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Structural basis for variation in adenovirus affinity for the cellular coxsackievirus and adenovirus receptor
Jason Howitt1, Maria C Bewley, Vito Graziano
1Biology Department, Brookhaven National Laboratory, Upton, New York 11973, USA.
Abstract:
The majority of adenovirus serotypes can bind to the coxsackievirus and adenovirus receptor (CAR) on human cells despite only limited conservation of the amino acid residues that comprise the receptor-binding sites of these viruses. Using a fluorescence anisotropy-based assay, we determined that the recombinant knob domain of the fiber protein from adenovirus serotype (Ad) 2 binds the soluble, N-terminal domain (domain 1 (D1)) of CAR with 8-fold greater affinity than does the recombinant knob domain from Ad12. Homology modeling predicted that the increased affinity of Ad2 knob for CAR D1 could result from additional contacts within the binding interface contributed by two residues, Ser408 and Tyr477, which are not conserved in the Ad12 knob. Consistent with this structural model, substitution of serine and tyrosine for the corresponding residues in the Ad12 knob (P417S and S489Y) increased the binding affinity by 4- and 8-fold, respectively, whereas the double mutation increased binding affinity 10-fold. X-ray structure analysis of Ad12 knob mutants P417S and S489Y indicated that both substituted residues potentially could form additional hydrogen bonds across the knob-CAR interface. Structural changes resulting from these mutations were highly localized, implying that the high tolerance for surface variation conferred by the stable knob scaffold can minimize the impact of antigenic drift on binding specificity and affinity during evolution of virus serotypes. Our results suggest that the interaction of knob domains from different adenovirus serotypes with CAR D1 can be accurately modeled using the Ad12 knob-CAR D1 crystal structure as a template.
Insights
Adenovirus serotypes bind human cells via the coxsackievirus and adenovirus receptor (CAR). Specific mutations in adenovirus type 2 (Ad2) fiber knob enhance binding affinity to CAR, revealing insights into viral evolution.
Area of Science:
- Virology
- Structural Biology
- Molecular Interactions
Background:
- Adenovirus serotypes exhibit variable binding to the coxsackievirus and adenovirus receptor (CAR) on human cells.
- Limited conservation exists in receptor-binding sites across different adenovirus serotypes.
- Understanding these interactions is crucial for viral entry mechanisms and serotype evolution.
Purpose of the Study:
- To investigate the differential binding affinities of adenovirus serotype 2 (Ad2) and adenovirus type 12 (Ad12) knob domains to the coxsackievirus and adenovirus receptor (CAR) domain 1 (D1).
- To elucidate the structural basis for enhanced Ad2 knob affinity to CAR D1.
- To explore the implications of these findings for adenovirus evolution and serotype diversification.
Main Methods:
- Utilized a fluorescence anisotropy-based assay to quantify binding affinities between recombinant knob domains and soluble CAR D1.
- Employed homology modeling to predict structural contributions to binding affinity.
- Performed site-directed mutagenesis on Ad12 knob domain residues (P417S, S489Y) and analyzed binding affinity changes.
- Conducted X-ray structure analysis of Ad12 knob mutants to examine structural alterations at the protein interface.
Main Results:
- Ad2 knob domain exhibited an 8-fold greater binding affinity to CAR D1 compared to Ad12 knob domain.
- Homology modeling suggested that residues Ser408 and Tyr477 in Ad2 knob contribute to increased affinity.
- Mutagenesis of Ad12 knob (P417S, S489Y) significantly increased binding affinity (4-fold for P417S, 8-fold for S489Y, 10-fold for double mutant).
- X-ray crystallography revealed that these mutations potentially form additional hydrogen bonds at the knob-CAR interface, with localized structural changes.
Conclusions:
- Specific amino acid substitutions in the adenovirus fiber knob domain can significantly enhance binding affinity to the coxsackievirus and adenovirus receptor (CAR).
- The stable knob scaffold tolerates surface variations, allowing for minimized impact of antigenic drift on binding specificity and affinity during serotype evolution.
- The Ad12 knob-CAR D1 crystal structure serves as a reliable template for modeling interactions of other adenovirus serotype knob domains with CAR D1.