Linkage of Multiequilibria in DNA Recognition by the D53H Escherichia coli cAMP Receptor Protein

Many allosteric proteins form homo-oligomeric complexes to regulate a biological function. In homo-oligomers, subunits establish communication pathways that are modulated by external stimuli like ligand binding. A challenge for dissecting the communication mechanisms in homo-oligomers is identifying intermediate liganded states, which are typically transiently populated. However, their identities provide the most mechanistic information on how ligand-induced signals propagate from bound to empty subunits. Here, we dissected the directionality and magnitude of subunit communication in a reengineered single-chain version of the homodimeric transcription factor cAMP receptor protein. By combining wild-type and mutant subunits in various asymmetric configurations, we revealed a linear relationship between the magnitude of cooperative effects and the number of mutant subunits. We found that a single mutation is sufficient to change the global allosteric behavior of the dimer even when one subunit was wild type. Dimers harboring two mutations with opposite cooperative effects had different allosteric properties depending on the arrangement of the mutations. When the two mutations were placed in the same subunit, the resulting cooperativity was neutral. In contrast, when placed in different subunits, the observed cooperativity was dominated by the mutation with strongest effects over cAMP affinity relative to wild type. These results highlight the distinct roles of intrasubunit interactions and intersubunit communication in allostery. Finally, dimers bound to either one or two cAMP molecules had similar DNA affinities, indicating that both asymmetric and symmetric liganded states activate DNA interactions. These studies have revealed the multiple communication pathways that homo-oligomers employ to transduce signals.Allosteric proteins are the basic building blocks in the transmission of biological signals, allowing communication between and within cells and from the extracellular environment to the cytosol (1). Because many allosteric proteins form homo-oligomeric complexes to modulate a ligand-induced biological response (2, 3), intersubunit communication must play a crucial role in the transduction of an allosteric signal (4, 5). Identifying the molecular mechanisms of intersubunit communication has important implications, from understanding how biological systems detect and transduce signals (6, 7) to reengineering of signaling proteins (8 -10) and to developing allosteric therapeutic modulators with enhanced affinities and specificities (11,12). Despite its importance, dissecting the mechanisms of transduction of allosteric signals from one protein subunit to another has proven difficult because it requires monitoring intermediate liganded states that are poorly populated, especially if the protein displays positive ligand binding cooperativity. Therefore, one of the remaining unresolved issues in allostery is dissecting the directionality of pathways of signal transmissions across protein subunits.A widely used s...

mentioning

confidence: 69%

Section: Role Of Asymmetric Conformations In the Mechanism Of Crp Actmentioning

confidence: 93%

Asymmetric configurations in a reengineered homodimer reveal multiple subunit communication pathways in protein allostery

Lanfranco

Gárate

Engdahl³

et al. 2017

“…These structures clearly demonstrate significant structural changes, unequivocally indicating that the structural isomerization reaction must be included as a finite step in the linked multiequilibria mechanism that describes the CRP function (1,32). The observed change in the secondary structure involving residues 126 -139 implies that perturbation of the equilibrium between the coil to helix and extension/contraction of the D-helix could perturb the equilibrium between the apo and holo isomerization reaction and in turn the allosteric behavior of CRP.…”

Section: Discussionmentioning

confidence: 91%

The N-terminal Capping Propensities of the D-helix Modulate the Allosteric Activation of the Escherichia coli cAMP Receptor Protein

Yu¹,

Maillard²,

Gribenko³

et al. 2012

Background: Residue 138 situates in the hinge region connecting the DNA-and cAMP-binding domains of CRP. Results: A correlation was established among N-capping propensities and cooperativity of cAMP binding and affinity for lac-DNA. Conclusion: Our results provide a quantitative characterization of the N-capping properties of residue 138 in the allosteric activation event. Significance: The results provide the thermodynamic basis to the structural model of allostery.

“…In principle, there are 5 possible conformers of CRP (with 0 ϳ 4 cAMP bound) that can interact with DNA (13). However, the apoCRP form has been reported to have very low DNA affinity that may not contribute to specific binding equilibrium (14).…”

Section: Methodsmentioning

confidence: 99%

A C-helix Residue, Arg-123, Has Important Roles in Both the Active and Inactive Forms of the cAMP Receptor Protein

Youn

Kerby

Koh

et al. 2007

The cAMP receptor protein (CRP) of Escherichia coli exists in an equilibrium between active and inactive forms, and the effector, cAMP, shifts that equilibrium to the active form, thereby allowing DNA binding. For this equilibrium shift, a C-helix repositioning around the C-helix residues Thr-127 and Ser-128 has been reported as a critical local event along with proper ␤4/␤5 positioning. Here we show that another C-helix residue, Arg-123, has a unique role in cAMP-dependent CRP activation in two different ways. First, Arg-123 is important for proper cAMP affinity, although it is not critical for the conformational change with saturating amounts of cAMP. Second, Arg-123 is optimal for stabilizing the inactive conformation of CRP when cAMP is absent, thereby allowing a maximal range of regulation by cAMP. However, Arg-123 does not appear to be critical for a functional response to cAMP, as has been proposed previously The cAMP receptor protein (CRP)2 of Escherichia coli is one of the best-studied transcriptional activators (1-3). It is a homodimeric protein that binds specific DNA sequences with high affinity in response to binding a molecule of cAMP to each monomer. In cases where it activates gene expression, the DNA-bound CRP interacts with RNA polymerase to stabilize polymerase binding and, thus, stimulate transcription.CRP is a classic allosteric protein, each subunit of which is composed of two distinct domains connected by a hinge region; this is, an N-terminal effector binding domain and a C-terminal DNA binding domain (4). cAMP binding to the effector binding domain triggers a conformational change in the protein that leads to a precise repositioning of the DNA binding domains that promotes binding to its target DNA sequence. CRP exists in equilibrium between an active form that can bind specific DNA target sequences and an inactive form that cannot. In this view, cAMP binding to the protein shifts the equilibrium toward the active form either by destabilizing the inactive form or by stabilizing the active form.Although an enormous amount is known about the behavior of the DNA binding form of CRP, relatively less is known about the process of activation by cAMP binding. The critical obstacle has been the absence of a crystal structure of the cAMP-free form, although several structures of active CRP are known both in the presence and absence of DNA (4 -6). A number of research groups have used other methods to better understand the cAMP-dependent activation of CRP protein (3). We have also contributed to this field by identifying two distinctive local effects of cAMP binding in the activation process (7); (i) C-helix repositioning through direct interaction of cAMP with two C-helix residues Thr-127 and Ser-128 and (ii) the concomitant reorientation of the ␤4/␤5 loop.Recently another group compared the structures of a large number of cAMP-binding proteins of quite diverse function (8). They showed that despite large functional differences there were underlying structural similarities in both cAMP sensing a...