Abstract
Minimalist hybrids comprising the DNA-binding domain of bHLH/PAS (basic-helix-loop-helix/Per-Arnt-Sim) protein Arnt fused to the leucine zipper (LZ) dimerization domain from bZIP (basic region-leucine zipper) protein C/EBP were designed to bind the E-box DNA site, CACGTG, targeted by bHLHZ (basic-helix-loop-helix-zipper) proteins Myc and Max, as well as the Arnt homodimer. The bHLHZ-like structure of ArntbHLH-C/EBP comprises the Arnt bHLH domain fused to the C/EBP LZ: i.e. swap of the 330 aa PAS domain for the 29 aa LZ. In the yeast one-hybrid assay (Y1H), transcriptional activation from the E-box was strong by ArntbHLH-C/EBP, and undetectable for the truncated ArntbHLH (PAS removed), as detected via readout from the HIS3 and lacZ reporters. In contrast, fluorescence anisotropy titrations showed affinities for the E-box with ArntbHLH-C/EBP and ArntbHLH comparable to other transcription factors (K d 148.9 nM and 40.2 nM, respectively), but only under select conditions that maintained folded protein. Although in vivo yeast results and in vitro spectroscopic studies for ArntbHLH-C/EBP targeting the E-box correlate well, the same does not hold for ArntbHLH. As circular dichroism confirms that ArntbHLH-C/EBP is a much more strongly α-helical structure than ArntbHLH, we conclude that the nonfunctional ArntbHLH in the Y1H must be due to misfolding, leading to the false negative that this protein is incapable of targeting the E-box. Many experiments, including protein design and selections from large libraries, depend on protein domains remaining well-behaved in the nonnative experimental environment, especially small motifs like the bHLH (60–70 aa). Interestingly, a short helical LZ can serve as a folding- and/or solubility-enhancing tag, an important device given the focus of current research on exploration of vast networks of biomolecular interactions.
Highlights
We utilized our minimalist design strategy to reduce the size and structural complexity of native transcription factors while maximizing retention of DNA-binding function
We focus on three families of transcriptional activators: basic region/leucine zipper, basic helix-loop-helix/leucine zipper, and basic helix-loophelix/Per-Arnt-Sim
Because of the importance of E-box regulation, we applied our minimalist strategy toward design of simplified proteins that target the E-box based on the basic region/leucine zipper (bZIP), basic helix-loop-helix/leucine zipper (bHLHZ), and bHLH/PAS scaffolds (Fig. 1): our aim is to generate smaller proteins of simplified structure compared to their native counterparts, while still retaining DNA-binding function
Summary
We utilized our minimalist design strategy to reduce the size and structural complexity of native transcription factors while maximizing retention of DNA-binding function. The ubiquitous bHLHZ Myc, Max, and Mad transcriptional activator network serves as a master regulator of the E-box site and is involved in 70% or more of known cancers and tumors [13] This network is a good starting point for design, for there exists much experimental data including highresolution structures [7,8,9]. Because of the importance of E-box regulation, we applied our minimalist strategy toward design of simplified proteins that target the E-box based on the bZIP, bHLHZ, and bHLH/PAS scaffolds (Fig. 1): our aim is to generate smaller proteins of simplified structure compared to their native counterparts, while still retaining DNA-binding function. Compared with the native Arnt bHLH/PAS domain at over 400 amino acids (fulllength Arnt is almost 1000 aa), our Arnt derivatives comprise 66 or 98 aa, and are accessible by either chemical synthesis or bacterial expression
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