A dual inhibition mechanism of herpesviral ICP47 arresting a conformationally thermostable TAP complex.

Valentina Herbring,Robert Tampé,Anja Bäucker,Simon Trowitzsch

doi:10.1038/srep36907

Abstract

As a centerpiece of antigen processing, the ATP-binding cassette transporter associated with antigen processing (TAP) became a main target for viral immune evasion. The herpesviral ICP47 inhibits TAP function, thereby suppressing an adaptive immune response. Here, we report on a thermostable ICP47-TAP complex, generated by fusion of different ICP47 fragments. These fusion complexes allowed us to determine the direction and positioning in the central cavity of TAP. ICP47-TAP fusion complexes are arrested in a stable conformation, as demonstrated by MHC I surface expression, melting temperature, and the mutual exclusion of herpesviral TAP inhibitors. We unveiled a conserved region next to the active domain of ICP47 as essential for the complete stabilization of the TAP complex. Binding of the active domain of ICP47 arrests TAP in an open inward facing conformation rendering the complex inaccessible for other viral factors. Based on our findings, we propose a dual interaction mechanism for ICP47. A per se destabilizing active domain inhibits the function of TAP, whereas a conserved C-terminal region additionally stabilizes the transporter. These new insights into the ICP47 inhibition mechanism can be applied for future structural analyses of the TAP complex.

Highlights

TAP complexes containing ICP47 fragments 1-78 and 1-88 are stable at 40 °C for 1 h. These findings suggest that the ICP47 fragments 1-35 and 1-50 fused to the elbow helix of either TAP1 or TAP2 did not allow an optimal positioning of the active domain in the binding cavity
This study reports on the identification of functionally arrested TAP complexes with high thermostability by fusing herpesviral ICP47 to TAP
Different lengths of ICP47 were chosen to map the optimal distance between the binding pocket and the N-terminal elbow helix of either TAP1 or TAP2

Summary

Introduction

The otherwise dynamic TAP complex is trapped in its open inward-facing conformation, impeding the interaction with US6 This strategy allowed us to detect distinct behaviors of ICP47 fragments of the same length regarding each TAP subunit as well as to compare effects of ICP47 fragments of different lengths towards transport inhibition and thermal stabilization of the TAP complex. We demonstrate that the C-terminal extended region of ICP47 is necessary for complete stabilization of the TAP complex, whereas the N-terminal active domain is sufficient for TAP inhibition. Based on these data, we propose a dual inhibition mechanism by ICP47, which explains the high sequence conservation of the extended C-terminal region of ICP47

Methods

Results

Conclusion