Modulation of allosteric behavior through adjustment of the differential stability of the two interacting domains in E. coli cAMP receptor protein

Abstract

The communication mechanism(s) responsible for the allosteric behavior of E.coli cAMP binding receptor protein, CRP, is still a subject of intense investigation. As a tool to explore the communication mechanism, the mutations at various positions in the cAMP-binding (K52N, D53H, S62F and T127L) or the DNA- binding (H159L) domain or both (K52N/H159L) were generated. The sites and specific nature of side chain substitutions were defined by earlier genetic studies, the results of which show that these mutants have a similar phenotype i.e. they are activated without exogenous cAMP. Presently, no significant changes in the structures of WT and mutant CRPs have been observed. Hence, the pressing issue is to identify a physical parameter that reflects the effects of mutations. In this study, the stability of these various CRP species in the presence of GuHCl was monitored by three spectroscopic techniques, namely, CD, tryptophan fluorescence and FT-IR which could provide data on the stability of α-helices and β-strands separately. Results of this study led to the following conclusions: 1. The α-helices can be grouped into two families with different stabilities. Mutations exert a differential effect on the stability of helices as demonstrated by a biphasic unfolding curve for the helices. 2. Regardless of the locations of mutations, the effects can be communicated to the other domain resulting in a perturbation of the stability of both domains, although the effects are more significantly expressed in the stability of the helices. 3. Although in an earlier study [Gekko, et al. Biochemistry 43 (2004) 3844] we showed that cooperativity of cAMP binding is generally correlated to the global dynamics of the protein and DNA binding affinity, in this study we found that generally there is no clear correlation between functional energetics and stability of secondary structures. Thus, results of this study imply that modulation of allostery in CRP is entropic in nature.

Figure 1

Structure of CRP. A. Monomeric structure of apo-CRP (PDB 3H1F). The cAMP- and DNA-binding domains are in green and yellow, respectively. The α-helices, the two sequence terminii and locations of the two tryptophan residues are labeled. The mutation sites are identified as colored spheres. The color codes are the same as in Fig.1B. B. Dimeric structure of holo-CRP (PDB 1G6N). The two subunits are in magenta and green, respectively. The locations of mutation sites are labeled.

Figure 1

Structure of CRP. A. Monomeric structure of apo-CRP (PDB 3H1F). The cAMP- and DNA-binding domains are in green and yellow, respectively. The α-helices, the two sequence terminii and locations of the two tryptophan residues are labeled. The mutation sites are identified as colored spheres. The color codes are the same as in Fig.1B. B. Dimeric structure of holo-CRP (PDB 1G6N). The two subunits are in magenta and green, respectively. The locations of mutation sites are labeled.

Figure 2

Chemical denaturation of CRP samples monitored by CD at 222 nm. The symbols and identity of CRP samples are: , D53H; , H159L; , S62F; □, T127L; , K52N; ■, K52N/H159L; ★, WT.

Figure 3

Chemical denaturation of CRP samples monitored by tryptophan fluorescence. The symbols and identity of CRP samples are the same as in Figure 2.

Figure 4

Fraction of folded and dimer of WT CRP. The symbols and identity of spectroscopic data are: ★, fraction of dimer as determined by sedimentation equilibrium; , fraction of folded α-helix as monitored by FT-IR; , fraction of folded β-strand as monitored by FT-IR; , fraction of folded CRP as monitored by fluorescence.

Figure 5

Fraction of folded and dimer of K52N CRP. The symbols and identity of spectroscopic data are the same as in Fig. 4.

Figure 6

Fraction of folded and dimer of H159L CRP. The symbols and identity of spectroscopic data are the same as in Fig. 4.

Figure 7

Fraction of folded and dimer of K52N/H159L CRP. The symbols and identity of spectroscopic data are the same as in Fig. 4.

Figure 8

Fraction of folded and dimer of D53H CRP. The symbols and identity of spectroscopic data are the same as in Fig. 4.

Figure 9

Fraction of folded and dimer of S62F CRP. The symbols and identity of spectroscopic data are the same as in Fig. 4.

Figure 10

Fraction of folded and dimer of T127I CRP. The symbols and identity of spectroscopic data are the same as in Fig. 4.