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

Abstract

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.

Keywords: allosteric regulation; cAMP receptor protein (CRP); conformational change; cooperativity; cyclic AMP (cAMP); single-chain dimer; subunit communication; transcription factor.

Figure 1.

Design and construction of a CRP single-chain dimer (CRP_SC). A, model of a CRP_SC that connects the two CRP subunits (cyan and orange) through a flexible polypeptide linker (red). The model was rendered in PyMOL based on the CRP structure bound to cyclic nucleotide (PDB 1G6N). B, structure of CRP in the unliganded (left, PDB 2WC2) and cAMP-bound conformations (right, PDB 1G6N). cAMP is shown in magenta.

Figure 2.

Biophysical and functional characterization of CRP_SC. A, SDS-PAGE showing the molecular mass (in kDa) of CRP_SC versus wild-type CRP (labeled CRP in all figure panels). B, size-exclusion chromatogram of CRP_SC and wild-type CRP. C, CD spectra of CRP_SC and wild-type CRP. D, chemical denaturation of CRP_SC and wild-type CRP monitored by changes in tryptophan fluorescence. The line corresponds to the fit of a two-state unfolding model as described under “Materials and Methods.” E, CRP-DNA interactions monitored by electrophoretic mobility shift assay using increasing lengths of the lac promoter in the absence and presence of 200 μm cAMP for CRP_SC and wild-type CRP.

Figure 3.

Quantification of the functional behavior of CRP_SC. A, cAMP binding to CRP_SC and wild-type CRP (labeled CRP in all figure panels) monitored by changes in ANS fluorescence. The solid lines represent the fit using a two-site binding model as described under “Materials and Methods.” B, binding of CRP_SC or wild-type CRP to a 32-bp fluorescein-labeled lac promoter monitored by changes in fluorescence anisotropy. The solid lines represent the fit as described in Ref. . Residuals of the fit for both experiments are shown below the titrations.

Figure 4.

Effect of symmetric and asymmetric mutations on cAMP binding affinity. A, cAMP titrations to CRP_SC^WT/WT (green circles) and the symmetric mutants CRP_SC^D/D (red circles) and CRP_SC^S/S (dark purple circles). B, cAMP titrations to the single asymmetric mutants CRP_SC^D/WT (light pink squares) and CRP_SC^S/WT (light purple squares). C, cAMP titrations to the double asymmetric mutants CRP_SC^S+D/WT (dark brown diamonds) and CRP_SC^S/D (beige diamonds). For comparison, the dashed lines corresponding to the fits of the parent symmetric proteins obtained from A were included in B and C. The solid lines in all three panels represent the fit using a two-site binding model as described under “Materials and Methods.” Error bars correspond to the standard deviation of at least three repeats.

Figure 5.

DNA-CRP_SC interactions using saturating and non-saturating cAMP concentrations. A, interaction of CRP_SC^S/D with the 32-bp lac promoter using 0, 30, and 1000 μm cAMP, which correspond to unbound (open squares), singly (gray triangles), and doubly (black circles) cAMP-bound states. The solid lines represent the fit as described in Ref. . B, DNA binding affinity constants of CRP_SC^S/D, CRP_SC^S/WT, and CRP_SC^S/S when the proteins are in the unbound (open squares), singly (gray triangles), and doubly (black circles) cAMP-bound states. C, DNA binding affinity constants obtained from fluorescence anisotropy experiments for symmetric and asymmetric CRP_SC in the absence (open squares) and presence of saturating cAMP concentrations (black circles). For CRP_SC^WT/WT, CRP_SC^D/D, and CRP_SC^D/WT, [cAMP] = 200 μm. For CRP_SC^S/S, [cAMP] = 2000 μm. For CRP_SC^S/D, CRP_SC^S/WT, and CRP_SC^S+D/WT, [cAMP] = 1000 μm.

Figure 6.

cAMP-binding energies and cooperativities of CRP_SC variants. Light and dark squares correspond to the cAMP-binding energy for the first (ΔG⁰₁) and second (ΔG⁰₂) sites, respectively. The order of CRP_SC mutants is sorted from most positive cooperativity (ΔG⁰₂ − ΔG⁰₁ < 0) to most negative (ΔG⁰₂ − ΔG⁰₁ > 0) as indicated by the dashed lines.

Figure 7.

Mechanism of CRP activation and interaction with DNA. In the absence of cAMP, CRP is in an inactive state. Binding of cAMP to one CRP subunit triggers a conformational change in the DNA-binding domain of the bound subunit, generating an asymmetric conformation within the dimer. The cAMP-bound subunit induces a re-orientation in the unliganded neighboring subunit that is compatible with strong DNA interactions. Binding of a second cAMP molecule can occur to either the singly cAMP-bound CRP or to the ternary complex DNA-CRP-cAMP.