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 virus (ASLV-A). The viral receptor function of Tva is determined by a 40-residue 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 other LDL-A modules, correct folding and structure of the Tva LDL-A module is Ca2+ dependent. When calcium was present during in vitro protein folding, the wt module was eluted as a single peak by reverse-phase high-pressure liquid 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 (Kd) of the Tva LDL-A module, determined by isothermal titration calorimetry, is approximately 40 microM. Characterization of several Tva mutants provided further evidence that calcium 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. Our results provide new insights regarding the structure and function of Tva in ASLV-A entry.
Wang
Q Y
QY
Department of Microbiology and Immunology, College of Medicine, University of Illinois at Chicago, Chicago, Illinois 60612, USA.
Dolmer
K
K
Huang
W
W
Gettins
P G
PG
Rong
L
L
D001355:Alpharetrovirus / Q000378:metabolism* / Q000472:pathogenicity; D000595:Amino Acid Sequence; D000818:Animals; D001353:Avian Leukosis / Q000821:virology; D030161:Avian Proteins; D002118:Calcium / Q000378:metabolism*; D002151:Calorimetry; D002851:Chromatography, High Pressure Liquid; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D011487:Protein Conformation; D017510:Protein Folding; D011973:Receptors, LDL / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D011991:Receptors, Virus / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001357:Sarcoma, Avian / Q000821:virology
D030161:Avian Proteins; D011973:Receptors, LDL; D011991:Receptors, Virus; C088745:Tva receptor; D002118:Calcium