Crystallographic structure of porcine adenovirus type 4 fiber head and galectin domains

Abstract

Adenovirus isolate NADC-1, a strain of porcine adenovirus type 4, has a fiber containing an N-terminal virus attachment region, shaft and head domains, and a C-terminal galectin domain connected to the head by an RGD-containing sequence. The crystal structure of the head domain is similar to previously solved adenovirus fiber head domains, but specific residues for binding the coxsackievirus and adenovirus receptor (CAR), CD46, or sialic acid are not conserved. The structure of the galectin domain reveals an interaction interface between its two carbohydrate recognition domains, locating both sugar binding sites face to face. Sequence evidence suggests other tandem-repeat galectins have the same arrangement. We show that the galectin domain binds carbohydrates containing lactose and N-acetyl-lactosamine units, and we present structures of the galectin domain with lactose, N-acetyl-lactosamine, 3-aminopropyl-lacto-N-neotetraose, and 2-aminoethyl-tri(N-acetyl-lactosamine), confirming the domain as a bona fide galectin domain.

FIG. 1.

(A) Schematic drawing of the domain organization of porcine adenovirus type 4 NADC-1 strain fiber. The predicted virus-binding tail is shown in black, the shaft domain in white, the head domain in light gray, the RGD-containing domain in white, and the tandem-repeat galectin domain in dark gray. The putative integrin-binding RGD sequence is indicated with an asterisk. Electron microscopy images of the fiber indicate flexibility between the head and C-terminal domains; therefore, the C-terminal domains are drawn in different orientations. (B) Sequence alignment of tandem-repeat galectins. Human galectin 4 (HGal-4; UNIPROT code P56470), human galectin 8 (H-Gal8; O00214), and human galectin 9 (HGal-9; O00182) are shown. Secondary structural elements of the porcine adenovirus 4 galectin domain are indicated.

FIG. 2.

Structure of the porcine adenovirus 4 (NADC-1 strain) fiber head. (A) The monomer is depicted with beta strands colored yellow, helices red, and loops green. Beta strands are labeled with letters, and alpha helices are numbered. (B) A view from the top along the 3-fold axis of the trimer. Monomers are colored red, green, and yellow. (C) Comparison of the porcine adenovirus type 4 fiber head structure (yellow) to that of the human adenovirus type 12 fiber head (blue) bound to the CAR D1 domain (red). (D) Comparison of the porcine adenovirus type 4 fiber head structure (yellow) to that of the human adenovirus type 11 fiber head (blue) bound to the CD46 short consensus repeats 1 and 2 (red). The DG, HI, and IJ loops of the adenovirus fiber head are shown and labeled; residues of the human adenovirus type 11 fiber head involved in the interaction also are labeled. (E and F) Electrostatic potential surfaces (blue, positive; red, negative) of human adenovirus type 37 fiber head in complex with sialyl-lactose (E; shown as sticks) and the porcine adenovirus type 4 fiber head (F).

FIG. 3.

Structure of the porcine adenovirus type 4 (NADC-1 isolate) fiber galectin domain. (A) The monomer is depicted with beta strands colored yellow, helices in red, and loops in green. N and C termini are labeled. (B) Superposition of the N-terminal (blue) onto the C-terminal (red) carbohydrate recognition domain. The S3-S4 (a), S4-S5 (b), and cF5-cS2 (c) loops are labeled.

FIG. 4.

Glycan binding specificity of adenovirus vectors. Screening of a glycan array with an adenovirus type 5-based vector (upper) and the same vector modified with the porcine adenovirus type 4, head, RGD, and galectin domains (lower). The plot shows the average relative fluorescence units (RFU; y axis) for the six addresses of each glycan versus the glycan number (x axis) as bars. Standard errors of the means in the fluorescence for the six addresses are indicated for each glycan. Carbohydrates giving a clear signal above the background level are drawn schematically as follows: black hexagons represent galactose, white hexagons represent glucose, and gray hexagons represent N-acetyl-glucosamine (the anomeric carbon is placed at the right side, and the numbering is clockwise). Thick and thin bars represent alpha and beta bonds, respectively.

FIG. 5.

Crystal structures of porcine adenovirus 4 galectin domain bound to lactose (A, E, I), N-acetyl-lactosamine (B, F, J), 3-aminopropyl-lacto-N-neotetraose (C, G, K), and 2-aminoethyl-tri(N-acetyl-lactosamine) (D, H, L, M). Panels A to D show unbiased electron density after molecular replacement contoured at 2σ (only the lactose molecule bound to the N-terminal carbohydrate domain is shown), panels E to H show overviews of the structures [only chain A of the structure with 2-aminoethyl-tri(N-acetyl-lactosamine) model is shown], and panels I to L show stick representations of the carbohydrates (carbon, green) and their interaction residues (carbon, white). Hydrogen bonds are shown as black dashed lines. Residues involved in the interaction and carbohydrate residues are labeled. (M) Surface representation of the galectin binding site for 2-aminoethyl-tri(N-acetyl-lactosamine).

FIG. 6.

Binding affinities of the porcine adenovirus type 4 galectin domain for carbohydrates obtained by surface plasmon resonance experiments. Sensorgrams and binding curves are shown for lacto-N-neotetraose (A), 3-aminopropyl-lacto-N-neotetraose (B), 2-azidoethyl-di(N-acetyl-lactosamine) (C), and 2-aminoethyl-tri(N-acetyl-lactosamine) (D). On the y axis, the SPR response in μRIU is plotted (normalized by division by the molecular weight of the carbohydrate and multiplied by 100); on the horizontal axis, the time in seconds for the sensorgrams and the carbohydrate concentration in mM for the binding curves are plotted. Calculated dissociation constants also are shown.

FIG. 7.

Comparison to other tandem-repeat galectins. (A) Structural superposition of the N-terminal carbohydrate recognition domain of human galectin-9 (blue) on the N-terminal carbohydrate recognition domain of porcine adenovirus 4 galectin (red), both in complex with tri(N-acetyl-lactosamine). The oxygen and nitrogen atoms of the carbohydrates are colored in red and blue, respectively, and the carbon atoms are green (for the carbohydrate bound to human galectin-9) or yellow (for the carbohydrate bound to porcine adenovirus 4 galectin). (B) Schematic drawing of homodimeric prototype galectin domains (left) of the current model for tandem-repeat galectin domains (middle) and our new model for tandem-repeat-type galectins (right) based on the structures determined.