Co-reporter:Jeong Hyun Lee;Gabriel Ozorowski;Andrew B. Ward
Science 2016 Vol 351(6277) pp:1043-1048
Publication Date(Web):04 Mar 2016
DOI:10.1126/science.aad2450
A more complete look at the HIV-1 envelope
HIV-1 uses its envelope protein (Env), a large glycoprotein present on the viral surface, to enter target cells. Env forms trimers on the viral surface. Structural studies of solubilized Env trimers have provided important insights into viral entry and antibody binding, but soluble trimers lack several important insoluble regions of the native protein. Lee et al. used cryo–electron microscopy to solve the structure of a trimeric Env protein of HIV-1, missing only its cytoplasmic tail, in complex with broadly neutralizing antibodies. A more complete understanding of Env's structure may aid in vaccine design ef orts.
Science, this issue p. 1043
Co-reporter:Jean-Philippe Julien;Alba Torrents de la Peña;Jeong Hyun Lee;Gabriel Ozorowski;Yuanzi Hua;Steven W. de Taeye;Travis Nieusma;Anila Yasmeen;Pavel Pugach;John P. Moore;Michael Golabek;Albert Cupo;Andrew B. Ward;P. J. Klasse;Rogier W. Sanders;Ian A. Wilson
PNAS 2015 Volume 112 (Issue 38 ) pp:11947-11952
Publication Date(Web):2015-09-22
DOI:10.1073/pnas.1507793112
A key challenge in the quest toward an HIV-1 vaccine is design of immunogens that can generate a broadly neutralizing antibody
(bnAb) response against the enormous sequence diversity of the HIV-1 envelope glycoprotein (Env). We previously demonstrated
that a recombinant, soluble, fully cleaved SOSIP.664 trimer based on the clade A BG505 sequence is a faithful antigenic and
structural mimic of the native trimer in its prefusion conformation. Here, we sought clade C native-like trimers with comparable
properties. We identified DU422 and ZM197M SOSIP.664 trimers as being appropriately thermostable (Tm of 63.4 °C and 62.7 °C,
respectively) and predominantly native-like, as determined by negative-stain electron microscopy (EM). Size exclusion chromatography,
ELISA, and surface plasmon resonance further showed that these trimers properly display epitopes for all of the major bnAb
classes, including quaternary-dependent, trimer-apex (e.g., PGT145) and gp120/gp41 interface (e.g., PGT151) epitopes. A cryo-EM
reconstruction of the ZM197M SOSIP.664 trimer complexed with VRC01 Fab against the CD4 binding site at subnanometer resolution
revealed a striking overall similarity to its BG505 counterpart with expected local conformational differences in the gp120
V1, V2, and V4 loops. These stable clade C trimers contribute additional diversity to the pool of native-like Env immunogens
as key components of strategies to induce bnAbs to HIV-1.
Co-reporter:Charles D. Murin;Xiangguo Qiu;Marnie L. Fusco;Larry Zeitlin;Erica Ollmann Saphire;Zachary A. Bornholdt;Andrew B. Ward;Gene G. Olinger;Gary P. Kobinger
PNAS 2014 Volume 111 (Issue 48 ) pp:17182-17187
Publication Date(Web):2014-12-02
DOI:10.1073/pnas.1414164111
Ebola virus (EBOV) and related filoviruses cause severe hemorrhagic fever, with up to 90% lethality, and no treatments are
approved for human use. Multiple recent outbreaks of EBOV and the likelihood of future human exposure highlight the need for
pre- and postexposure treatments. Monoclonal antibody (mAb) cocktails are particularly attractive candidates due to their
proven postexposure efficacy in nonhuman primate models of EBOV infection. Two candidate cocktails, MB-003 and ZMAb, have
been extensively evaluated in both in vitro and in vivo studies. Recently, these two therapeutics have been combined into
a new cocktail named ZMapp, which showed increased efficacy and has been given compassionately to some human patients. Epitope
information and mechanism of action are currently unknown for most of the component mAbs. Here we provide single-particle
EM reconstructions of every mAb in the ZMapp cocktail, as well as additional antibodies from MB-003 and ZMAb. Our results
illuminate key and recurring sites of vulnerability on the EBOV glycoprotein and provide a structural rationale for the efficacy
of ZMapp. Interestingly, two of its components recognize overlapping epitopes and compete with each other for binding. Going
forward, this work now provides a basis for strategic selection of next-generation antibody cocktails against Ebola and related
viruses and a model for predicting the impact of ZMapp on potential escape mutations in ongoing or future Ebola outbreaks.
Co-reporter:Jean-Philippe Julien;Albert Cupo;Devin Sok;Robyn L. Stanfield;Dmitry Lyumkis;Marc C. Deller;Per-Johan Klasse;Dennis R. Burton;Rogier W. Sanders;John P. Moore;Andrew B. Ward;Ian A. Wilson
Science 2013 Volume 342(Issue 6165) pp:1477-1483
Publication Date(Web):20 Dec 2013
DOI:10.1126/science.1245625
Knowing the Enemy
Infection of host cells by HIV-1 is mediated by an envelope glycoprotein (Env) trimeric spike on the surface of the virus. Proteins comprising the Env trimer must be cleaved for infectivity, and thus viral fusion involves three Env conformations. The flexibility of the Env trimer has made it a challenge to determine a high-resolution structure, although such a structure is key both for understanding trimer function and for guiding vaccine design. Lyumkis et al. (p. 1484) and Julien et al. (p. 1477) studied soluble cleaved trimers stabilized by specific mutations but that have kept a near-native antigenicity profile. Lyumkis et al. present a high-resolution structure of the trimer in complex with a broadly neutralizing antibody, and Julien et al. present a crystal structure of the trimer in complex with another broadly neutralizing antibody.
Co-reporter:Dmitry Lyumkis;Jean-Philippe Julien;Natalia de Val;Albert Cupo;Clinton S. Potter;Per-Johan Klasse;Dennis R. Burton;Rogier W. Sanders;John P. Moore;Bridget Carragher;Ian A. Wilson;Andrew B. Ward
Science 2013 Volume 342(Issue 6165) pp:1484-1490
Publication Date(Web):20 Dec 2013
DOI:10.1126/science.1245627
Knowing the Enemy
Infection of host cells by HIV-1 is mediated by an envelope glycoprotein (Env) trimeric spike on the surface of the virus. Proteins comprising the Env trimer must be cleaved for infectivity, and thus viral fusion involves three Env conformations. The flexibility of the Env trimer has made it a challenge to determine a high-resolution structure, although such a structure is key both for understanding trimer function and for guiding vaccine design. Lyumkis et al. (p. 1484) and Julien et al. (p. 1477) studied soluble cleaved trimers stabilized by specific mutations but that have kept a near-native antigenicity profile. Lyumkis et al. present a high-resolution structure of the trimer in complex with a broadly neutralizing antibody, and Julien et al. present a crystal structure of the trimer in complex with another broadly neutralizing antibody.
Co-reporter:Erick Giang;Kristin E. Cogburn;Xiaoping Dai;Robyn L. Stanfield;Yuanzi Hua;Leopold Kong;Travis Nieusma;Rameshwar U. Kadam;Andrew B. Ward;Dennis R. Burton;Ian A. Wilson;Mansun Law
Science 2013 Volume 342(Issue 6162) pp:1090-1094
Publication Date(Web):29 Nov 2013
DOI:10.1126/science.1243876
Deciphering Hepatitis C
Hepatitis C virus is a major cause of liver disease and cancer. Two envelope glycoproteins, E1 and E2, form a heterodimer that facilitates infection. The envelope proteins have been difficult to crystallize, hindering vaccine development. Kong et al. (p. 1090) designed an E2 core glycoprotein construct and solved the crystal structure of the glycosylated protein in complex with a broadly neutralizing antibody. The host cell receptor binding site was identified by electron microscopy and mutagenesis. The findings should help in future drug and vaccine design.
Co-reporter:Jean-Philippe Julien;Albert Cupo;Jeong Hyun Lee;Ronald Derking;Simon Hoffenberg;Charles D. Murin;C. Richter King;Per Johan Klasse;Rogier W. Sanders;Ian A. Wilson;Michael J. Caulfield;Andre J. Marozsan;John P. Moore;Andrew B. Ward
PNAS 2013 Volume 110 (Issue 11 ) pp:4351-4356
Publication Date(Web):2013-03-12
DOI:10.1073/pnas.1217537110
PG9 is the founder member of an expanding family of glycan-dependent human antibodies that preferentially bind the HIV (HIV-1)
envelope (Env) glycoprotein (gp) trimer and broadly neutralize the virus. Here, we show that a soluble SOSIP.664 gp140 trimer
constructed from the Clade A BG505 sequence binds PG9 with high affinity (∼11 nM), enabling structural and biophysical characterizations
of the PG9:Env trimer complex. The BG505 SOSIP.664 gp140 trimer is remarkably stable as assessed by electron microscopy (EM)
and differential scanning calorimetry. EM, small angle X-ray scattering, size exclusion chromatography with inline multiangle
light scattering and isothermal titration calorimetry all indicate that only a single PG9 fragment antigen-binding (Fab) binds
to the Env trimer. An ∼18 Å EM reconstruction demonstrates that PG9 recognizes the trimer asymmetrically at its apex via contact
with two of the three gp120 protomers, possibly contributing to its reported preference for a quaternary epitope. Molecular
modeling and isothermal titration calorimetry binding experiments with an engineered PG9 mutant suggest that, in addition
to the N156 and N160 glycan interactions observed in crystal structures of PG9 with a scaffolded V1/V2 domain, PG9 makes secondary
interactions with an N160 glycan from an adjacent gp120 protomer in the antibody–trimer complex. Together, these structural
and biophysical findings should facilitate the design of HIV-1 immunogens that possess all elements of the quaternary PG9
epitope required to induce broadly neutralizing antibodies against this region.
Co-reporter:Andrew Ward, Sheila Mulligan, Bridget Carragher, Geoffrey Chang, Ronald A. Milligan
Journal of Structural Biology (March 2009) Volume 165(Issue 3) pp:169-175
Publication Date(Web):1 March 2009
DOI:10.1016/j.jsb.2008.11.006
Bacterial ATP binding cassette (ABC) exporters fulfill a wide variety of transmembrane transport roles and are homologous to the human multidrug resistance P-glycoprotein. Recent X-ray structures of the exporters MsbA and Sav1866 have begun to describe the conformational changes that accompany the ABC transport cycle. Here we present cryo-electron microscopy structures of MsbA reconstituted into a lipid bilayer. Using ATPase inhibitors, we captured three nucleotide transition states of the transporter that were subsequently reconstituted into helical arrays. The enzyme–substrate complex (trapped by ADP-aluminum fluoride or AMPPNP) crystallized in a different helical lattice than the enzyme–product complex (trapped by ADP-vanadate). ∼20 Å resolution maps were calculated for each state and revealed MsbA to be a dimer with a large channel between the membrane spanning domains, similar to the outward facing crystal structures of MsbA and Sav1866. This suggests that while there are likely structural differences between the nucleotide transition states, membrane embedded MsbA remains in an outward facing conformation while nucleotide is bound.
Co-reporter:Jeong Hyun Lee, Natalia de Val, Dmitry Lyumkis, Andrew B. Ward
Structure (6 October 2015) Volume 23(Issue 10) pp:1943-1951
Publication Date(Web):6 October 2015
DOI:10.1016/j.str.2015.07.020
•Natively glycosylated HIV-1 Env trimer solved to 4.36 Å resolution by cryo-EM•Complete description of the epitope of broadly neutralizing HIV-1 antibody PGT128•Multi-branched oligomannose glycans can be visualized and modeled by cryo-EMSecretory and membrane proteins from mammalian cells undergo post-translational modifications, including N-linked glycosylation, which can result in a large number of possible glycoforms. This sample heterogeneity can be problematic for structural studies, particularly X-ray crystallography. Thus, crystal structures of heavily glycosylated proteins such as the HIV-1 Env viral spike protein have been determined by removing the majority of glycans. This step is most frequently carried out using Endoglycosidase H (EndoH) and requires that all expressed glycans be in the high-mannose form, which is often not the native glycoform. With significantly improved technologies in single-particle cryoelectron microscopy, we demonstrate that it is now possible to refine and build natively glycosylated HIV-1 Env structures in solution to 4.36 Å resolution. At this resolution we can now analyze the complete epitope of a broadly neutralizing antibody (bnAb), PGT128, in the context of the trimer expressed with native glycans.
Co-reporter:Claudia Blattner, Jeong Hyun Lee, Kwinten Sliepen, Ronald Derking, ... Andrew B. Ward
Immunity (15 May 2014) Volume 40(Issue 5) pp:669-680
Publication Date(Web):15 May 2014
DOI:10.1016/j.immuni.2014.04.008
•PGT151 binds an interprotomer epitope formed by gp41 and gp120 on cleaved Env trimers•The PGT151 epitope does not overlap with any other epitope described so far•PGT151 enables isolation of functional cleaved Env from the cell membrane•Membrane-extracted and soluble SOSIP.664 Env trimers are structurally similarAll previously characterized broadly neutralizing antibodies to the HIV-1 envelope glycoprotein (Env) target one of four major sites of vulnerability. Here, we define and structurally characterize a unique epitope on Env that is recognized by a recently discovered family of human monoclonal antibodies (PGT151–PGT158). The PGT151 epitope is comprised of residues and glycans at the interface of gp41 and gp120 within a single protomer and glycans from both subunits of a second protomer and represents a neutralizing epitope that is dependent on both gp120 and gp41. Because PGT151 binds only to properly formed, cleaved trimers, this distinctive property, and its ability to stabilize Env trimers, has enabled the successful purification of mature, cleaved Env trimers from the cell surface as a complex with PGT151. Here we compare the structural and functional properties of membrane-extracted Env trimers from several clades with those of the soluble, cleaved SOSIP gp140 trimer.Download high-res image (332KB)Download full-size image
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