Diabetes Mutations Delineate an Atypical POU Domain in HNF-1α Young-In Chi, J.Daniel Frantz, Byung-Chul Oh, Lone Hansen, Sirano Dhe-Paganon, Steven E. Shoelson  Molecular Cell  Volume 10, Issue 5, Pages 1129-1137 (November 2002) DOI: 10.1016/S1097-2765(02)00704-9

Figure 1 Mutation Rate and Structure-Based Sequence Alignment (A) Mutations per codon (vertical black bars) are plotted versus linear sequence. Mutation rate (colored boxes) was plotted per element of predicted structure (blue, dimerization domain [Dim]; green, DNA binding domains; red, linkers and trans-activation domain). (B) Schematic representation of HNF-1α domain organization. (C) Structure-based sequence alignment of POU-homeodomains: human HNF-1α and HNF-1β (this work), human Oct-1 (Klemm et al., 1994), and rat Pit-1 (Jacobson et al., 1997). Individual α helices are labeled (POUS, Pα1–5; POUH, Hα1–3) and shaded green. Key contacts with DNA that are conserved between the HNF-1α, HNF-1β, Oct-1, and Pit-1 are white on a black background. Red stars denote residues of HNF-1α that interact at the POUS domain/POUH domain interface. MODY3 mutations are noted in black above the HNF-1α sequence; mutation A241T in HNF-1β is shown in red. Three additional sequences on the light gray background, from human (accession number AAH09259), mouse (AAH02212), and C. elegans (T33839), are predicted to contain POU domains based on predicted structural similarity to HNF-1α and conservation of relevant key residues (see Figure 5). Molecular Cell 2002 10, 1129-1137DOI: (10.1016/S1097-2765(02)00704-9)

Figure 2 Structure of the HNF-1α/DNA Complex (A) Two protein molecules (1 and 2) are bound to dsDNA. The five α helices of each POUS domain and three α helices in each POUH domain are labeled Pα1–5 and Hα1–3, respectively, as in Figure 1. (B) Comparison of the HNF-1α, Oct-1, and Pit-1 domains. Domains from HNF-1α, colored mauve, are positioned as they occur in the structure. POUH and POUS domains from Oct-1 in blue and Pit-1 in turquoise were independently superimposed. Arrows point to “atypical” extensions from the HNF-1α domains, not present in Oct-1 and Pit-1, that interact with each other. Molecular Cell 2002 10, 1129-1137DOI: (10.1016/S1097-2765(02)00704-9)

Figure 3 The Interface between POUS and POUH Domains (A) Interfaces viewed as “open books.” POUS and POUH domains were rotated 90° along a vertical axis, in opposite directions, to expose the buried surfaces between them. Contact residues are numbered and colored blue, or red if associated with MODY3. HNF-1α is at the top; Pit-1 is in complex with the growth hormone (GH) or prolactin (Prl) promoters (Scully et al., 2000). (B) Transcriptional activation by HNF-1α and variants with substitutions at the POUS-POUH domain interface. Standard luciferase-based transcriptional reporter assays were conducted using HeLa cells transfected with 0.1 or 0.5 μg of wild-type (wt) or mutated pcDNA(HNF-1α) and the luciferase reporter construct. Data from multiple experiments (± SEM) were plotted as relative luciferase activity according to position of the substituted residue in the POUS domain, flexible interdomain (ID), or POUH domain. MODY3 mutations are indicated. The Western blot (top) compares expression levels of wt and mutated forms of HNF-1α. Molecular Cell 2002 10, 1129-1137DOI: (10.1016/S1097-2765(02)00704-9)

Figure 4 DNA Interactions and MODY3 Mutations (A) Schematic summary of protein-DNA contacts. Residues from molecules 1 and 2 are in pink and green, respectively. POUH and POUS domain residues are in rectangles and ovals, respectively; filled shapes are MODY3 mutations. DNA bases within the consensus recognition motif are colored blue. (B) MODY3 mutations. Ribbon representation of HNF-1α in pink and DNA in orange. Side chains of residues affected by diabetes-associated missense mutations are displayed in green (predominantly affecting DNA binding), yellow (interdomain interactions), or blue (protein stability). Molecular Cell 2002 10, 1129-1137DOI: (10.1016/S1097-2765(02)00704-9)

Figure 5 Electrostatic Surface Potentials of HNF-1α and Additional Putative POU Domains Models of human HNF-1β, human AAH09259, and C. elegans T33839 were based on the structure of HNF-1α using the program MODELLER. Electrostatic surface potentials were shaded red, −7 kt/e to blue, +7 kt/e, using GRASP. The structure of HNF-1α bound to DNA and models of HNF-1β, AAH09259, and T33839 are similarly oriented, with putative DNA binding surfaces either facing the viewer (top panels) or rotated away by 180° (bottom panels). The modeled structure of murine AAH02212 (data not shown) is highly similar to that of AAH09259. Molecular Cell 2002 10, 1129-1137DOI: (10.1016/S1097-2765(02)00704-9)