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.
Copyright © 2010 Elsevier Ltd. All rights reserved.
Publication types
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Research Support, N.I.H., Extramural
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Research Support, N.I.H., Intramural
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Research Support, U.S. Gov't, Non-P.H.S.
MeSH terms
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Crystallography
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DNA-Binding Proteins / chemistry*
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DNA-Binding Proteins / isolation & purification
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DNA-Binding Proteins / metabolism
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Endosomal Sorting Complexes Required for Transport / chemistry*
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Endosomal Sorting Complexes Required for Transport / isolation & purification
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Endosomal Sorting Complexes Required for Transport / metabolism
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ErbB Receptors / metabolism
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HIV-1 / chemistry*
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HeLa Cells
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Humans
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Hydrogen Bonding
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Models, Molecular*
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Molecular Dynamics Simulation*
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Protein Conformation*
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Protein Structure, Tertiary / genetics
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RNA Interference
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Transcription Factors / chemistry*
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Transcription Factors / isolation & purification
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Transcription Factors / metabolism
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gag Gene Products, Human Immunodeficiency Virus / chemistry*
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gag Gene Products, Human Immunodeficiency Virus / isolation & purification
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gag Gene Products, Human Immunodeficiency Virus / metabolism
Substances
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DNA-Binding Proteins
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Endosomal Sorting Complexes Required for Transport
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Transcription Factors
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Tsg101 protein
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gag Gene Products, Human Immunodeficiency Virus
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ErbB Receptors