Probing P(T/S)XP peptides for novel structural interactions with Tsg101 UEV

To complete their lifecycle, enveloped viruses such as human immunodeficiency virus type-1 (HIV-1) recruit host machinery to facilitate budding and egress. For HIV-1, this involves an interaction between a PTAP motif in the HIV-1 Gag polyprotein and the ubiquitin E2 variant (UEV) domain of tumor susceptibility gene 101 (Tsg101). The latter is part of the native endosomal sorting complex required for transport (ESCRT) machinery, which functions in endosomal cargo sorting but is hijacked by HIV-1 for plasma membrane budding. The interaction between the PTAP signal of HIV-1 Gag and the UEV domain of Tsg101 is well characterized biochemically and structurally, with the PTAP motif binding into a groove in the UEV domain. The HIV-1 PTAP sequence emulates similar P(T/S)XP signals that drive host protein interactions involved with ESCRT, where some host proteins may contain several potential P(T/S)XP motifs. This suggested potential competitive or cooperative action on the UEV surface, and so we screened two additional P(T/S)XP motifs from the ESCRT-0 protein Hrs and several motifs identified in other viruses for interaction with the Tsg101 UEV domain using solution nuclear magnetic resonance. In the process, we identified an additional interaction mode for P(T/S)XP peptides with the Tsg101 UEV, involving both a charged surface and a cluster of aromatic residues, neither of which are implicated in the previously characterized PTAP interaction. We identified several binding determinants of this P(T/S)XP interaction using alanine mutant peptides and combined chemical shift perturbations with distance data from paramagnetic relaxation enhancement experiments using spin-labeled peptides to build a structural model of the interaction on the UEV surface. This additional P(T/S)XP interaction may reflect a novel viral strategy to recruit Tsg101 and by extension ESCRT for egress, or hijacking of a different endogenous role of Tsg101 altogether.