title: Identification and characterization of PWWP domain residues critical for LEDGF/p75 chromatin binding and human immunodeficiency virus type 1 infectivity.
abstract: Lens epithelium-derived growth factor (LEDGF)/p75 functions as a bimodal tether during lentiviral DNA integration: its C-terminal integrase-binding domain interacts with the viral preintegration complex, whereas the N-terminal PWWP domain can bind to cellular chromatin. The molecular basis for the integrase-LEDGF/p75 interaction is understood, while the mechanism of chromatin binding is unknown. The PWWP domain is homologous to other protein interaction modules that together comprise the Tudor clan. Based on primary amino acid sequence and three-dimensional structural similarities, 24 residues of the LEDGF/p75 PWWP domain were mutagenized to garner essential details of its function during human immunodeficiency virus type 1 (HIV-1) infection. Mutating either Trp-21 or Ala-51, which line the inner wall of a hydrophobic cavity that is common to Tudor clan members, disrupts chromatin binding and virus infectivity. Consistent with a role for chromatin-associated LEDGF/p75 in stimulating integrase activity during infection, recombinant W21A protein is preferentially defective for enhancing integration into chromatinized target DNA in vitro. The A51P mutation corresponds to the S270P change in DNA methyltransferase 3B that causes human immunodeficiency, centromeric instability, and facial anomaly syndrome, revealing a critical role for this amino acid position in the chromatin binding functions of varied PWWP domains. Our results furthermore highlight the requirement for a conserved Glu in the hydrophobic core that mediates interactions between other Tudor clan members and their substrates. This initial systematic mutagenesis of a PWWP domain identifies amino acid residues critical for chromatin binding function and the consequences of their changes on HIV-1 integration and infection.
authors.affiliation: Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute and Division of AIDS, Harvard Medical School, Boston, MA 02115, USA.
authors.forename: Ming-Chieh
authors.initials: MC
authors.lastname: Shun
authors.forename: Yaïr
authors.initials: Y
authors.lastname: Botbol
authors.forename: Xiang
authors.initials: X
authors.lastname: Li
authors.forename: Francesca
authors.initials: F
authors.lastname: Di Nunzio
authors.forename: Janet E
authors.initials: JE
authors.lastname: Daigle
authors.forename: Nan
authors.initials: N
authors.lastname: Yan
authors.forename: Judy
authors.initials: J
authors.lastname: Lieberman
authors.forename: Marc
authors.initials: M
authors.lastname: Lavigne
authors.forename: Alan
authors.initials: A
authors.lastname: Engelman
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000737:chemistry / Q000502:physiology*; D000818:Animals; D002460:Cell Line; D002843:Chromatin / Q000378:metabolism*; D015497:HIV-1 / Q000472:pathogenicity*; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000737:chemistry / Q000502:physiology*; D051379:Mice; D017434:Protein Structure, Tertiary; D014157:Transcription Factors / Q000737:chemistry / Q000502:physiology*; D016662:Virus Integration
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D002843:Chromatin; D036341:Intercellular Signaling Peptides and Proteins; C504649:PSIP1 protein, human; D014157:Transcription Factors; C119380:lens epithelium-derived growth factor
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