Vaccinia viral protein complex A17/A27 - its specific molecular interaction and binding mechanism

• Main Authors: Guo-Chian Li, 李國乾
• Other Authors: Wenlung Chen
• Format: Others
• Language: zh-TW
• Published: 2011
• Online Access: http://ndltd.ncl.edu.tw/handle/49418428558948247831

碩士 === 國立嘉義大學 === 應用化學系研究所 === 99 === Vaccinia virus (VACV) is the prototype of the Orthopoxvirus genus of the Poxviridae family, which infects various cell lines and animals. The viral genome is 190 kb in size that encodes more than 200 proteins. Among them, A27 and H3 bind to heparan sulfate, whereas D8 binds to chondroitin sulfate specifically facilitate virus attachment to cell surface. Heparin or heparan sulfate (HS) binding protein A27 is an extracellular domain viral protein that associates with membrane protein A17 to be able to expose to outer-membrane. A27 tends to self-assemble forming oligomers and A17 membrane protein precipitates that obstacle the studies of A27/A17 interaction. Till now, the A27/A17 binding sites and its binding mechanism remain largely unclear. A17 (203 aa) consists of transmembrane domains (61-161) and extracellular domains at N- and C-terminal region (1-60 and 162-203), respectively. A27 (110 aa) consists of a random coil (1-32), containing HS binding domain, a coil-coiled (43-84) and a Leucine zipper domain (85-110). To unravel the A27/A17 binding sites and its binding mechanism, we expressed two A17 segments, namely A17 N-terminus (A17-N) and A17 C-terminus (A17-C), in order to overcome sample precipitation difficulty. In addition, a scramble mutant A17-S was expressed and purified, in which the sequence of 24-34 residues was scrambled. By surface plasmon resonance (SPR), we measured the binding affinities of the A27/A17-N and A27/A17-C interactions separately. The binding affinities (KD) of the A27/A17-N and A27/A17-C interactions are in the range of 10-9 and 10-6 M-1, respectively. And the 6 binding affinity (KD) of the A27/A17-S is around 10-6 M-1, nearly three orders of magnitude less than the parental. As revealed, the 24-34 residues in A17-N are responsible for the A27 interaction. Here we propose that A27 interacts with A17 membrane protein is mediated by a two-step binding mode, involving in a hydrophobic binding site (S1) and an electrostatic charge binding site (S2), as evidenced by 2D HSQC NMR spectra. A26 has been reported playing an important role in the virus entry, acting as a membrane fusion suppressor in the virus entry that interacts with A27. To challenge the A17 binding sites, we expressed an A26 fragment: A26-aa (441-472 a.a. of WT-A26 and mutate C441A and C442A). A26-aa is homologous to one of A27 coiled-coil domain (71-110). As shown by SPR and NMR, A26-aa is able to compete with the S2 binding site, not the S1, in competition with the A17/A27 interaction. All together, we here conclude that the S1 site in A17 is the major binding site responsible for specific interaction with A27 whereas the S2 site is the other binding site binding to A27 and is regulated by A26. And a molecular model was proposed to indicate the binding events at the atomic level that is useful for understanding different virus entry mechanisms.