Crystallographic and functional analysis of the ESCRT-I /HIV-1 Gag PTAP interaction

Structure. 2010 Nov 10;18(11):1536-47. doi: 10.1016/j.str.2010.08.010.

Abstract

Budding of HIV-1 requires the binding of the PTAP late domain of the Gag p6 protein to the UEV domain of the TSG101 subunit of ESCRT-I. The normal function of this motif in cells is in receptor downregulation. Here, we report the 1.4-1.6 Å structures of the human TSG101 UEV domain alone and with wild-type and mutant HIV-1 PTAP and Hrs PSAP nonapeptides. The hydroxyl of the Thr or Ser residue in the P(S/T)AP motif hydrogen bonds with the main chain of Asn69. Mutation of the Asn to Pro, blocking the main-chain amide, abrogates PTAP motif binding in vitro and blocks budding of HIV-1 from cells. N69P and other PTAP binding-deficient alleles of TSG101 did not rescue HIV-1 budding. However, the mutant alleles did rescue downregulation of endogenous EGF receptor. This demonstrates that the PSAP motif is not rate determining in EGF receptor downregulation under normal conditions.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, N.I.H., Intramural
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Crystallography
  • DNA-Binding Proteins / chemistry*
  • DNA-Binding Proteins / isolation & purification
  • DNA-Binding Proteins / metabolism
  • Endosomal Sorting Complexes Required for Transport / chemistry*
  • Endosomal Sorting Complexes Required for Transport / isolation & purification
  • Endosomal Sorting Complexes Required for Transport / metabolism
  • ErbB Receptors / metabolism
  • HIV-1 / chemistry*
  • HeLa Cells
  • Humans
  • Hydrogen Bonding
  • Models, Molecular*
  • Molecular Dynamics Simulation*
  • Protein Conformation*
  • Protein Structure, Tertiary / genetics
  • RNA Interference
  • Transcription Factors / chemistry*
  • Transcription Factors / isolation & purification
  • Transcription Factors / metabolism
  • gag Gene Products, Human Immunodeficiency Virus / chemistry*
  • gag Gene Products, Human Immunodeficiency Virus / isolation & purification
  • gag Gene Products, Human Immunodeficiency Virus / metabolism

Substances

  • DNA-Binding Proteins
  • Endosomal Sorting Complexes Required for Transport
  • Transcription Factors
  • Tsg101 protein
  • gag Gene Products, Human Immunodeficiency Virus
  • ErbB Receptors