The thermodynamics of the binding of cyclic adenosine monophosphate (cAMP) and its non-functional analog, cyclic guanosine monophosphate (cGMP), to cyclic AMP receptor protein (CRP) and its T127L mutant were investigated by isothermal titration calorimetry (

The three-dimensional model of the Escherichia coli cyclic AMP (cAMP) receptor protein (CRP) shows that several amino acids are involved as chemical contacts for binding cAMP. We have constructed and characterized mutants at four of these positions, E72, R82, S83, and R123. The mutations were made in wild-type crp as well as a cAMP-independent crp, crp*. The activities of the mutant proteins were characterized in vivo for their ability to activate the lac operon. These results provide genetic evidence to support that E72 CRP is a transcriptional regulatory protein which acts both positively and negatively in E. coli (for reviews, see references 4, 11, 19, and 26). It activates transcription at catabolite-sensitive operons such as the lactose, galactose, maltose, and arabinose operons and represses its own transcription. CRP directly responds to cAMP in the cell. Upon binding the ligand, the protein undergoes a conformational change which allows it to bind to specific DNA sequences located in the promoter regions of these operons.CRP is a 45-kDa dimeric protein. The dimer consists of two identical subunits of 209 amino acids. The three-dimensional crystal structure of CRP complexed with cAMP was first determined to 2.9 A (0.29 nm) by McKay and Steitz (15) and has since been further refined to 2.5 A (0.25 nm) (28).The structure of CRP-cAMP bound to DNA was recently determined by Schultz et al. (24). The CRP-cAMP structure suggests that each subunit can bind one molecule of cAMP and one-half of the symmetrical 22-bp DNA operator site. Each subunit is made up of two domains (Fig. 1). The smaller carboxy-terminal domain provides the DNA-binding specificity through a helix-turn-helix motif characteristic of many prokaryotic DNA-binding proteins (17,25). The E and F a-helices are oriented such that the F a-helix fits into the major groove and the E a-helix lies across the minor groove of the operator DNA. Several CRP-DNA interactions have been characterized by mutagenesis experiments (5-7, 13, 29).The amino-terminal domain of CRP provides the cAMP binding specificity. The structure suggests a ,B-basket-type pocket with an a-helix leading into eight 1-strands. off this pocket is the long C a-helix which connects the two domains. Six specific amino acids in each subunit have been suggested as important protein side chain contacts for binding cAMP (Fig. 2). Hydrogen bonds have been proposed between the ribose and phosphate oxygens of cAMP and glutamic acid 72 (E72) and serine 83 (S83). In this model, E72 is stabilized by ionic interaction with arginine 123 (R123). In addition, the cAMP phosphate interacts with arginine 82 (R82). Two other side chains, threonine 127 (T127) and serine 128 (S128), are proposed to form one and two hydrogen bonds, respectively, with the adenine of cAMP. The S128 contact is provided from the opposite subunit to each cAMP binding pocket. T127 and S128 were not tested in the present work.In this study, mutations were made in crp at the codons for the amino acid residues E72, R82, S83, and R123...

Relationships of ten heliconiine butterflies (genera Dryas and Heliconius, family Nymphalidae) were elucidated by phylogenetic analysis of characters based on ribosomal DNA restriction site variation and morphology. Agraulis vanillae, also a heliconiine, was used as the outgroup species. Although neither the morphological nor the molecular data unambiguously resolve relationships among the heliconiines, a combined analysis of both data sets results in a tree that is similar to traditional systematic arrangements and previous views of radiation in the group. Both pupal‐mating and nonpupal‐mating species group as clades in the combined analysis. However, the restriction site data alone do not support the monophyly of the pupal‐mating clade, and the morphological data alone do not support the monophyly of the non‐pupal‐mating clade. Furthermore, relationships of H. melpomene, H. cydno and the silvaniform species depart from traditional arrangements based on morphology and reproductive compatibility experiments. All trees support the independent evolution of similar wing patterns of species previously suggested to be members of mimicry complexes. Several mimicry complexes appear to have a member in each of the two major monophyletic groups (pupal‐mating and non‐pupal‐mating clades).

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