Stable binding of the herpes simplex virus ICP47 protein to the peptide binding site of TAP.
The herpes simplex virus (HSV) ICP47 protein inhibits the MHC class I antigen presentation pathway by inhibiting the transporter associated with antigen presentation (TAP) which translocates peptides across the endoplasmic reticulum membrane. At present, ICP47 is the only inhibitor of TAP. Here, we show that ICP47 produced in bacteria can block human, but not mouse, TAP, and that heat denaturation of ICP47 has no effect on its ability to block TAP. ICP47 inhibited peptide binding to TAP without affecting ATP binding, consistent with previous observations that the peptide binding and ATP binding sites of TAP are distinct. ICP47 bound to TAP with a higher affinity (KD approximately 5 x 10(-8) M) than did peptides, and ICP47 did not dissociate from TAP. ICP47 was not transported by TAP and remained sensitive to proteases added from the cytosolic surface of the membrane. Peptides acted as competitive inhibitors of ICP47 binding to TAP, and this inhibition required a 100- to 1000-fold molar excess of peptide. These results demonstrate that ICP47 binds to a site which includes the peptide binding domain of TAP and remains bound to this site in a stable fashion.
Tomazin
R
R
Cancer Research Group, Molecular Biology and Biotechnology Program, McMaster University, Hamilton, Ontario, Canada.
Hill
A B
AB
Jugovic
P
P
York
I
I
van Endert
P
P
Ploegh
H L
HL
Andrews
D W
DW
Johnson
D C
DC
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001667:Binding, Competitive; D001692:Biological Transport; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D003001:Cloning, Molecular; D004721:Endoplasmic Reticulum / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D051379:Mice; D008861:Microsomes / Q000378:metabolism; D010455:Peptides / Q000378:metabolism*; D011485:Protein Binding; D018411:Spodoptera; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*
D002352:Carrier Proteins; D010455:Peptides; D014764:Viral Proteins; D000255:Adenosine Triphosphate