Volume 15, Issue 4, Pages 647-657 (August 2004) An Atomic Resolution Model for Assembly, Architecture, and Function of the Dr Adhesins  Kirstine L. Anderson, Jason Billington, David Pettigrew, Ernesto Cota, Peter Simpson, Pietro Roversi, Ho An Chen, Petri Urvil, Laurence du Merle, Paul N. Barlow, M.Edward Medof, Richard A.G. Smith, Bogdan Nowicki, Chantal Le Bouguénec, Susan M. Lea, Stephen Matthews  Molecular Cell  Volume 15, Issue 4, Pages 647-657 (August 2004) DOI: 10.1016/j.molcel.2004.08.003

Figure 1 Characterization and Definition of the Constructs Used and Their Binding to DAF (A) Gel filtration profile of AfaE-III. Elution positions for predicted AfaE-III multimers are indicated. WT AfaE-III and AfaE-dsc traces are shown in red (top) and blue (bottom), respectively. (B) High-field methyl region of the 1D 1H NMR spectrum for AfaE-III. WT AfaE-III and AfaE-dsc are shown as red (top) and blue (bottom) profiles, respectively. (C) Primary amino acid sequences of AfaE-dsc, AfaE-III, and DraE showing the numbering schemes chosen. Amino acid positions (identified from chemical shift mapping, mutagenesis experiments, or both) implicated in DAF binding are shown in green, orange, and purple, respectively. The natural differences between AfaE-III and DraE are highlighted in blue, and an additional point mutation introduced into DraE during cloning for large-scale production is shown in yellow. The complementing strand is shaded in gray, and the engineered turn is underlined. (D) Microscopic examination of AfaE-dsc-coated beads interacting with HeLa cells. AfaE-dsc-coated beads associated with cells are visualized by light (middle) and immunofluorescence (right) microscopy. Control experiment with BSA-coated beads visualized by light microscopy (left). Molecular Cell 2004 15, 647-657DOI: (10.1016/j.molcel.2004.08.003)

Figure 2 Three-Dimensional Structure of AfaE-dsc (A) Stereoview showing Cα traces representing the ensemble of NMR-derived structures. (B) Ribbon representation of a representative structure for AfaE-dsc. All β strands are shown in blue, with the exception of the self-complementing strand, which is blue/white. (C) Sequence comparison of the engineered C-terminal strand of AfaE-dsc with the N-terminal residues of members of the Dr family of adhesins. The conserved G, T, and L are highlighted. (D) The binding cleft of the self-complementing strand Gd. The body of the protein is represented using a surface map, with the exception of strand Gd in AfaE-dsc, which is shown as a ball and stick representation. (E) Plot of 2D 1H-15N steady-state heteronuclear NOE value against sequence number for AfaE-dsc. Molecular Cell 2004 15, 647-657DOI: (10.1016/j.molcel.2004.08.003)

Figure 3 Topology Diagrams of the Atomic Structure and Proposed Topology for All Dr Family Adhesin Polymers (A) AfaE-dsc. All β strands are shown in sky blue, with the exception of the self-complementing strand, which is sky blue/white. 310 helices are shown in dark blue, and loops are shown as black lines. (B) Topology diagram for the proposed DSC/DSE-induced AfaE-III oligomer. Molecular Cell 2004 15, 647-657DOI: (10.1016/j.molcel.2004.08.003)

Figure 4 Dr Family Adhesin Architecture (A) Sequence comparison of a putative AfaD-III strand with F β strand from AfaE-dsc together with the AfaE-III donating strand Gd in register. (B) Gel filtration and 1D NMR spectra (right) illustrating the removal of AfaD-III aggregates upon interaction with a peptide corresponding to the N-terminal 18 amino acids of AfaE-III (AfaEN1–18) and engineering of a self-complemented AfaD (AfaD-dsc). (C) Electron micrograph of “thin” fimbrial Dr adhesin together with schematic representations. Molecular Cell 2004 15, 647-657DOI: (10.1016/j.molcel.2004.08.003)

Figure 5 The Interaction between DAF and AfaE (A) Binding experiments illustrating the interaction between DAF and AfaE/AfaE-dsc. SPR binding curves of the interaction between DAF1234 and AfaE. The inset shows a Scatchard plot of equilibrium responses derived from the data presented in the panel. A linear fit of these data is shown and yields an estimate for the dissociation constant (Kd) of 16 μM. (B) AfaE-dsc competition for DAF binding. DAF (12.5 μM) binding to AfaE-III in the absence (−) and presence (+) of AfaE-dsc (50 μM). The data presented are the results of three independent experiments over two independent AfaE-III surfaces with ∼5800 RU (left) and ∼5300 RU (right) coupled on each. Molecular Cell 2004 15, 647-657DOI: (10.1016/j.molcel.2004.08.003)

Figure 6 Identification of the Mutual Interaction Surfaces for AfaE-dsc and DAF23 (A) A region of 2D 1H-15N HNCO spectra for 15N,13C AfaE-dsc (black) and in the presence of 15N-labeled DAF23 at the molar ratio 1:1 (red). Resonances with perturbed line widths or resonance positions are indicated. (B) Region of 2D 1H-15N HSQC spectra for 15N-labeled DAF23 (black) in presence of unlabeled AfaE-dsc at the molar ratio 1:1 (red). Resonances with perturbed line widths or resonance positions are indicated. (C) Solvent accessible surface representations of AfaE-dsc with perturbed residues colored in green. Selective assignments are labeled in order to delineate the binding surface. (D) Solvent accessible surface representation of DAF1234 (PDB code 1OJV) with perturbed residues colored in orange and assigned. Selective assignments are labeled. (E) Alternative pathway C3 convertase hemolytic assay. Data represent an average of three experiments (Z = 1.9; DAF234 = 50 ng/ml). Levels of inhibition in the presence of DAF together with increasing concentrations of AfaE-dsc are shown in blue, while data for AfaE-dsc alone are in red. Molecular Cell 2004 15, 647-657DOI: (10.1016/j.molcel.2004.08.003)