Structural variations in species B adenovirus fibers impact CD46 association - PubMed (original) (raw)
Structural variations in species B adenovirus fibers impact CD46 association
Lars Pache et al. J Virol. 2008 Aug.
Abstract
A majority of species B adenoviruses (Ads) use CD46 as their primary receptor; however, the precise mechanisms involved in the binding of different Ad types to CD46 have not been resolved. Although previous studies indicate close similarities between two members of species B2 Ads in their usage of CD46, our current investigations revealed a surprisingly low CD46 binding affinity of the species B1 Ad16 fiber knob (equilibrium dissociation constant of 437 nM). We determined the crystal structure of the Ad16 fiber knob and constructed a model of this protein in complex with CD46. A comparison of this model to that of the CD46-Ad11 complex revealed structural differences in the FG and IJ loops that are part of the CD46 binding site. An analysis of a panel of recombinant fiber knobs with mutations targeting these regions in Ad16 and Ad11 uncovered a major contribution of the FG loop on CD46 binding. Two extra residues in the FG loop of the Ad16 fiber significantly reduce receptor interaction. Although avidity effects permit the use of CD46 on host cells by Ad16, virus binding occurs with lower efficiency than with B2 Ad types. The longer FG loop of the Ad16 fiber knob also is shared by other species B1 Ad fibers and, thus, may contribute to the low CD46 binding efficiencies observed for these Ad types. Our findings provide a better understanding of how different Ad types associate with CD46 and could aid in the selection of specific Ad fibers for more efficient Ad gene delivery vectors.
Figures
FIG. 1.
Biacore analysis of sCD46 binding to Ad fiber knob protein. sCD46 was injected at various concentrations over a sensor surface containing the immobilized Ad11 (A) or Ad16 (B) fiber knob. Measured response curves were overlaid with a global fit to a 1:1 interaction model to obtain kinetic constants. Sensograms show one of duplicate sets of injections used for curve fitting. (C) The amount of sCD46 bound to the Ad16 fiber knob at equilibrium was plotted against the concentration of injected sCD46. The KD was obtained by nonlinear curve fitting of the Langmuir binding model. Values in parentheses represent the error of the fitting procedure in the final digit.
FIG. 2.
Functional analysis of fiber knob binding to CD46 on cells. (A) Stably transfected CHO-K1 cells expressing either CD46 or an empty vector (NULL) were incubated in the presence of 30,000 Ad11 or Ad5.16F virus particles per cell. The mean fluorescence intensity, measured by flow cytometry, was normalized to 100 for CD46-expressing cells incubated with virus. (B and C) CD46-expressing CHO cells were preincubated with different amounts of the Ad11 (FK11) or Ad16 (FK16) fiber knob prior to incubation with Ad11 (B) or Ad5.16F (C) virus. After subtracting the background fluorescence of cells in the absence of virus, the fluorescence of cells incubated with virus in the absence of fiber knob was defined as 100%. Data points represent the means from triplicate samples. Error bars represent standard deviations.
FIG. 3.
Structural comparison of the Ad16 and Ad11 fiber knobs. (A) Ribbon diagram showing the crystal structure of the Ad16 fiber knob trimer viewed down the threefold axis. (B) Stereo diagram showing the quality of 2_Fo_−Fc electron density for residues 289 to 293 of the Ad16 fiber contoured at 1.0σ. (C) A ribbon diagram showing two subunits of the Ad16 fiber knob (red) superimposed on the complex of the Ad11 fiber knob (green) and SCR1-SCR2 of CD46 (blue). FG, HI, and IJ loops of Ad16 are indicated. (D) Structure-based alignment of the amino acids comprising the exposed regions of three surface loops that likely are involved in CD46 binding.
FIG. 4.
Sequences of recombinant fiber knobs containing mutations. Wild-type and mutant recombinant fiber knob proteins used in the virus binding competition experiments are shown in a structure-based alignment. The expression and trimerization of fiber knob proteins was verified by seminative gel electrophoresis. Mutated residues are highlighted. F16K, Ad16 fiber knob; F11K, Ad11 fiber knob.
FIG. 5.
Comparison of Ad16 fiber knobs binding to CD46. (A, C, and E) Structural comparison of the CD46 binding loops with associated residues in Ad16 and Ad11 fiber knobs (FK16 and FK11, respectively). Corresponding Ad11 residues are indicated in parentheses. CD46 is shown in dark gray, Ad16 in black, and Ad11 in light gray. (A) Parts of the HI loop in complex with E63 of CD46, R309 and R310 of Ad16, and the corresponding arginines of Ad11, R279 and R280, shown as stick representations. (C) IJ loop with R69 and L72 of CD46; T212 of Ad16; and V304, Q305, and M184 of Ad11 shown as stick representations. (E) FG loop with T64 of CD46 and Y275 and A276 of Ad16 shown as stick representations. (B, D, and F) Functional analysis of Ad16 fiber knob mutants. CHO cells expressing CD46 were preincubated with various concentrations of fiber knob mutants targeting the HI (B), IJ (D), or FG (F) loop of the Ad16 fiber knob prior to the addition of 30,000 Ad5.16F virus particles per cell. The mean fluorescence intensity was measured by flow cytometry. The background fluorescence of cells in the absence of virus was subtracted, and the fluorescence of cells incubated in the presence of virus without preincubation with a fiber knob was defined as 100%. Data points represent the means from triplicate experiments, with standard deviations shown by error bars. The plots of FG loop mutants were overlaid with a curve (dashed line) showing inhibition by the Ad11 fiber knob for comparison.
FIG. 6.
Effect of mutations in the DG loop on Ad11 binding to CD46. (A) Virus binding competition experiments with insertion mutants targeting the DG loop of the Ad11 fiber knob were performed as described in the legend to Fig. 5. Data points represent the means from triplicate experiments, with standard deviations shown by error bars. (B) Comparison of the kinetic constants of the Ad11 and Ad16 fiber knobs (FK11 and FK16, respectively) to those of FK11 246AA247. The overall affinity (KD) and kinetics (ka and Kd) of FK11 246AA247 were analyzed by Biacore, as described for the Ad16 fiber knob. Kinetic constants were obtained by curve fitting using a 1:1 interaction model. The KD calculated from association and dissociation rates was verified by equilibrium binding analysis. Values in parentheses represent the errors of the fitting procedure in the final digit.
FIG. 7.
Sequence comparison of species B Ad fiber knobs. (A) Phylogenetic tree diagram of the amino acid sequence of species B Ad fiber knob domains. Sequences were aligned from the TLWT sequence that marks the start of the knob domain to the stop codon. (B) Sequence alignment of the partial FG loop of the Ad16 fiber knob (residues 275 to 281) with the corresponding sequences of other species B Ads. Ad16, Ad3, and Ad7h differ from the other Ad serotypes by having two extra residues in the FG loop.
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