Probing Disordered Region Influence on Pdx1 Binding to Natural Promoters and Near‐Consensus Sites

Abstract

Pdx1 is a well‐known protein in the set of β‐cell‐specific transcription factors (TFs), involved in the production of insulin and islet amyloid polypeptide (IAPP) hormones. Like many TFs, Pdx1 contains regions of disorder whose function in transcription has not been adequately studied. It is known that the Pdx1 homeodomain (HD) binds to a core DNA recognition sequence containing the tetranucleotide TAAT, for which its consensus binding site is reported as 5´‐CTCTAAT(T/G)AG‐3´. Naturally, insulin is expressed in much higher levels than IAPP, perhaps owing to the affinity of Pdx1 to its target sites. Alignment of human insulin and iapp promoter elements highlights the single nucleobase substitution from 5´‐CTAAT‐3´ to 5´‐TTAAT‐3´, respectively. We hypothesize that the interactions mediated by the disordered tails provide additional target specificity by interacting with DNA sequences upstream of the TAAT motif. Here we report a combination of ITC, NMR spectroscopy, and multi‐microsecond MD calculations of Pdx1 variants that define its interactions with a panel of natural promoter and consensus‐derived sequences. Our results point to a small preference of the Pdx1 HD for 5´‐CTAAT‐3´. Molecular mechanics calculations, corroborated by experimental NMR data, lead to a rational explanation for sequence discrimination at this position. More significantly, our computational studies suggest that interactions between the disordered region of Pdx1 and the major groove of the DNA are responsible for sequence identification upstream of the core motif. Taken together, our results suggest a molecular mechanism where the disordered regions of Pdx1 are responsible for establishing differential affinity to elements from the insulin and iapp promoter sequences.