Role of calcium in protein folding and function of Tva, the receptor of subgroup A avian sarcoma and leukosis virus

Tva is the cellular receptor for subgroup A avian sarcoma and leukosis viru s (ASLV-A). The viral receptor function of Tva is determined by a 40-residu e cysteine-rich motif called the LDL-A module. In this study, we expressed and purified the wild-type (wt) Tva LDL-A module as well as several mutants and examined their in vitro folding properties. We found that, as for othe r LDL-A modules, correct folding and structure of the Tva LDL-A module is C a2+ dependent. When calcium was present during in vitro protein folding, th e wt module was eluted as a single peak by reverse-phase high-pressure liqu id chromatography. Furthermore, two-dimensional nuclear magnetic resonance (NMR) spectroscopy gave well-dispersed spectra in the presence of calcium. In contrast, the same protein folded in vitro in the absence of calcium was eluted as multiple broad peaks and gave a poorly dispersed NMR spectrum in the presence of calcium. The calcium affinity (K-d) of the Tva LDL-A modul e, determined by isothermal titration calorimetry, is approximately 40 muM. Characterization of several Tva mutants provided further evidence that cal cium is important in protein folding and function of Tva, Mutations of the Ca2+-binding residues (D46A and E47A) completely abrogated the Ca2+-binding ability of Tva, and the proteins were not correctly folded. Interestingly, mutations of two non-calcium-binding residues (W48A and L34A) also exerted adverse effect on Ca2+-dependent folding, albeit to a much less extent. Ou r results provide new insights regarding the structure and function of Tva in ASLV-A entry.