Structural Analysis of the Mechanism of Adenovirus Binding to Its Human Cellular Receptor, CAR
10.1126/science.286.5444.1579
Crossref journal-article
American Association for the Advancement of Science (AAAS)
Science (221)
Abstract

Binding of virus particles to specific host cell surface receptors is known to be an obligatory step in infection even though the molecular basis for these interactions is not well characterized. The crystal structure of the adenovirus fiber knob domain in complex with domain I of its human cellular receptor, coxsackie and adenovirus receptor (CAR), is presented here. Surface-exposed loops on knob contact one face of CAR, forming a high-affinity complex. Topology mismatches between interacting surfaces create interfacial solvent-filled cavities and channels that may be targets for antiviral drug therapy. The structure identifies key determinants of binding specificity, which may suggest ways to modify the tropism of adenovirus-based gene therapy vectors.

Bibliography

Bewley, M. C., Springer, K., Zhang, Y.-B., Freimuth, P., & Flanagan, J. M. (1999). Structural Analysis of the Mechanism of Adenovirus Binding to Its Human Cellular Receptor, CAR. Science, 286(5444), 1579–1583.

Authors 5
  1. Maria C. Bewley (first)
  2. Karen Springer (additional)
  3. Yian-Biao Zhang (additional)
  4. Paul Freimuth (additional)
  5. John M. Flanagan (additional)
References 32 Referenced 373
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  4. The knob fiber protein (Ad12 knob) and the NH 2 -terminal fragment (residues 22 to 125) of the cellular receptor (CAR D1) were expressed in E. coli and purified as described previously (3). Purified proteins were proteolysed separately with trypsin (10 mg/ml). The 1:3 (trimeric knob: CAR D1) complex was formed at room temperature and purified by anion exchange chromatography. Crystals of Ad12 knob were grown at room temperature with the sitting drop vapor diffusion method from an Ad12 knob solution of 20 mg/ml suspended over a reservoir of 26% polyethylene glycol (PEG) 3350. Showers of small poorly ordered crystals grew over the course of a week and were used to seed a 10-μl drop containing equal volumes of Ad12 knob and 26% PEG 3350 over a reservoir of 26% PEG 3350. Typically crystals grew overnight as rhombohedral plates (0.5 mm by 0.5 mm by 0.2 mm). They were flash cooled at 99 K with 50% PEG 3350 as a cryoprotectant. Crystals of the complex were grown at room temperature with the sitting drop vapor diffusion method from 0.9 M ammonium sulfate in 100 mM MES (pH 6.2). Mercury was introduced into the Ad12 knob–CAR D1 complex by soaking a single CAR D1-knob complex crystal in 10 mM thimerosal for 6 hours. Crystals were flash cooled at 99 K with 50% ethylene glycol as a cryoprotectant. Data were processed with the HKL Program Suite (22). The structure of Ad12 knob was solved by molecular replacement (23) with a monomer of Ad5 knob [Protein Data Bank (PDB) accession number 1KNB.PDB] as a search model. Six monomers were placed and their positions were refined with rigid body refinement. Simulated annealing protocols in CNS (24) with the use of tight NCS restraints were punctuated by rounds of model building. The refinement statistics are shown in Table 1. The structure of the Ad12 knob–CAR D1 complex was determined with a combination of single isomorphous replacement (SIR) solvent flattening and molecular replacement. The refined structure of the Ad12 knob monomer was used as a search model in molecular replacement. A single clear solution was found corresponding to a monomer in the asymmetric unit such that the biological three-fold axis was coincident with the crystallographic axis. The heavy atom position was determined by visual inspection of a difference map with model phases and its position was refined with MLPHARE (23). Phase combination with the use of the Ad12 knob structure and the experimental SIR phases followed by solvent flattening with the program DM (23) resulted in a map with a mean figure of merit (FOM) of 0.74. The structure was refined with CNS punctuated by rounds of model building.
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  11. Single-letter abbreviations for the amino acid residues are as follows: A Ala; C Cys; D Asp; E Glu; F Phe; G Gly; H His; I Ile; K Lys; L Leu; M Met; N Asn; P Pro; Q Gln; R Arg; S Ser; T Thr; V Val; W Trp; and Y Tyr.
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  32. The authors thank V. Graziano for analyzing the binding affinities of the knob variants; M. Rossmann D. Engelman C. Anderson J. Dunn and J. Kuryian for critically reading the manuscript and suggesting improvements; and L. Berman R. Sweet and J. Berendsen for access to beamlines X25 X12C and X8C respectively. This research was supported by NIH grant AI36251 to P.F. and by the Office of Biological and Environmental Research of the U.S. Department of Energy under Prime Contract DE-AC02-98CH10886 with Brookhaven National Laboratory. The macromolecular crystallography beamlines X25 X12C and X8C at the National Synchrotron Light Source are also supported by NSF and by NIH grant 1P41 RR12408-01A1. Coordinates have been deposited in the Protein Data Bank with accession codes 1NOB and 1KAC for Ad12 knob and Ad12 knob in complex with CAR D1 respectively.
Dates
Type When
Created 23 years, 1 month ago (July 27, 2002, 5:37 a.m.)
Deposited 1 year, 7 months ago (Jan. 13, 2024, 5:03 a.m.)
Indexed 3 weeks, 2 days ago (Aug. 6, 2025, 8:46 a.m.)
Issued 25 years, 9 months ago (Nov. 19, 1999)
Published 25 years, 9 months ago (Nov. 19, 1999)
Published Print 25 years, 9 months ago (Nov. 19, 1999)
Funders 0

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@article{Bewley_1999, title={Structural Analysis of the Mechanism of Adenovirus Binding to Its Human Cellular Receptor, CAR}, volume={286}, ISSN={1095-9203}, url={http://dx.doi.org/10.1126/science.286.5444.1579}, DOI={10.1126/science.286.5444.1579}, number={5444}, journal={Science}, publisher={American Association for the Advancement of Science (AAAS)}, author={Bewley, Maria C. and Springer, Karen and Zhang, Yian-Biao and Freimuth, Paul and Flanagan, John M.}, year={1999}, month=nov, pages={1579–1583} }