National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)
UM1 AI100663
米国
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)
P01 AI110657
米国
引用
ジャーナル: Nature / 年: 2017 タイトル: Open and closed structures reveal allostery and pliability in the HIV-1 envelope spike. 著者: Gabriel Ozorowski / Jesper Pallesen / Natalia de Val / Dmitry Lyumkis / Christopher A Cottrell / Jonathan L Torres / Jeffrey Copps / Robyn L Stanfield / Albert Cupo / Pavel Pugach / John P ...著者: Gabriel Ozorowski / Jesper Pallesen / Natalia de Val / Dmitry Lyumkis / Christopher A Cottrell / Jonathan L Torres / Jeffrey Copps / Robyn L Stanfield / Albert Cupo / Pavel Pugach / John P Moore / Ian A Wilson / Andrew B Ward / 要旨: For many enveloped viruses, binding to a receptor(s) on a host cell acts as the first step in a series of events culminating in fusion with the host cell membrane and transfer of genetic material for ...For many enveloped viruses, binding to a receptor(s) on a host cell acts as the first step in a series of events culminating in fusion with the host cell membrane and transfer of genetic material for replication. The envelope glycoprotein (Env) trimer on the surface of HIV is responsible for receptor binding and fusion. Although Env can tolerate a high degree of mutation in five variable regions (V1-V5), and also at N-linked glycosylation sites that contribute roughly half the mass of Env, the functional sites for recognition of receptor CD4 and co-receptor CXCR4/CCR5 are conserved and essential for viral fitness. Soluble SOSIP Env trimers are structural and antigenic mimics of the pre-fusion native, surface-presented Env, and are targets of broadly neutralizing antibodies. Thus, they are attractive immunogens for vaccine development. Here we present high-resolution cryo-electron microscopy structures of subtype B B41 SOSIP Env trimers in complex with CD4 and antibody 17b, or with antibody b12, at resolutions of 3.7 Å and 3.6 Å, respectively. We compare these to cryo-electron microscopy reconstructions of B41 SOSIP Env trimers with no ligand or in complex with either CD4 or the CD4-binding-site antibody PGV04 at 5.6 Å, 5.2 Å and 7.4 Å resolution, respectively. Consequently, we present the most complete description yet, to our knowledge, of the CD4-17b-induced intermediate and provide the molecular basis of the receptor-binding-induced conformational change required for HIV-1 entry into host cells. Both CD4 and b12 induce large, previously uncharacterized conformational rearrangements in the gp41 subunits, and the fusion peptide becomes buried in a newly formed pocket. These structures provide key details on the biological function of the type I viral fusion machine from HIV-1 as well as new templates for inhibitor design.
凍結剤: ETHANE / チャンバー内温度: 277 K / 装置: HOMEMADE PLUNGER 詳細: 3 uL of sample applied to a holey carbon grid on glow discharged face and blotted manually on sample side until filter paper detached from grid, followed by immediate plunging.
詳細
B41 SOSIP.664 was incubated with a 6X molar excess of soluble CD4 and 17b Fab overnight at room temperature, purified by size exclusion chromatography, and concentrated prior to grid application
Homology model of B41 SOSIP.664 created using PDB 5CEZ as initial model. sCD4 and 17b coordinates taken from PDB 1GC1. Performed fragment based refinement using Rosetta, followed by relaxed refinement in Rosetta. Modeled glycans using Chimera and performed final refinements using Phenix.
精密化
空間: REAL / プロトコル: FLEXIBLE FIT / 当てはまり具合の基準: EMRinger
得られたモデル
PDB-5vn3: Cryo-EM model of B41 SOSIP.664 in complex with soluble CD4 (D1-D2) and fragment antigen binding variable domain of 17b