Thermodynamic and Structural Determinants of Differential Pdx1 Binding to Elements from the Insulin and IAPP Promoters

Abstract

In adult mammals, the production of insulin and other peptide hormones, such as the islet amyloid polypeptide (IAPP), is limited to β-cells due to tissue-specific expression of a set of transcription factors, the best known of which is Pdx1. Like many homeodomain transcription factors, Pdx1 binds to a core DNA recognition sequence containing the tetranucleotide 5´-TAAT-3´; its consensus recognition element is 5´-CTCTAAT(T/G)AG-3´. Currently, a complete thermodynamic profile of Pdx1 binding to near-consensus and native promoter sequences has not been established, obscuring the mechanism of target site selection by this critical transcription factor. Strikingly, while Pdx1 responsive elements in the human insulin promoter conform to the pentanucleotide 5´-CTAAT-3´ sequence, the Pdx1 responsive elements in the human iapp promoter all contain a substitution to 5-TTAAT-3´. The crystal structure of Pdx1 bound to the consensus nucleotide sequence does not explain how Pdx1 identifies this natural variation, if it does at all. Here we report a combination of isothermal calorimetric titrations, NMR spectroscopy, and extensive multi-microsecond molecular dynamics calculations of Pdx1 that define its interactions with a panel of natural promoter elements and consensus-derived sequences. Our results show a small preference of Pdx1 for a Cyt base 5´ relative to the core TAAT promoter element. Molecular mechanics calculations, corroborated by experimental NMR data, lead to a rational explanation for sequence discrimination at this position. Taken together, our results suggest a molecular mechanism for differential Pdx1 affinity to elements from the insulin and iapp promoter sequences.

Generating NMR chemical shift assignments of intrinsically disordered proteins using carbon-detected NMR methods

“…Pdx1 is a transcription factor, known to play an important role in pancreatic development and β-cell maturation, as well as the regulation of insulin production in mature organisms. [26–31] The well folded homeodomain component of Pdx1 is well studied,[16, 29–31] but only represents a small fraction of the total polypeptide. Full length Pdx1 includes both Nand C-terminal intrinsically disordered regions that are responsible for mediating known protein-protein interactions[32, 33] and which include sites of polymorphism that correlate with type-2 diabetes in humans.…”

“…[45] As a first step towards evaluating the structure of Pdx1-C, we have calculated its secondary structure propensity (SSP), [20] based on the chemical shift assignments we report here, and compared the results to those we have previously reported for the cooperatively folding homeodomain of Pdx1. [16] Figure 5 shows the difference between the offset from random coil for the 13 C α and 13 C β nuclei (ΔδCα - ΔδCβ) for each residue in Pdx1-homeodomain (Fig. 5A) and Pdx1-C (Fig.…”

“…The recombinant plasmid was transformed into BL21(DE3) competent cells for protein overexpression. Cell growth conditions and the protein purification protocol for Pdx1-C were identical to our previously reported procedures for the Pdx1-homeodomain,[16] except as noted below. As a final step to ensure full purification, Pdx1-C was subjected to size exclusion chromatography using a HiPrep 26/60 Sephacryl S-200 HR column (GE Life Sciences) in 50 mM Tris pH 7.5, 150 mM NaCl, 5 mM β-mercaptoethanol, and 1 mM EDTA.…”

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