Hossein Fazelinia
Shuang Yan Tang
Computational design of AraC protein with
novel effector specificity
We are currently focusing our attention on the AraC regulatory protein belonging to the AraC/XylS family of transcriptional regulators [5,6]. This well-studied E. coli protein tightly regulates transcription from two cognate promoters, ParaBAD and ParaC, in response to the substrate L-arabinose, as shown in Figure 2. Promoter ParaBAD controls the genes involved in L-arabinose catabolism (araB, araA, and araD), while ParaC controls the araC gene. AraC is composed of a C-terminal, DNA-binding domain and an N-terminal effector-binding and dimerization domain, and the protein exists primarily as a homodimer in solution. In the absence of L-arabinose, the AraC dimer binds to two distant half-sites on the arabinose operon separated by 210 bases resulting in the formation of a DNA loop, effectively repressing transcription from both ParaBAD and ParaC. In the presence of arabinose, the N-terminal domain binds the sugar resulting in a conformational change that causes the AraC dimer to switch from binding the two distant operon sites to binding two contiguous half-sites where it activates transcription from the adjacent ParaBAD promoter (Figure 2) [7-10].
The availability of high resolution atomic-level X-ray crystal structures of the effector-binding/dimerization domain of AraC in the presence and absence of arabinose make it a viable candidate for modeling novel effector recognition by computationally redesigning the binding pocket [11]. The AraC structures indicate that the N-terminal arm (residues 7 to 18) plays a critical role in binding arabinose and closing the binding pocket (see Figure 3), and this arm movement is believed to cause the altered dimer conformation and subsequent DNA binding affinity [12]. We propose that engineering the AraC binding pocket to bind new molecules such that the lowest energy conformation for this N-terminal arm involves critical contacts with the molecule of interest ensures that the designed binding event will elicit the conformational change and consequent transcriptional activation response.