Solution structure of the viral receptor domain of Tva and its implications in viral entry

Abstract

Tva is the cellular receptor for subgroup A avian sarcoma and leukosis virus (ASLV-A). The viral receptor function of Tva is determined by a 40-residue, cysteine-rich motif called the LDL-A module. Here we report the solution structure of the LDL-A module of Tva, determined by nuclear magnetic resonance (NMR) spectroscopy. Although the carboxyl terminus of the Tva LDL-A module has a structure similar to those of other reported LDL-A modules, the amino terminus adopts a different conformation. The LDL-A module of Tva does not contain the signature antiparallel beta-sheet observed in other LDL-A modules, and it is more flexible than other reported LDL-A modules. The LDL-A structure of Tva provides mechanistic insights into how a simple viral receptor functions in retrovirus entry. The side chains of H38 and W48 of Tva, which have been identified as viral contact residues by mutational analysis, are solvent exposed, suggesting that they are directly involved in EnvA binding. However, the side chain of L34, another potential viral contact residue identified previously, is buried inside of the module and forms the hydrophobic core with other residues. Thus L34 likely stabilizes the Tva structure but is not a viral interaction determinant. In addition, we propose that the flexible amino-terminal region of Tva plays an important role in determining specificity in the Tva-EnvA interaction.

FIG. 1.

Stereo view of the 20 selected structures of the Tva LDL-A module with the lowest target function from the final DYANA calculations.

FIG. 2.

Structure comparison of the Tva LDL-A module and LRP CR8. Left, N terminus; right, C terminus. Cyan, β-sheet; red and yellow, α-helix. Bottom right, calcium-binding site; straight lines, three disulfide bonds. (A) Ribbon representation of the final structure of the Tva LDL-A module, based on the 20 best structures shown in Fig. 1. (B) Ribbon representation of the three-dimensional solution structure of CR8. (C) Superimposition of the backbone of the Tva LDL-A module (blue) and that of CR8 (red).

FIG. 3.

(A) Calcium-coordinating site of the Tva LDL-A module. (B) Electrostatic surface of the Tva LDL-A module. The structure is in the same orientation as the structure in Fig. 2A; red, negative charge; blue, positive charge.

FIG. 4.

Amino acid sequence alignment of the Tva LDL-A module, LDL-A22 of LRP2, and all LDL-A modules with known structures: LDL-A5, LDL-A1, LDL-A2, LDL-A6, LRP CR3, and CR8. Conserved amino acids are boxed, and the six residues involved in calcium coordination are shaded. The residues of the Tva LDL-A module from both quail and chickens are numbered from 11 to 50 according to the mature Tva (2).

FIG. 5.

Squares of the generalized order parameters by a model-free analysis of internal motions, S², of the Tva LDL-A module (A) and LRP CR8 (B). For the Tva LDL-A module, relaxation parameters were determined for 37 nonproline residues. The remaining four nonproline residues could not be determined due to spectrum overlapping or a weak signal. For LRP CR8, relaxation parameters were determined for 42 nonproline residues. Note that the residues at the N and C termini of the recombinant Tva module are derived from the vector and are designated − or +, respectively. The residues for CR8 are numbered as described by Huang et al. (18). Bar, residues between the second and the third cysteines of Tva with a low S² value.

FIG. 6.

Molecular model of Tva as the RSV-A receptor. The side chains of the putative ligand contact residues (green) are surface exposed, whereas the side chains of the residues involved in the formation of the hydrophobic core (red) are buried. In this model, it is proposed that the conformation of the more flexible N terminus of Tva is rigidified upon EnvA binding (induced fit).