A Turn-like Structure 'KKPE' Segment Mediates the Specific Binding of Viral Protein A27 to Heparin and Heparan Sulfate on Cell Surfaces
| Autor: | Yu Ho, Da-Rong Wang, Su-Ching Lin, Steve S.-F. Yu, Wen Chang, Der-Lii M. Tzou, Ping-Chen Shih, Min-Shiang Yang, Jye-Chian Hsiao |
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| Rok vydání: | 2009 |
| Předmět: |
Models
Molecular Viral protein Amino Acid Motifs Mutation Missense Vaccinia virus Plasma protein binding medicine.disease_cause Biochemistry Mice chemistry.chemical_compound Viral envelope medicine Animals Humans Chondroitin sulfate Binding site Nuclear Magnetic Resonance Biomolecular Molecular Biology Binding selectivity chemistry.chemical_classification Binding Sites Heparin Chemistry Membrane Proteins Cell Biology Heparan sulfate Molecular biology Amino acid Amino Acid Substitution Protein Structure and Folding Mutagenesis Site-Directed Biophysics Heparitin Sulfate Carrier Proteins Viral Fusion Proteins HeLa Cells Protein Binding |
| Zdroj: | Journal of Biological Chemistry. 284:36535-36546 |
| ISSN: | 0021-9258 |
| DOI: | 10.1074/jbc.m109.037267 |
| Popis: | Vaccinia viral envelope protein A27 (110 amino acids) specifically interacts with heparin (HP) or heparan sulfate (HS) proteoglycans for cell surface attachment. To examine the binding mechanism, a truncated soluble form of A27 (sA27-aa; residues 21-84 of A27) with Cys(71) and Cys(72) 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 (residues 43-84), both essential for the specific binding. As shown by surface plasmon resonance (SPR), the binding affinity of sA27-aa for HP (K(A) = 1.25 x 10(8) m(-1)) was approximately 3 orders of magnitude stronger than that for nonspecific binding, such as to chondroitin sulfate (K(A) = 1.65 x 10(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) = 1.9 x 10(11) m(-1)). Importantly, T22K/A25K retained the binding specificity for HP and HS but not chondroitin sulfate, 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. |
| Databáze: | OpenAIRE |
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