Cryo-electron microscopy structure of an adenovirus-integrin complex indicates conformational changes in both penton base and integrin

Abstract

A structure of adenovirus type 12 (HAdV12) complexed with a soluble form of integrin alphavbeta5 was determined by cryo-electron microscopy (cryoEM) image reconstruction. Subnanometer resolution (8 A) was achieved for the icosahedral capsid with moderate resolution (27 A) for integrin density above each penton base. Modeling with alphavbeta3 and alpha(IIb)beta3 crystal structures indicates that a maximum of four integrins fit over the pentameric penton base. The close spacing (approximately 60 A) of the RGD protrusions on penton base precludes integrin binding in the same orientation to neighboring RGD sites. Flexible penton-base RGD loops and incoherent averaging of bound integrin molecules explain the moderate resolution observed for the integrin density. A model with four integrins bound to a penton base suggests that integrin might extend one RGD-loop in the direction that could induce a conformational change in the penton base involving clockwise untwisting of the pentamer. A global conformational change in penton base could be one step on the way to the release of Ad vertex proteins during cell entry. Comparison of the cryoEM structure with bent and extended models for the integrin ectodomain reveals that integrin adopts an extended conformation when bound to the Ad penton base, a multivalent viral ligand. These findings shed further light on the structural basis of integrin binding to biologically relevant ligands, as well as on the molecular events leading to HAdV cell entry.

FIG. 1.

Integrin domains and conformations. (A) Structural domains of integrin αv and β chains, including the extracellular domains, transmembrane-spanning regions, and small cytoplasmic domains, shown in extended schematic forms. The domains are represented as 10Å-resolution density maps based on crystallographic coordinates. The membrane is represented by a gray bar. (Modified from Stewart and Nemerow (32) and reprinted with permission from Elsevier.) (B) Models for soluble αvβ5 integrin with Fos/Jun dimerization domains. Each chain has a six residue glycine-rich linker between the ectodomain and the Fos or Jun dimerization domain. The model of a bent integrin conformation (left) was built as a composite of αvβ3 integrin crystal structures, PDB-IDs 1L5G and 1U8C (42, 43), and the crystal structure of c-Fos/c-Jun bound to DNA, PDB-ID 1FOS (6). The model of an extended integrin conformation (right) is similar to the extended model docked into the HAdV12/αvβ5 cryo structure (Fig. 8B).

FIG. 2.

CryoEM structure of the HAdV12/αvβ5 integrin complex. (A) Full structure viewed along a twofold icosahedral axis and shown as three radial shells: icosahedral capsid (300 to 463 Å, blue), integrin ring and fiber (443 to 515 Å, gold), and diffuse integrin density (515 to 600 Å, red). The icosahedral capsid shell is shown filtered to 8 Å with an applied B-factor of −300 Ų, the integrin ring and fiber filtered to 19 Å with an applied B-factor of −500 Ų, and the diffuse integrin density filtered to 33 Å with an applied B-factor of −500 Ų. (B) Enlarged top and side views of the vertex region colored as in panel A with the fiber shaft in green. The diffuse integrin density (red) is shown with a lower isosurface contour level in panel B (1.2σ versus 1.7σ in panel A) to display a fuller extent of this density. Scale bars, 100 Å. (C) An FSC plot indicating a resolution range for the HAdV12/αvβ5 icosahedral capsid (radial shell, 300 to 463 Å) of 8.3 to 6.9Å (8.3 Å, FSC = 0.5; 7.5 Å, FSC = 0.3; 6.9 Å, FSC = 0.143) (blue); the HAdV12/αvβ5 radial shell (443-515Å) including the integrin ring and fiber above the penton base of 27Å to 19Å (27 Å, FSC = 0.5; 24 Å, FSC = 0.3; 19 Å, FSC = 0.143) (gold); and the HAdV12/αvβ5 radial shell (515-600Å) with diffuse integrin density of 85Å to 33Å (85 Å, FSC = 0.5; 50 Å, FSC = 0.3; 33 Å, FSC = 0.143) (red).

FIG. 3.

Penton base in the HAdV12/αvβ5 integrin complex and models for the integrin-binding RGD loop. (A) Top view of the HAdV12 penton base cryoEM density (mesh) with the docked crystal structure of the HAdV2 penton base pentamer and fiber peptide (PDB-ID 1X9T) (48). (B) Side view of the same but with the cryoEM density shown at a higher isosurface contour level to reveal α-helical density rods. The arrows indicate the longest α-helix in the penton base monomer. The rectangle indicates the α-helical density below the penton base assigned to protein IIIa (29). Each penton base monomer is in a different color with the fiber peptide at the top of the penton base in green. (C and D) Top and side views of the HAdV2 penton base crystal structure (gray) with 10 different models for the HAdV12 RGD loop (amino acids 296 to 312) built with Rosetta (27).

FIG. 4.

The RGD-binding integrin domains form the ring of density over the penton base in the HAdV12/αvβ5 structure. (A) The two RGD-binding integrin domains, αv chain β-propeller (blue) and β chain I domain (red), together with the RGD residues (green) from the αvβ3/RGD crystal structure (PDB-ID 1L5G), are shown modeled over the HAdV2 penton base crystal structure (PDB-ID 1X9T). One monomer of penton base is shown in gold, the rest in gray, and the fiber peptides are depicted as wide black ribbons. The missing residues in the penton base RGD loop are represented by dashed lines. (B) The penton base is shown with four docked copies of the two-domain integrin unit. (C) The penton base is shown with simulated density (light blue) generated from the integrin models in panel B with fivefold averaging and filtered to 27-Å resolution.

FIG. 5.

The integrin headpiece forms the ring and connector regions stretching away from the penton base in the HAdV12/αvβ5 structure. (A) The additional integrin headpiece domains are labeled. The RGD-binding integrins domains are modeled above the penton base as in Fig. 4. This is a composite integrin headpiece model built from PDB-IDs 1L5G and chain B of 2VDK with a 69° angle between the β-chain I and hybrid domains (38). (B) The penton base is shown with four docked copies of the integrin headpiece. (C) The penton base is shown with simulated density (light blue) generated from the integrin models in panel B with fivefold averaging and filtered to 27-Å resolution.

FIG. 6.

Natural twist of penton base and possible untwisting by integrin. (A) Space-filling representation of the penton base pentamer (PDB-ID 1X9T) with each subunit in a different color. The natural twist of one subunit from the bottom of the pentamer to the top solvent accessible surface in the virion is represented by the arrow. (B) Side view of four superimposed penton base monomers with four copies of the RGD residues (magenta, cyan, blue, and green) as modeled in Fig. 4 and 5. Note that the magenta copy of the RGD residues extends the RGD loop counter to the natural twist of the penton base monomer. (C) Side view of the penton base pentamer shown with one monomer in magenta, fiber peptides in green, and the modeled RGD-binding integrin domains (blue and red) with the magenta copy of the RGD residues. (D) The integrin domains are removed, and an arrow indicates the direction that an integrin bound in the manner of panel C would tend to extend the RGD loop of penton base and lead to untwisting. (E) Top view of the penton base pentamer with a curved arrow indicating clockwise untwisting of the penton base.

FIG. 7.

The penton base has low partial occupancy in the HAdV12/αvβ5 cryoEM structure. (A) Thin (∼20-Å) slab of the Ad35f cryoEM structure (29) with docked crystal structures of hexon (cyan) (PDB-ID 1P30) and penton base (gold) with the N-terminal fiber peptide (green). The cryoEM density (mesh) is isocontoured at 1.7σ and shows rods for α-helices in both hexon and penton base. (B) Thin slab of the HAdV12/αvβ5 cryoEM structure with the same docked atomic resolution structures. The cryoEM density is isocontoured at 2.8σ and shows rods for α-helices in hexon and almost no density for the penton base. (C) Same as panel B but with the HAdV12/αvβ5 cryoEM density isocontoured at 2.3σ showing rods for α-helices in penton base similar to those shown for Ad35f in panel A, but with significantly more hexon density than in panel A. Both maps are shown filtered to 7.5 Å with the same applied B factor (−300 Ų).

FIG. 8.

Comparison of HAdV12/αvβ5 integrin density with bent and extended integrin models. (A) The cryoEM integrin density (mesh) is shown with four copies of the integrin ectodomain in a bent conformation. This is a composite integrin model built from PDB-IDs 1L5G (43) and 18UC (42). (B) The penton base is shown with four copies of the integrin ectodomain in an extended conformation. This is a composite integrin model built from the domains shown in Fig. 5 with the remaining domains modeled to approximate the cryoEM density in the outermost radial shell (515 to 600 Å) of the HAdV12/αvβ5 structure. In both panels the αv chains are blue, the β chains are red, the bound RGD peptides are green, and the penton base is gold. Note that the gap between the upper and lower cryoEM integrin density regions is an artifact from calculating the density in separate radial shells.