| Title | A turn-like structure "KKPE" segment mediates the specific binding of viral envelope protein A27 to heparin and heparan sulfate on the cell surfaces. | | Author(s) | Shih PC, Yang MS, Lin SC, Ho Y, Hsiao JC, Wang DR, Yu SS, Chang W, Tzou DL | | Institution | Institute of Chemistry, Academia Sinica, Taiwan; | | Source | J Biol Chem 2009 Oct 26. | | Abstract | Vaccinia viral envelope protein A27 (110 amino acids) specifically interacts with heparin (HP) or heparan sulfate proteoglycans (HS) for cell surface attachment. To examine the binding mechanism, a truncated soluble form of A27 (sA27-aa; residues 21-84 of A27) with Cys71 and Cys72 mutated to Ala was used as the parent molecule. sA27-aa consists of two structurally distinct domains, a flexible Arg/Lys-rich heparin-binding site (HBS) (residues 21-32; (21)STKAAKKPEAKR(32)) and a rigid coiled-coil domain (43-84), both essential for the specific binding. As shown by SPR, the binding affinity of sA27-aa for HP (K(A) = 1.25x10(8) M(-1)) was approximately three orders of magnitude stronger than that for nonspecific binding, such as to chondroitin sulfate (CS) (K(A) = 1.65x10(5) M(-1)). Using site-directed mutagenesis of HBS and solution NMR, we identified a "KKPE" segment with a turn-like conformation that mediates specific HP binding. In addition, a double mutant T22K/A25K in which the "KKPE" segment remained intact showed an extremely high affinity for HP (K(A) = 1.9x10(11) M(-1)). Importantly, T22K/A25K retained the binding specificity for HP and HS but not CS, as shown by in vitro SPR and in vivo cell adhesion and competitive binding assays. Molecular modeling of the HBS was performed by dynamics simulations and provides an explanation of the specific binding mechanism in good agreement with the site-directed mutagenesis and SPR results. We conclude that a turn-like structure introduced by the "KKPE" segment in vaccinia viral envelope protein A27 is responsible for its specific binding to HP and to HS on cell surfaces. | | Language | ENG | | Pub Type(s) | JOURNAL ARTICLE
| | PubMed ID | 19858217 |
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