions. It has been proposed that indirect readout is involved in CRP-DNA binding, in which specificity for this base pair is primarily related to sequence effects on the energetic susceptibility of the DNA to kink formation. In the current study, the possibility of contributions to indirect readout by water-mediated hydrogen bonding of CRP with the T:A base pair was investigated. A 1.0 ns molecular dynamics simulation of the CRP-cAMP-DNA complex in explicit solvent was performed, and assessed for water-mediated CRP-DNA hydrogen bonds; results were compared to several X-ray crystal structures of comparable complexes. While several water-mediated CRP-DNA hydrogen bonds were identified, none of these involved the T:A base pair at position 6. Therefore, the sequence specificity for this base pair is not likely enhanced by water-mediated hydrogen bonding with the CRP.     

State-selective frustration as a key driver of allosteric pluripotency

Allosteric pluripotency arises when an allosteric effector switches from agonist to antagonist depending on the experimental conditions. For example, the Rp-cAMPS ligand of Protein Kinase A (PKA) switches from agonist to antagonist as the MgATP concentration increases and/or the kinase substrate affinity or concentration decreases. Understanding allosteric pluripotency is essential to design effective allosteric therapeutics with minimal side effects. Allosteric pluripotency of PKA arises from divergent allosteric responses of two homologous tandem cAMP-binding domains, resulting in a free energy landscape for the Rp-cAMPS-bound PKA regulatory subunit R1a in which the ground state is kinase inhibition-incompetent and the kinase inhibition-competent state is excited. The magnitude of the free energy difference between the ground non-inhibitory and excited inhibitory states (ΔG_R,Gap) relative to the effective free energy of R1a binding to the catalytic subunit of PKA (ΔG_R:C) dictates whether the antagonism-to-agonism switch occurs. However, the key drivers of ΔG_R,Gap are not fully understood. Here, by analyzing an R1a mutant that selectively silences allosteric pluripotency, we show that a major determinant of ΔG_R,Gap unexpectedly arises from state-selective frustration in the ground inhibition-incompetent state of Rp-cAMPS-bound R1a. Such frustration is caused by steric clashes between the phosphate-binding cassette and the helices preceding the lid, which interact with the phosphate and base of Rp-cAMPS, respectively. These clashes are absent in the excited inhibitory state, thus reducing the ΔG_R,Gap to values comparable to ΔG_R:C, as needed for allosteric pluripotency to occur. The resulting model of allosteric pluripotency is anticipated to assist the design of effective allosteric modulators.

The Rp-cAMPS ligand of protein kinase A switches from agonist to antagonist depending on metabolite and proteomic contexts. We show that the state-selective frustration is a key driver of this allosteric pluripotency phenomenon.     

The discretized flow on domains of attraction: a structural stability result

It is well known that, for stepsize sufficiently small, compactattractors of ordinary differential equations persist underdiscretization. The present paper describes the structure ofthe discrete-time dynamical system obtained via discretizationon A(M_h)\M_h where M_h is the approximate attractor and A(M_h)is its domain of attraction. The existence of a smooth embeddinginto a continuous-time parallelizable flow is proved. The constructioncan be used to define sections for discretizations and can beinterpreted as a justification of the method of modified equations.