Structure and functional interactions of the Tsg101 UEV domain
Abstract
Human Tsg101 plays key roles in HIV budding and in cellular vacuolar protein sorting (VPS). In performing these functions, Tsg101 binds both ubiquitin (Ub) and the PTAP tetrapeptide ‘late domain’ motif located within the viral Gag protein. These interactions are mediated by the N-terminal domain of Tsg101, which belongs to the catalytically inactive ubiquitin E2 variant (UEV) family. We now report the struc ture of Tsg101 UEV and chemical shift mapping of the Ub and PTAP binding sites. Tsg101 UEV resembles canonical E2 ubiquitin conjugating enzymes, but has an additional N-terminal helix, an extended β-hairpin that links strands 1 and 2, and lacks the two C-terminal helices normally found in E2 enzymes. PTAP-containing peptides bind in a hydrophobic cleft exposed by the absence of the C-terminal helices, whereas ubiquitin binds in a novel site surrounding the β-hairpin. These studies provide a structural framework for understanding how Tsg101 mediates the protein–protein interactions required for HIV budding and VPS.
<TAD> is the ensemble of 20 lowest-penalty structures calculated using the program DYANA (Güntert et al., 1997). <CNS> is the same ensemble after 1000 steps (15 ps each) of simulated annealing at 2000 K, 1000 slow-cooling steps to 0 K and 2000 steps of restrained Powell minimization in cartesian space (anneal.inp protocol) (Brünger et al., 1998).
Only meaningful and non-redundant restraints as determined by the DYANA CALIBA function.
Two upper-limit distance restraints were used to define each hydrogen bond.
Energies for structures input into CNS (from DYANA) were estimated within the generate_easy.inp program after the first regularization without restraints.
Violation energies from DYANA have units of Å or °, while energies from CNS are in kcal/mol.
Determined using PROCHECK-NMR (Laskowski et al., 1996).
Superposition and overall r.m.s.ds were calculated using the program MOLMOL (Koradi et al., 1996).
All structured residues minus the vestigial active site loop (residues 91–123).
Affinities of wild-type Tsg101 UEV domain for p6, p6–Ub and Ub are reported as dissociation constants (Kd), averaged from 16, 8 and 3 independent measurements, respectively. Affinities of mutant constructs are reported as fold decreases relative to wild-type binding.
Extrapolated to 50% binding.
Measured at 20 mM sodium phosphate pH 7.2, 150 mM NaCl. All mutant binding affinities were measured under these conditions.
Measured at 20 mM sodium phosphate pH 7.2, 50 mM NaCl.
Measured at 20 mM sodium phosphate pH 7.2, 0 mM NaCl.
Measured at 20 mM sodium phosphate pH 6, 50 mM NaCl, the conditions used for NMR chemical shift perturbation.
Acknowledgements
We thank Brittany Chamberlin, Lianghuey Liu, Peter Stenlund and Dennis Edwards for technical support, Bob Schackmann for peptide synthesis, and Eric Ross for computer support. We thank Andrew VanDemark for providing the coordinates of the model for the Ubc13-Mms2/Ub2 ternary complex, and Chris Hill, Andrew VanDemark and Marty Rechsteiner for critical reading of the manuscript. This work was supported by National Institutes of Health (NIH) funding to W.I.S. The Utah Biomolecular NMR Facility is supported by grants from the NIH and the National Science Foundation. Structure coordinates and chemical shifts have been deposited at the Protein Data Bank (1KPP, CNS ensemble; 1KPQ, DYANA ensemble).