Two-state allosteric modeling suggests protein equilibrium as an integral component for cyclic AMP (cAMP) specificity in the cAMP receptor protein of Escherichia coli

Activation of the cAMP receptor protein (CRP) from Escherichia coli is highly specific to its allosteric ligand, cAMP. Ligands such as adenosine and cGMP, which are structurally similar to cAMP, fail to activate wild-type CRP. However, several cAMP-independent CRP variants (termed CRP*) exist that can be further activated by both adenosine and cGMP, as well as by cAMP. This has remained a puzzle because the substitutions in many of these CRP* variants lie far from the cAMP-binding pocket (>10 A) and therefore should not directly affect that pocket. Here we show a surprising similarity in the altered ligand specificity of four CRP* variants with a single substitution in D53S, G141K, A144T, or L148K, and we propose a common basis for this phenomenon. The increased active protein population caused by an equilibrium shift in these variants is hypothesized to preferentially stabilize ligand binding. This explanation is completely consistent with the cAMP specificity in the activation of wild-type CRP. The model also predicts that wild-type CRP should be activated even by the lower-affinity ligand, adenosine, which we experimentally confirmed. The study demonstrates that protein equilibrium is an integral factor for ligand specificity in an allosteric protein, in addition to the direct effects of ligand pocket residues.     

Structure and expression of gene <Emphasis Type="Italic">vfr</Emphasis> in <Emphasis Type="Italic">Pseudomonas chlororaphis</Emphasis> 449

Gene vfr of Pseudomonas chlororaphis 449 previously described only in Pseudomonas aeruginosa was identified, cloned, and sequenced; its localization in the chromosome was determined. Amino acid sequence of the protein encoded by gene vfr in P. chlororaphis 449 was shown to have a 83% identity with the Vfr protein of P. aeruginosa PAO1 and a 63% identity with the CRP protein of Escherichia coli. Amino acid residues that ensure the most important structural properties of the CRP protein, i.e., its binding to cAMP, RNA polymerase, and DNA, were identical or highly conserved in Vfr proteins of P. aeruginosa and P. chlororaphis 449. The cloned vfr gene of P. chlororaphis 449 was complemented partially the mutation at gene crp in cells of E. coli AM306 enhancing ten times synthesis of β-galactosidase dependent on the CRP protein. Unlike P. aeruginosa, the Vfr protein in cells of P. chlororaphis 449 does not participate in the regulation of synthesis of N-acyl-homoserine lactones.     

Dynamic Fluctuations Provide the Basis of a Conformational Switch Mechanism in Apo Cyclic AMP Receptor Protein

Escherichia coli cyclic AMP Receptor Protein (CRP) undergoes conformational changes with cAMP binding and allosterically promotes CRP to bind specifically to the DNA. In that, the structural and dynamic properties of apo CRP prior to cAMP binding are of interest for the comprehension of the activation mechanism. Here, the dynamics of apo CRP monomer/dimer and holo CRP dimer were studied by Molecular Dynamics (MD) simulations and Gaussian Network Model (GNM). The interplay of the inter-domain hinge with the cAMP and DNA binding domains are pre-disposed in the apo state as a conformational switch in the CRP''s allosteric communication mechanism. The hinge at L134-D138 displaying intra- and inter-subunit coupled fluctuations with the cAMP and DNA binding domains leads to the emergence of stronger coupled fluctuations between the two domains and describes an on state. The flexible regions at K52-E58, P154/D155 and I175 maintain the dynamic coupling of the two domains. With a shift in the inter-domain hinge position towards the N terminus, nevertheless, the latter correlations between the domains loosen and become disordered; L134-D138 dynamically interacts only with the cAMP and DNA binding domains of its own subunit, and an off state is assumed. We present a mechanistic view on how the structural dynamic units are hierarchically built for the allosteric functional mechanism; from apo CRP monomer to apo-to-holo CRP dimers.