Molecular mechanism and structural aspects of transporter associated with antigen processing inhibition by the cytomegalovirus protein US6.
The human cytomegalovirus (HCMV) has evolved a set of elegant strategies to evade host immunity. The HCMV-encoded type I glycoprotein US6 inhibits peptide trafficking from the cytosol into the endoplasmic reticulum and subsequent peptide loading of major histocompatibility complex I molecules by blocking the transporter associated with antigen processing (TAP). We studied the molecular mechanism of TAP inhibition by US6 in vitro. By using purified US6 and human TAP co-reconstituted in proteoliposomes, we demonstrate that the isolated endoplasmic reticulum (ER)-luminal domain of US6 is essential and sufficient to block TAP-dependent peptide transport. Neither the overall amount of bound peptides nor the peptide affinity of TAP is affected by US6. Interestingly, US6 causes a specific arrest of the peptide-stimulated ATPase activity of TAP by preventing binding of ATP but not ADP. The affinity of the US6-TAP interaction was determined to 1 microm. The ER-luminal domain of US6 is monomeric in solution and consists of 19% alpha-helices, 25% beta-sheets, and 27% beta-turns. All eight cysteine residues are involved in forming a stabilizing network of four intramolecular disulfide bridges. Glycosylation of US6 is not required for function. These findings point to fascinating mechanistic and structural properties, by which specific binding of US6 at the ER-luminal loops of TAP signals across the membrane to the nucleotide-binding domains to prevent ATP hydrolysis of TAP.
Kyritsis
C
C
Institute of Physiological Chemistry, Philipps-University Marburg, Karl-von-Frisch-Strasse 1, 35033 Marburg, Germany.
Gorbulev
S
S
Hutschenreiter
S
S
Pawlitschko
K
K
Abele
R
R
Tampé
R
R
D018528:ATP-Binding Cassette Transporters / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D000244:Adenosine Diphosphate / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D017931:DNA Primers; D004721:Endoplasmic Reticulum / Q000378:metabolism; D007313:Insecta; D011487:Protein Conformation; D016601:RNA-Binding Proteins / Q000502:physiology*; D011994:Recombinant Proteins / Q000378:metabolism; D014764:Viral Proteins / Q000502:physiology*
D018528:ATP-Binding Cassette Transporters; D017931:DNA Primers; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; C432877:US6 protein, Human cytomegalovirus; D014764:Viral Proteins; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases