Structural analysis of the extracellular domain of vaccinia virus envelope protein, A27L, by NMR and CD spectroscopy.
This study presents the molecular structure of the extracellular domain of vaccinia virus envelope protein, A27L, determined by NMR and CD spectroscopy. A recombinant protein, eA27L-aa, containing this domain in which cysteines 71 and 72 were replaced with alanine, was constructed to prevent self-assembly due to intermolecular disulfide bonds between these two cysteines. The soluble eA27L-aa protein forms an oligomer resembling that of A27L on vaccinia virions. Heteronuclear correlation NMR spectroscopy was carried out on eA27L-aa in the presence or absence of urea to determine backbone resonance assignments. Chemical shift index (CSI) propensity analysis showed that eA27L-aa has two distinct structural domains, a relatively flexible 22-amino acid random coil in the N-terminal region and a fairly rigid alpha-helix structure in the remainder of the structure. Binding interaction studies using isothermal titration calorimetry suggest that a 12-amino acid lysine/arginine-rich segment in the N-terminal region is responsible for glycosaminoglycan binding. The rigid alpha-helix portion of eA27L-aa is probably involved in the intrinsic self-assembly, and CSI propensity analysis suggests that region N37-E49, with a residual alpha-helix tendency, is probably the self-assembly core. Self-assembly was ascribed to three hydrophobic leucine residues (Leu(41), Leu(45), and Leu(48)) in this segment. The folding mechanism of eA27L-aa was analyzed by CD spectroscopy, which revealed a two-step transition with a Gibbs free energy of 2.5 kcal/mol in the absence of urea. Based on these NMR and CD studies, a residue-specific molecular model of the extracellular domain of A27L is proposed. These studies on the molecular structure of eA27L-aa will help in understanding how vaccinia virus enters cells.
Lin
Ta-Hsien
TH
Institute of Biochemistry, National Yang-Ming University, Shih-pai, Taipei 11221, Taiwan.
Chia
Chih-Ming
CM
Hsiao
Jye-Chian
JC
Chang
Wen
W
Ku
Chiao-Chu
CC
Hung
Shang-Cheng
SC
Tzou
Der-Lii M
DL
D002151:Calorimetry; D002942:Circular Dichroism; D006493:Heparin / Q000378:metabolism; D019906:Nuclear Magnetic Resonance, Biomolecular; D011485:Protein Binding; D017433:Protein Structure, Secondary; D014616:Vaccinia virus / Q000737:chemistry*; D014764:Viral Proteins / Q000737:chemistry* / Q000378:metabolism
C110360:A27L protein, Orthopoxvirus; D014764:Viral Proteins; D006493:Heparin