The Intrinsically Disordered Loop in the USF1 bHLHZ Domain Modulates Its DNA-Binding Sequence Specificity in Hereditary Asthma

Abstract

USF1, a basic region/helix–loop–helix/leucine zipper (bHLHZ) transcription factor, binds to the E-box in the PAI-1 (plasminogen activator inhibitor) promoter. Two alleles containing the E-box control PAI-1 transcription; these alleles are termed “4G” and “5G” based on the G tract flanking E-box. USF1-governed transcription of PAI-1 is elevated in heritable asthma sufferers: the 4G/4G genotype has the highest plasma levels of PAI-1. While USF1 uses its basic region to bind E-box, we found that it uses its 12 amino-acid loop to recognize the flanking sequence and discern the single-nucleotide difference between the alleles. We used the bacterial one-hybrid and electrophoretic mobility shift assays to assess protein–DNA recognition, and circular dichroism to examine protein secondary structure. We mutated Ser233 and Thr234 in the USF1 bHLHZ loop to Ala to generate S233A and T234A. Interestingly, USF1 bHLHZ, S233A, and T234A prefer the 5G sequence (USF1 bHLHZ K d values 4.1 ± 0.3 nM and 7.0 ± 0.4 nM for 5G and 4G, respectively), whereas studies in stimulated human mast cells showed a preference for 4G. We replaced the 8 amino-acid loop of transcription factor Max bHLHZ with the 12 amino-acid USF1 loop: this mutant now distinguishes the 4G/5G polymorphismwhile Max bHLHZ does notconfirming that USF1 differentiation of the 4G/5G is driven by the loop.

Combining rational design and continuous evolution on minimalist proteins that target DNA

“…S2 for more details). 53,55 After extensive exploration of spacer lengths ranging from 7 to 25 base pairs (bp), we established that the -7GC E-box reporter was appropriate for use with ME47 in the context of PACE, and that this reporter was only activated by ME47 and not by any endogenous cellular proteins (Figs. 1, S2, S3, S4; "7" refers to spacer length in bp, "GC" refers to flanking sequence variants 2 bp outside of the E-box, Fig.…”

“…65,66 The bHLH and bHLHZ TFs possess significant intrinsic order, 49 including the HLH loop, which can critically contribute to a TF's DNA-binding target affinity and specificity. 55 Disordered protein structures have endured the rigorous process of natural selection and demonstrated their utility; for successful protein design, it is essential for us to exploit intrinsically disordered regions and proteins. Evolutionary methods that mimic the natural selection process, which has chosen intrinsically disordered regions/proteins time and again, are vital to successful protein design.…”

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

“…65,66 The bHLH and bHLHZ TFs possess significant intrinsic order, 49 including the HLH loop, which can critically contribute to a TF's DNA-binding target affinity and specificity. 55 and Ser29, for example; thus, mutants are named "ME47/XX" and list the 12th and 29th amino acids after the backslash. (We note that ME47 could be termed "ME47/RC" in this nomenclature;…”

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

“…S2 for more details). 53,55 After extensive exploration of spacer lengths ranging from 7 to 25 base pairs (bp), we established that the -7GC E-box reporter was appropriate for use with ME47 in the context of PACE, and that this reporter was only activated by ME47 and not by any endogenous cellular proteins (Figs. 1, S2, S3, S4; "7" refers to spacer length in bp, "GC" refers to flanking sequence variants 2 bp outside of the E-box, Fig.…”