Interaction of bacteriophage Lambda with its cell surface receptor: An in vitro study of binding of the viral tail protein gpJ to LamB (Maltoporin)
Interaction of bacteriophage Lambda with its cell surface receptor: An in vitro study of binding of the viral tail protein gpJ to LamB (Maltoporin)
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DOI：
10.1021/bi051800v
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发表时间：
2006-02-28
期刊：
影响因子：
2.9
通讯作者：
Benz, R
中科院分区：
文献类型：
--
作者：
Berkane, E;Orlik, F;Benz, R
The cell surface receptor for bacteriophage Lambda is LamB (maltoporin). Responsible for phage binding to LamB is the C-terminal part, gpJ, of phage tail protein J. To study the interaction between LamB and gpJ, a chimera protein composed of maltose binding protein (MBP or Ma1E) connected to the C-terminal part of J (gpJ, amino acids 684-1131) of phage tail protein J of bacteriophage Lambda was expressed in Escherichia coli and purified to homogeneity. The interaction of the MBP-gpJ chimera protein with reconstituted LamB and its mutants LamB Y118G and the loop deletion mutant LamB Delta 4+Delta 6+Delta 9v was studied using planar lipid bilayer membranes on a single-channel and multichannel level. Titration with the MBP-gpJ chimera blocked completely the ion current through reconstituted LamB when it was added to the cis side, the extracellular side of LamB; with a half-saturation constant of similar to 6 nM in 1 M KC1. Control experiments with LamB Delta 4+Delta 6+Delta 9v from which all major external loops had been removed showed similar blocking, whereas MBP alone caused no visible effect. Direct conductance measurement with His(6)-gpJ that contained a hexahistidyl tag (His(6) tag) at the N-terminal end of the protein for easy purification revealed no blocking of the ion current, requiring other measurements for the binding constant. However, when maltoporin was preincubated with His-gpJ, MBP-gpJ could not block the channel, which indicated that also His(6)-gpJ bound to the channel. High-molecular mass bands on SDS-PAGE and Western blots, confirming the planar lipid bilayer experiment results, also demonstrated stable complex formation between His(6)-gpJ and LamB or LamB mutants. The results revealed that phage Lambda binding includes not only the extracellular loops.
