Structural Analysis of the Extracellular Domain of Vaccinia Virus Envelope Protein, A27L, by NMR and CD Spectroscopy

• Published in: The Journal of biological chemistry 2002-06, Vol.277 (23), p.20949-20959
• Main Authors: Lin, Ta-Hsien, Chia, Chih-Ming, Hsiao, Jye-Chian, Chang, Wen, Ku, Chiao-Chu, Hung, Shang-Cheng, Tzou, Der-Lii M.
• Format: Article
• Language: English
• Subjects: Calorimetry; Circular Dichroism; Heparin - metabolism; Nuclear Magnetic Resonance, Biomolecular; Protein Binding; Protein Structure, Secondary; Vaccinia virus - chemistry; Viral Proteins - chemistry; Viral Proteins - metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M110403200

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 α-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 α-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 α-helix tendency, is probably the self-assembly core. Self-assembly was ascribed to three hydrophobic leucine residues (Leu41, Leu45, and Leu48) 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.