title: Reengineering natural design by rational design and in vivo library selection: the HLH subdomain in bHLHZ proteins is a unique requirement for DNA-binding function.
abstract: To explore the role of the HLH subdomain in bHLHZ proteins, we designed sets of minimalist proteins based on bHLHZ protein Max, bHLH/PAS protein Arnt and bZIP protein C/EBP. In the first, the Max bHLH and C/EBP leucine zipper were fused such that the leucine heptad repeats were not in register; therefore, the protein dimerization interface was disrupted. Max1bHLH-C/EBP showed little ability to activate transcription from the E-box (5'-CACGTG) in the yeast one-hybrid assay, and no E-box binding by quantitative fluorescence anisotropy. Max1bHLH-C/EBP's activity was significantly improved after library selection (three amino acids randomized between HLH and leucine zipper), despite the Max bHLH and C/EBP zipper still being out of register: a representative mutant gave a high nanomolar K(d) value for E-box binding. Thus, selection proved to be a powerful tool for salvaging the flawed Max1bHLH-C/EBP, although the out-of-register mutants still did not achieve the strong DNA-binding affinities displayed by their in-register counterparts. ArntbHLH-C/EBP hybrids further demonstrated the importance of maintaining register, as out-of-register mutants showed no E-box-responsive activity, whereas the in-register hybrid showed moderate activity. In another design, we eliminated the HLH altogether and fused the Max basic region to the C/EBP zipper to generate bZIP-like hybrids. Despite numerous designs and selections, these hybrids possessed no E-box-responsive activity. Finally, we tested the importance of the loop sequence in MaxbHLHZ by fluorescence and circular dichroism. In one mutant, the loop was shortened by two residues; in the other, the Lys57:DNA-backbone interaction was abolished by mutation to Gly57. Both showed markedly decreased E-box-binding relative to MaxbHLHZ. Our results suggest that, in contrast to the more rigid bZIP, the HLH is capable of significant conformational adaptation to enable gene-regulatory function and is required for protein dimerization and positioning the basic region for DNA recognition.
authors.affiliation: Department of Chemistry, University of Toronto, Mississauga, ON L5L1C6, Canada.
authors.forename: Jing
authors.initials: J
authors.lastname: Xu
authors.forename: Antonia T
authors.initials: AT
authors.lastname: De Jong
authors.forename: Gang
authors.initials: G
authors.lastname: Chen
authors.forename: Hiu-Kwan
authors.initials: HK
authors.lastname: Chow
authors.forename: Christopher O
authors.initials: CO
authors.lastname: Damaso
authors.forename: Adrian
authors.initials: A
authors.lastname: Schwartz Mittelman
authors.forename: Jumi A
authors.initials: JA
authors.lastname: Shin
mesh_terms: D051784:Aryl Hydrocarbon Receptor Nuclear Translocator / Q000235:genetics* / Q000378:metabolism; D050976:Basic-Leucine Zipper Transcription Factors / Q000235:genetics* / Q000378:metabolism; D002942:Circular Dichroism; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D005454:Fluorescence Polarization; D018257:Helix-Loop-Helix Motifs / Q000235:genetics*; D009154:Mutation / Q000235:genetics; D011487:Protein Conformation; D015202:Protein Engineering / Q000379:methods*; D020798:Two-Hybrid System Techniques
chemical_list: D050976:Basic-Leucine Zipper Transcription Factors; D004268:DNA-Binding Proteins; C067850:Myc associated factor X; D051784:Aryl Hydrocarbon Receptor Nuclear Translocator