Crystallographic and functional analysis of the ESCRT-I /HIV-1 Gag PTAP interaction.
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.
Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892-0580, USA.
Young Jun
YJ
Im
Lillian
L
Kuo
Xuefeng
X
Ren
Patricia V
PV
Burgos
Xue Zhi
XZ
Zhao
Fa
F
Liu
Terrence R
TR
Burke
Juan S
JS
Bonifacino
Eric O
EO
Freed
James H
JH
Hurley
D003461:Crystallography; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D056827:Endosomal Sorting Complexes Required for Transport / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D066246:ErbB Receptors / Q000378:metabolism; D015497:HIV-1 / Q000737:chemistry*; D006367:HeLa Cells; D006801:Humans; D006860:Hydrogen Bonding; D008958:Models, Molecular; D056004:Molecular Dynamics Simulation; D011487:Protein Conformation; D017434:Protein Structure, Tertiary / Q000235:genetics; D034622:RNA Interference; D014157:Transcription Factors / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D054301:gag Gene Products, Human Immunodeficiency Virus / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D000099177:Tsg101 Protein
D004268:DNA-Binding Proteins; D056827:Endosomal Sorting Complexes Required for Transport; D066246:ErbB Receptors; D014157:Transcription Factors; D054301:gag Gene Products, Human Immunodeficiency Virus; D000099177:Tsg101 Protein