Directed Evolution of the Escherichia coli cAMP Receptor Protein at the cAMP Pocket

Sanjiva M Gunasekara,Matt N Hicks,Jin Park,Cory L Brooks,Jose Serate,Cameron V Saunders,Simranjeet K Grover,Joy J Goto,Jin-Won Lee,Hwan Youn

doi:10.1074/jbc.m115.678474

Abstract

The Escherichia coli cAMP receptor protein (CRP) requires cAMP binding to undergo a conformational change for DNA binding and transcriptional regulation. Two CRP residues, Thr(127) and Ser(128), are known to play important roles in cAMP binding through hydrogen bonding and in the cAMP-induced conformational change, but the connection between the two is not completely clear. Here, we simultaneously randomized the codons for these two residues and selected CRP mutants displaying high CRP activity in a cAMP-producing E. coli. Many different CRP mutants satisfied the screening condition for high CRP activity, including those that cannot form any hydrogen bonds with the incoming cAMP at the two positions. In vitro DNA-binding analysis confirmed that these selected CRP mutants indeed display high CRP activity in response to cAMP. These results indicate that the hydrogen bonding ability of the Thr(127) and Ser(128) residues is not critical for the cAMP-induced CRP activation. However, the hydrogen bonding ability of Thr(127) and Ser(128) was found to be important in attaining high cAMP affinity. Computational analysis revealed that most natural cAMP-sensing CRP homologs have Thr/Ser, Thr/Thr, or Thr/Asn at positions 127 and 128. All of these pairs are excellent hydrogen bonding partners and they do not elevate CRP activity in the absence of cAMP. Taken together, our analyses suggest that CRP evolved to have hydrogen bonding residues at the cAMP pocket residues 127 and 128 for performing dual functions: preserving high cAMP affinity and keeping CRP inactive in the absence of cAMP.

Highlights

Thr127 and Ser128 are important for the function of the Escherichia coli cAMP receptor protein (CRP)
Various CRP Mutants Altered at Positions 127 and 128 Are Active in Vivo in a cAMP-producing E. coli—A total of 400 amino acid combinations are possible for the two CRP 127 and 128 positions
Initial screening of the mutant pool using isopropyl ␤-D-1-thiogalactopyranoside (IPTG) concentrations higher than 1 ␮M resulted in a high proportion of blue colonies, suggesting that many combinations generated active CRP phenotype in the cAMP-producing E. coli strain

Summary

Background

Thr127 and Ser128 are important for the function of the Escherichia coli CRP (cAMP receptor protein). In vitro DNA-binding analysis confirmed that these selected CRP mutants display high CRP activity in response to cAMP These results indicate that the hydrogen bonding ability of the Thr127 and Ser128 residues is not critical for the cAMP-induced CRP activation. We interpreted the ligand behaviors of CRP and CRP mutants using the MWC (MonodWyman-Changeux) model, which is proven to accurately describe the behaviors of many allosteric proteins (20 –22), and found that the ligand behaviors of CRP can be described as well by the MWC model According to this model, CRP exists in equilibrium between the active and inactive forms in the absence of cAMP (Fig. 2A). We show through directed evolution and computational analysis that the dyad Thr127 and Ser128 have an additional role in keeping CRP inactive in the absence of cAMP, in addition to attaining high cAMP affinity via a hydrogen bonding network

Experimental Procedures

Results

Hydrogen bonds

Discussion