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Combining Rational Design and Continuous Evolution on Minimalist Proteins That Target DNA

submitted on 21.02.2020, 14:45 and posted on 24.02.2020, 06:00 by Jumi Shin, Ichiro Inamoto, Inder Sheoran, Serban Popa, Montdher Hussain
We designed MEF to mimic the basic region/helix-loop-helix/leucine zipper (bHLHZ) domain of transcription factors Max and Myc, which bind with high DNA sequence specificity and affinity to the E-box motif (enhancer box, CACGTG). To make MEF, we started with our rationally designed ME47, a hybrid of the Max basic region and E47 HLH, that effectively inhibited tumor growth in a mouse model of breast cancer. ME47, however, displays propensity for instability and misfolding. We therefore sought to improve ME47's structural and functional features. We used phage-assisted continuous evolution (PACE) to uncover "nonrational" changes to complement our rational design. PACE mutated Arg12 that contacts the DNA phosphodiester backbone. We would not have rationally made such a change, but this mutation improved ME47's stability with little change in DNA-binding function. We mutated Cys29 to Ser and Ala in ME47's HLH to eliminate undesired disulfide formation; these mutations reduced E-box binding activity. To compensate, we fused the designed FosW leucine zipper to ME47 to increase the dimerization interface and improve protein stability and E-box targeting activity. This "franken-protein" MEF comprises the Max basic region, E47 HLH, and FosW leucine zipper—plus mutations that arose during PACE and rational design—and is a tractable, reliable protein in vivo and in vitro. Compared with ME47, MEF gives three-fold stronger binding to Ebox with four-fold increased specificity for E-box over nonspecific DNA. Generation of MEF demonstrates that combining rational design and continuous evolution can be a powerful tool for designing proteins with robust structure and strong DNA-binding function.




Email Address of Submitting Author


University of Toronto



ORCID For Submitting Author


Declaration of Conflict of Interest

No conflict

Version Notes

submitted and now in review (as of last night) for JACS