Differential modulation of energy landscapes of cyclic AMP receptor protein (CRP) as a regulatory mechanism for class II CRP-dependent promoters

Abstract

The Escherichia coli cAMP receptor protein, CRP, is a homodimeric global transcription activator that employs multiple mechanisms to modulate the expression of hundreds of genes. These mechanisms require different interfacial interactions among CRP, RNA, and DNA of varying sequences. The involvement of such a multiplicity of interfaces requires a tight control to ensure the desired phenotype. CRP-dependent promoters can be grouped into three classes. For decades scientists in the field have been puzzled over the differences in mechanisms between class I and II promoters. Using a new crystal structure, IR spectroscopy, and computational analysis, we defined the energy landscapes of WT and 14 mutated CRPs to determine how a homodimeric protein can distinguish nonpalindromic DNA sequences and facilitate communication between residues located in three different activation regions (AR) in CRP that are ϳ30 Å apart. We showed that each mutation imparts differential effects on stability among the subunits and domains in CRP. Consequently, the energetic landscapes of subunits and domains are different, and CRP is asymmetric. Hence, the same mutation can exert different effects on ARs in class I or II promoters. The effect of a mutation is transmitted through a network by long-distance communication not necessarily relying on physical contacts between adjacent residues. The mechanism is simply the sum of the consequences of modulating the synchrony of dynamic motions of residues at a distance, leading to differential effects on ARs in different subunits. The computational analysis is applicable to any system and potentially with predictive capability. The Escherichia coli cAMP receptor protein (CRP), 5 is a homodimeric global transcription activator that controls the transcription of numerous genes involving carbon utilization (1, 2). The CRP-dependent promoters can be grouped into three classes (3-5). In class I promoters, CRP binds upstream of RNA polymerase (RNAP), at a site centered close to position Ϫ61.5 on the DNA (3, 6). In class II promoters, CRP is sandwiched between the ␣-CTD and 70 subunit of RNAP, at a site centered close to position Ϫ41.5 on the DNA (4, 7). In class III promoters, two or more CRP dimers bind at multiple sites through class I or a combination of class I/class II CRP-dependent promoters (5, 6). Fig. 1 shows the models of class I and II promoters with respect to the relative positions of CRP, RNAP, and the two DNA half-sites for CRP binding. Three surfaceexposed regions of CRP, designated as activating regions 1-3 (AR 1 , AR 2 , and AR 3), modulate the contacts with RNAP (4). The contacts between ARs and RNAP are promoter-dependent. Fig. 2A shows the relative locations of these surfaces in CRP and the distances between key residues in these surfaces. The architectural designs of these promoters and the extensive results of mutation studies of class II promoters (4-6, 8-13) enabled the field to conclude that RNAP contacts AR 1 of the CRP upstream subunit. Furthermore, through analogous modeling, ...

cAMP is an allosteric modulator of DNA-binding specificity in the cAMP receptor protein from Mycobacterium tuberculosis

“…Given that the structures of the CRP MTB in the apo-state and cAMP-bound state are similar, it is plausible that protein dynamics ( 16 , 18 , 21 , 53 , 54 , 55 , 56 ) also play an important role in how cAMP allosterically reduces nonspecific DNA interaction. Our cAMP-binding studies show differences in ANS fluorescence between apo-state and cAMP-bound state ( Fig.…”

“…It is well documented that the CRP Ecoli exhibits positive cooperativity between the two cAMP-binding sites, wherein the first binding reaction is exothermic and the second is endothermic ( 18 , 19 , 24 , 33 ). In contrast, by using two orthogonal techniques (ITC and fluorescence), we find that the CRP MTB displays negative cAMP-binding cooperativity ( Fig.…”

“…Additional differences are observed in the homodimer symmetry. In the CRP Ecoli , the two subunits in the apo-state are symmetric, but the cAMP-bound state shows asymmetry between the DNA-binding domains' conformation ( 18 ) ( Fig. 1 D , top ).…”

“…The CRP Ecoli and CRP MTB show structural differences, and their functional response to cAMP binding also differs. In the CRP Ecoli , cAMP binding enhances the affinity of the protein to tightly interact with pseudopalindromic gene promoter sequences involved in carbohydrate metabolism ( 18 , 19 , 20 , 21 , 22 , 23 ). In the CRP MTB , the affinity for DNA promoters with and without cAMP appears to be similar ( 12 , 20 , 24 ).…”

“…The CRP Ecoli and CRP MTB show structural differences, and their functional response to cAMP binding also differs. In the CRP Ecoli , cAMP binding enhances the affinity of the protein to tightly interact with pseudopalindromic gene promoter sequences involved in carbohydrate metabolism (18)(19)(20)(21)(22)(23). In the The CRP has a conserved structural organization with two identical subunits, each one harboring a cAMP-binding domain in the N terminus (pale cyan in the CRP Ecoli ; tan in the CRP MTB ) and a DNA-binding domain in the C terminus (dark teal in the CRP Ecoli ; dark brown in the CRP MTB ).…”