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 ∼30 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.
Highlights
Change in protein dynamics as a function of mutation is consistent with our earlier study showing a linear correlation between protein dynamics and magnitudes of allostery in cAMP binding [36]
An allosteric protein consists of a pre-existing connectivity pattern among residues
Allosteric behavior is the consequence of modulation of the roughness of the energetic landscape
Summary
Downstream CRP subunits instead of just the downstream CRP subunit as in class I promoters. Same mutations are defective for class II but not class I This observation implies that these mutations exert differential effects on CRP subunits. How do the same regulatory residues seemingly exert differential effects on the CRP dimer subunits of identical sequence? There is still no clear understanding of the underlying principles that govern the long-range effects exerted by these ARs that physically occupy different spaces on the CRP surface. In a more in-depth analysis, we computationally simulated the change in energy landscapes of CRP domains induced by 14 mutants that have been investigated in a series of detailed studies to elucidate the mutual allosteric regulatory mechanism employed by these ARs. In this study, we provide evidence that the CRP subunits are energetically asymmetric and that mutations exert their effects differentially between the two subunits through networks of long-range communication. The different allosteric behavior between class I and II CRP-dependent promoters is the consequence of differential modulation of the roughness of the energetic landscape
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