Owen's Papers | Barbie's Papers


D. M. Dawidziak, J. G. Sanchez, J. M. Wagner, B. K. Ganser-Pornillos, and O. Pornillos (2017) “Structure and catalytic activation of the TRIM23 RING E3 ubiquitin ligase,” Proteins, accepted for publication (Pubmed) (PDB: 5VZV) (PDB: 5VZW) (Abstract) Tripartite motif (TRIM) proteins comprise a large family of RING-type ubiquitin E3 ligases that regulate important biological processes. An emerging general model is that TRIMs form elongated antiparallel coiled-coil dimers that prevent interaction of the two attendant RING domains. The RING domains themselves bind E2 conjugating enzymes as dimers, implying that an active TRIM ligase requires higher-order oligomerization of the basal coiled-coil dimers. Here, we report crystal structures of the TRIM23 RING domain in isolation and in complex with an E2-ubiquitin conjugate. Our results indicate that TRIM23 enzymatic activity requires RING dimerization, consistent with the general model of TRIM activation.

P. Ł. Janczyk, K. A. Skorupka, J. G. Tooley, D. R. Matson, C. A. Kestner, T. West, O. Pornillos, and P. T. Stukenberg (2017) “Mechanism of Ska recruitment by Ndc80 complexes to kinetochores,” Dev Cell 41, 438-449 (Pubmed) (Abstract) Yeast use the ring-shaped Dam1 complex to slide down depolymerizing microtubules to move chromosomes, but current models suggest that other eukaryotes do not have a sliding ring. We visualized Ndc80 and Ska complexes on microtubules by electron microscopic tomography to identify the structure of the human kinetochore-microtubule attachment. Ndc80 recruits the Ska complex so that the V shape of the Ska dimer interacts along protofilaments. We identify a mutant of the Ndc80 tail that is deficient in Ska recruitment to kinetochores and in orienting Ska along protofilaments in vitro. This mutant Ndc80 binds microtubules with normal affinity but is deficient in clustering along protofilaments. We propose that Ska is recruited to kinetochores by clusters of Ndc80 proteins and that our structure of Ndc80 and Ska complexes on microtubules suggests a mechanism for metazoan kinetochores to couple the depolymerization of microtubules to power the movement of chromosomes.


M. J. Bayro, B. K. Ganser-Pornillos, K. K. Zadrozny, M. Yeager, and R. Tycko (2016) “Helical conformation in the CA-SP1 junction of the immature HIV-1 lattice determined from solid-state NMR of virus-like particles,” J Am Chem Soc 138, 12029-12032 (Pubmed) (Abstract) Maturation of HIV-1 requires disassembly of the Gag polyprotein lattice, which lines the viral membrane in the immature state, and subsequent assembly of the mature capsid protein lattice, which encloses viral RNA in the mature state. Metastability of the immature lattice has been proposed to depend on the existence of a structurally ordered, α-helical segment spanning the junction between capsid (CA) and spacer peptide 1 (SP1) subunits of Gag, a segment that is dynamically disordered in the mature capsid lattice. We report solid state nuclear magnetic resonance (ssNMR) measurements on the immature lattice in noncrystalline, spherical virus-like particles (VLPs) derived from Gag. The ssNMR data provide definitive evidence for this critical α-helical segment in the VLPs. Differences in ssNMR chemical shifts and signal intensities between immature and mature lattice assemblies also support a major rearrangement of intermolecular interactions in the maturation process, consistent with recent models from electron cryomicroscopy and X-ray crystallography.

J. G. Sanchez, J. J. Chiang, K. M. Sparrer, S. L. Alam, M. Chi, M. D. Roganowicz, B. Sankaran, M. U. Gack, and O. Pornillos (2016) “Mechanism of TRIM25 catalytic activation in the antiviral RIG-I pathway,” Cell Rep 16, 1315-1325 (Pubmed) (PDB: 5EYA) (Abstract) Antiviral response pathways induce interferon by higher-order assembly of signaling complexes called signalosomes. Assembly of the RIG-I signalosome is regulated by K63-linked polyubiquitin chains, which are synthesized by the E3 ubiquitin ligase, TRIM25. We have previously shown that the TRIM25 coiled-coil domain is a stable, antiparallel dimer that positions two catalytic RING domains on opposite ends of an elongated rod. We now show that the RING domain is a separate self-association motif that engages ubiquitin-conjugated E2 enzymes as a dimer. RING dimerization is required for catalysis, TRIM25-mediated RIG-I ubiquitination, interferon induction, and antiviral activity. We also provide evidence that RING dimerization and E3 ligase activity are promoted by binding of the TRIM25 SPRY domain to the RIG-I effector domain. These results indicate that TRIM25 actively participates in higher-order assembly of the RIG-I signalosome and helps to fine-tune the efficiency of the RIG-I-mediated antiviral response.

J. M. Wagner, K. K. Zadrozny, J. Chrustowicz, M. D. Purdy, M. Yeager, B. K. Ganser-Pornillos, and O. Pornillos (2016) “Crystal structure of an HIV assembly and maturation switch,” eLife 5, e17063 (Pubmed) (PDB: 5I4T) (Abstract) Virus assembly and maturation proceed through the programmed operation of molecular switches, which trigger both local and global structural rearrangements to produce infectious particles. HIV-1 contains an assembly and maturation switch that spans the C-terminal domain (CTD) of the capsid (CA) region and the first spacer peptide (SP1) of the precursor structural protein, Gag. The crystal structure of the CTD-SP1 Gag fragment is a goblet-shaped hexamer in which the cup comprises the CTD and an ensuing type II β-turn, and the stem comprises a 6-helix bundle. The β-turn is critical for immature virus assembly and the 6-helix bundle regulates proteolysis during maturation. This bipartite character explains why the SP1 spacer is a critical element of HIV-1 Gag but is not a universal property of retroviruses. Our results also indicate that HIV-1 maturation inhibitors suppress unfolding of the CA-SP1 junction and thereby delay access of the viral protease to its substrate.

J. M. Wagner, M. Roganowicz, K. Skorupka, S. L. Alam, D. E. Christensen, G. L. Doss, Y. Wan, G. A. Frank, B. K. Ganser-Pornillos, W. I. Sundquist, and O. Pornillos (2016) “Mechanism of B-box 2 domain-mediated higher-order assembly of the retroviral restriction factor TRIM5α,” eLife 5, e16309 (Pubmed) (PDB: 5EIU) (PDB: 5F7T) (PDB: 5IEA) (Abstract) Restriction factors and pattern recognition receptors are important components of intrinsic cellular defenses against viral infection. Mammalian TRIM5α proteins are restriction factors and receptors that target the capsid cores of retroviruses and activate ubiquitin-dependent antiviral responses upon capsid recognition. Here, we report crystallographic and functional studies of the TRIM5α B-box 2 domain, which mediates higher-order assembly of TRIM5 proteins. The B-box can form both dimers and trimers, and the trimers can link multiple TRIM5α proteins into a hexagonal net that matches the lattice arrangement of capsid subunits and enables avid capsid binding. Two modes of conformational flexibility allow TRIM5α to accommodate the variable curvature of retroviral capsids. B-box mediated interactions also modulate TRIM5α's E3 ubiquitin ligase activity, by stereochemically restricting how the N-terminal RING domain can dimerize. Overall, these studies define important molecular details of cellular recognition of retroviruses, and how recognition links to downstream processes to disable the virus.

Y.-L. Li, V. Chandrasekaran, S. D. Carter, C. L. Woodward, D. Christensen, K. A. Dryden, O. Pornillos, M. Yeager, B. K. Ganser-Pornillos, G. J. Jensen, and W. I. Sundquist (2016) “Primate TRIM5 proteins form hexagonal nets on HIV-1 capsids,” eLife 5, e16269 (Pubmed) (Abstract) TRIM5 proteins are restriction factors that block retroviral infections by binding viral capsids and preventing reverse transcription. Capsid recognition is mediated by C-terminal domains on TRIM5α (SPRY) or TRIMCyp (cyclophilin A), which interact weakly with capsids. Efficient capsid recognition also requires the conserved N-terminal tripartite motifs (TRIM), which mediate oligomerization and create avidity effects. To characterize how TRIM5 proteins recognize viral capsids, we developed methods for isolating native recombinant TRIM5 proteins and purifying stable HIV-1 capsids. Biochemical and EM analyses revealed that TRIM5 proteins assembled into hexagonal nets, both alone and on capsid surfaces. These nets comprised open hexameric rings, with the SPRY domains centered on the edges and the B-box and RING domains at the vertices. Thus, the principles of hexagonal TRIM5 assembly and capsid pattern recognition are conserved across primates, allowing TRIM5 assemblies to maintain the conformational plasticity necessary to recognize divergent and pleomorphic retroviral capsids.

J. M. Grime, J. F. Dama, B. K. Ganser-Pornillos, C. L. Woodward, G. J. Jensen, M. Yeager, and G. A. Voth (2016) “Coarse-grained simulation reveals key features of HIV-1 capsid self-assembly,” Nat Commun 7, 11568 (Pubmed) (Abstract) The maturation of HIV-1 viral particles is essential for viral infectivity. During maturation, many copies of the capsid protein (CA) self-assemble into a capsid shell to enclose the viral RNA. The mechanistic details of the initiation and early stages of capsid assembly remain to be delineated. We present coarse-grained simulations of capsid assembly under various conditions, considering not only capsid lattice self-assembly but also the potential disassembly of capsid upon delivery to the cytoplasm of a target cell. The effects of CA concentration, molecular crowding, and the conformational variability of CA are described, with results indicating that capsid nucleation and growth is a multi-stage process requiring well-defined metastable intermediates. Generation of the mature capsid lattice is sensitive to local conditions, with relatively subtle changes in CA concentration and molecular crowding influencing self-assembly and the ensemble of structural morphologies.


A. T. Gres, K. A. Kirby, V. N. KewalRamani, J. J. Tanner, O. Pornillos, and S. G. Sarafianos (2015) “X-ray crystal structures of native HIV-1 capsid protein reveal conformational variability,” Science 349, 99-103 (Pubmed) (Abstract) The detailed molecular interactions between native HIV-1 capsid protein (CA) hexamers that shield the viral genome and proteins have been elusive. We report crystal structures describing interactions between CA monomers related by 6-fold symmetry within hexamers (intra-hexamer), and 3-fold and 2-fold symmetry between neighboring hexamers (inter-hexamer). The structures describe how CA builds hexagonal lattices, the foundation of mature capsids. Lattice structure depends on an adaptable hydration layer modulating interactions among CA molecules. Disruption of this layer alters inter-hexamer interfaces, highlighting an inherent structural variability. A CA-targeting antiviral affects capsid stability by binding across CA molecules and altering inter-hexamer interfaces remote to the ligand-binding site. Inherent structural plasticity, hydration layer rearrangement, and effector-binding affect capsid stability and have functional implications for the retroviral life-cycle.


A. Bhattacharya, S. L. Alam, T. Fricke, K. Zadrozny, J. Sedzicki, A. B. Taylor, B. Demeler, O. Pornillos, B. K. Ganser-Pornillos, F. Diaz-Griffero, D. N. Ivanov, and M. Yeager (2014) “Structural basis of HIV-1 capsid recognition by PF74 and CPSF6,” Proc Natl Acad Sci USA 111, 18625-18630 (Pubmed) (PDB: 4QNB) (Abstract) Upon infection of susceptible cells by HIV-1, the conical capsid formed by ∼250 hexamers and 12 pentamers of the CA protein is delivered to the cytoplasm. The capsid shields the RNA genome and proteins required for reverse transcription. In addition, the surface of the capsid mediates numerous host-virus interactions, which either promote infection or enable viral restriction by innate immune responses. In the intact capsid, there is an intermolecular interface between the N-terminal domain (NTD) of one subunit and the C-terminal domain (CTD) of the adjacent subunit within the same hexameric ring. The NTD-CTD interface is critical for capsid assembly, both as an architectural element of the CA hexamer and pentamer and as a mechanistic element for generating lattice curvature. Here we report biochemical experiments showing that PF-3450074 (PF74), a drug that inhibits HIV-1 infection, as well as host proteins cleavage and polyadenylation specific factor 6 (CPSF6) and nucleoporin 153 kDa (NUP153), bind to the CA hexamer with at least 10-fold higher affinities compared with nonassembled CA or isolated CA domains. The crystal structure of PF74 in complex with the CA hexamer reveals that PF74 binds in a preformed pocket encompassing the NTD-CTD interface, suggesting that the principal inhibitory target of PF74 is the assembled capsid. Likewise, CPSF6 binds in the same pocket. Given that the NTD-CTD interface is a specific molecular signature of assembled hexamers in the capsid, binding of NUP153 at this site suggests that key features of capsid architecture remain intact upon delivery of the preintegration complex to the nucleus.

J. G. Sanchez, K. Okreglicka, V. Chandrasekaran, J. M. Welker, W. I. Sundquist, and O. Pornillos (2014) “The tripartite motif coiled-coil is an elongated antiparallel hairpin dimer,” Proc Natl Acad Sci USA 111, 2494-2499 (Pubmed) (PDB: 4LTB) (Abstract) Tripartite motif (TRIM) proteins comprise a large family of coiled-coil containing RING E3 ligases that function in many cellular processes, and particularly in innate antiviral response pathways. The TRIM coiled-coil mediates dimer formation. Both dimerization and high-order assembly are important elements of TRIM protein function, but the atomic details of TRIM quaternary structure have not been defined. Here, we present crystallographic and biochemical analyses showing that TRIM proteins dimerize by forming interdigitating antiparallel helical hairpins. The dimer core comprises an antiparallel coiled-coil with a distinctive, symmetric pattern of heptad and hendecad repeats that appears to be conserved across the entire TRIM family. Our studies reveal how the coiled-coil positions the N-terminal catalytic RING domain and C-terminal substrate-binding domain of the TRIM25 protein to facilitate polyubiquitylation of the RIG-I/viral RNA recognition complex, and how dimers of the TRIM5α protein are organized into hexagonal arrays that recognize the HIV-1 capsid lattice and restrict retroviral replication.


M. Yeager, K. A. Dryden, and B. K. Ganser-Pornillos (2013) “Lipid monolayer and sparse matrix screening for growing two-dimensional crystals for electron crystallography: methods and examples,” Methods Mol Biol 955, 527-537 (Pubmed) (Abstract) Electron microscopy provides an efficient method for rapidly assessing whether a solution of macromolecules is homogeneous and monodisperse. If the macromolecules can be induced to form two-dimensional crystals that are a single layer in thickness, then electron crystallography of frozen-hydrated crystals has the potential of achieving three-dimensional density maps at sub-nanometer or even atomic resolution. Here we describe the lipid monolayer and sparse matrix screening methods for growing two-dimensional crystals and present successful applications to soluble macromolecular complexes: carboxysome shell proteins and HIV CA, respectively. Since it is common to express recombinant proteins with poly-His tags for purification by metal affinity chromatography, the monolayer technique using bulk lipids doped with Ni(2+) lipids has the potential for broad application. Likewise, the sparse matrix method uses screening conditions for three-dimensional crystallization and is therefore of broad applicability.


B. K. Ganser-Pornillos, M. Yeager, and O. Pornillos (2012) “Assembly and maturation of HIV,” Adv Exp Med Biol 726, 441-465 (Pubmed)


O. Pornillos, B. K. Ganser-Pornillos, and M. Yeager (2011) “Atomic-level modelling of the HIV capsid,” Nature 469, 424-427 (PDF) (Pubmed) (PDB: 3P05) (PDB: 3P0A) (Abstract) The mature capsids of human immunodeficiency virus type 1 (HIV-1) and other retroviruses are fullerene shells, composed of the viral CA protein, that enclose the viral genome and facilitate its delivery into new host cells. Retroviral CA proteins contain independently folded amino (N)- and carboxy (C)-terminal domains (NTD and CTD) that are connected by a flexible linker. The NTD forms either hexameric or pentameric rings, whereas the CTD forms symmetric homodimers that connect the rings into a hexagonal lattice. We previously used a disulphide crosslinking strategy to enable isolation and crystallization of soluble HIV-1 CA hexamers. Here we use the same approach to solve the X-ray structure of the HIV-1 CA pentamer at 2.5 Å resolution. Two mutant CA proteins with engineered disulphides at different positions (P17C/T19C and N21C/A22C) converged onto the same quaternary structure, indicating that the disulphide-crosslinked proteins recapitulate the structure of the native pentamer. Assembly of the quasi-equivalent hexamers and pentamers requires remarkably subtle rearrangements in subunit interactions, and appears to be controlled by an electrostatic switch that favours hexamers over pentamers. This study completes the gallery of substructures describing the components of the HIV-1 capsid and enables atomic-level modelling of the complete capsid. Rigid-body rotations around two assembly interfaces appear sufficient to generate the full range of continuously varying lattice curvature in the fullerene cone.

B. K. Ganser-Pornillos, V. Chandrasekaran, O. Pornillos, J. G. Sodroski, W. I. Sundquist, and M. Yeager (2011) “Hexagonal assembly of a restricting Trim5α protein,” Proc Natl Acad Sci USA 108, 534-539 (PDF) (Pubmed) (Abstract) TRIM5α proteins are restriction factors that protect mammalian cells from retroviral infections by binding incoming viral capsids, accelerating their dissociation, and preventing reverse transcription of the viral genome. Individual TRIM5 isoforms can often protect cells against a broad range of retroviruses, as exemplified by rhesus monkey TRIM5α and its variant, TRIM5-21R, which recognize HIV-1 as well as several distantly related retroviruses. Although capsid recognition is not yet fully understood, previous work has shown that the C-terminal SPRY/B30.2 domain of dimeric TRIM5α binds directly to viral capsids, and that higher-order TRIM5α oligomerization appears to contribute to the efficiency of capsid recognition. Here, we report that recombinant TRIM5-21R spontaneously assembled into two-dimensional paracrystalline hexagonal lattices comprising open, six-sided rings. TRIM5-21R assembly did not require the C-terminal SPRY domain, but did require both protein dimerization and a B-box 2 residue (Arg121) previously implicated in TRIM5α restriction and higher-order assembly. Furthermore, TRIM5-21R assembly was promoted by binding to hexagonal arrays of the HIV-1 CA protein that mimic the surface of the viral capsid. We therefore propose that TRIM5α proteins have evolved to restrict a range of different retroviruses by assembling a deformable hexagonal scaffold that positions the capsid-binding domains to match the symmetry and spacing of the capsid surface lattice. Capsid recognition therefore involves a synergistic combination of direct binding interactions, avidity effects, templated assembly, and lattice complementarity.


O. Pornillos, B. K. Ganser-Pornillos, S. Banumathi, Y. Hua, and M. Yeager (2010) “Disulfide bond stabilization of the hexameric capsomer of human immunodeficiency virus,” J Mol Biol 401, 985-995 (Pubmed) (PDB: 3MGE)

D. Zhu, J. Hensel, R. Hilgraf, M. Abbasian, O. Pornillos, G. Deyanat-Yazdi, X. H. Hua, and S. Cox (2010) “Inhibition of protein kinase CK2 expression and activity blocks tumor cell growth in vitro,” Mol Cell Biochem 333, 159-167 (Pubmed)


O. Pornillos, B. K. Ganser-Pornillos, B. N. Kelly, Y. Hua, F. G. Whitby, C. D. Stout, W. I. Sundquist, C. P. Hill, and M. Yeager (2009) “X-ray structures of the hexameric building block of the HIV capsid,” Cell 137, 1282-1292 (Pubmed) (PDB: 3GV2) (PDB: 3H4E) (PDB: 3H47)


B. K. Ganser-Pornillos, M. Yeager, and W. I. Sundquist (2008) “The structural biology of HIV assembly,” Curr Opin Struct Biol 18, 203-217 (Pubmed) (Abstract) HIV assembly and replication proceed through the formation of morphologically distinct immature and mature viral capsids that are organized by the Gag polyprotein (immature) and by the fully processed CA protein (mature). The Gag polyprotein is composed of three folded polypeptides (MA, CA, and NC) and three smaller peptides (SP1, SP2, and p6) that function together to coordinate membrane binding and Gag-Gag lattice interactions in immature virions. Following budding, HIV maturation is initiated by proteolytic processing of Gag, which induces conformational changes in the CA domain and results in the assembly of the distinctive conical capsid. Retroviral capsids are organized following the principles of fullerene cones, and the hexagonal CA lattice is stabilized by three distinct interfaces. Recently identified inhibitors of viral maturation act by disrupting the final stage of Gag processing, or by inhibiting the formation of a critical intermolecular CA-CA interface in the mature capsid. Following release into a new host cell, the capsid disassembles and host cell factors can potently restrict this stage of retroviral replication. Here, we review the structures of immature and mature HIV virions, focusing on recent studies that have defined the global organization of the immature Gag lattice, identified sites likely to undergo conformational changes during maturation, revealed the molecular structure of the mature capsid lattice, demonstrated that capsid architectures are conserved, identified the first capsid assembly inhibitors, and begun to uncover the remarkable biology of the mature capsid.


Y.-J. Chen, O. Pornillos, S. Lieu, C. Ma, A. P. Chen, and G. Chang (2007) “X-ray structure of EmrE supports dual topology model,” Proc Natl Acad Sci USA 104, 18999-19004 (PDF) (Pubmed) (PDB: 3B5D) (PDB: 3B61) (PDB: 3B62) (Abstract) EmrE, a multidrug transporter from Escherichia coli, functions as a homodimer of a small four-transmembrane protein. The membrane insertion topology of the two monomers is controversial. Although the EmrE protein was reported to have a unique orientation in the membrane, models based on electron microscopy and now defunct x-ray structures, as well as recent biochemical studies, posit an antiparallel dimer. We have now reanalyzed our x-ray data on EmrE. The corrected structures in complex with a transport substrate are highly similar to the electron microscopy structure. The first three transmembrane helices from each monomer surround the substrate binding chamber, whereas the fourth helices participate only in dimer formation. Selenomethionine markers clearly indicate an antiparallel orientation for the monomers, supporting a "dual topology" model.

B. K. Ganser-Pornillos, A. Cheng, and M. Yeager (2007) “Structure of full-length HIV-1 CA: a model for the mature capsid lattice,” Cell 131, 70-79 (Pubmed) (PDB: 3DIK) (Abstract) The capsids of mature retroviruses perform the essential function of organizing the viral genome for efficient replication. These capsids are modeled as fullerene structures composed of closed hexameric arrays of the viral CA protein, but a high-resolution structure of the lattice has remained elusive. A three-dimensional map of two-dimensional crystals of the R18L mutant of HIV-1 CA was derived by electron cryocrystallography. The docking of high-resolution domain structures into the map yielded the first unambiguous model for full-length HIV-1 CA. Three important protein-protein assembly interfaces are required for capsid formation. Each CA hexamer is composed of an inner ring of six N-terminal domains and an outer ring of C-terminal domains that form dimeric linkers connecting neighboring hexamers. Interactions between the two domains of CA further stabilize the hexamer and provide a structural explanation for the mechanism of action of known HIV-1 assembly inhibitors.

M. A. Hanson, A. Brooun, K. A. Baker, V. P. Jaakola, C. Roth, E. Y. Chien, A. Alexandrov, J. Velasquez, L. Davis, M. Griffith, K. Moy, B. K. Ganser-Pornillos, Y. Hua, P. Kuhn, S. Ellis, M. Yeager, and R. C. Stevens (2007) “Profiling of membrane protein variants in a baculovirus system by coupling cell-surface detection with small-scale parallel expression,” Protein Expr Purif 56, 85-92 (Pubmed) (Abstract) Production of structure-grade mammalian membrane proteins in substantial quantities has been hindered by a lack of methods for effectively profiling multiple constructs expression in higher eukaryotic systems such as insect or mammalian cells. To address this problem, a specialized small-scale eukaryotic expression platform by Thomson Instrument Company (Vertiga-IM) was developed and used in tandem with a Guava EasyCyte microcapillary 96-well cytometer to monitor cell density and health and evaluate membrane protein expression. Two proof of concept experiments were conducted using the human beta(2)-adrenergic receptor (beta(2)AR) and the gap junction protein connexin26 (Cx26) in a baculovirus expression system. First, cell surface expression was used to assess the expression levels of 14 beta(2)AR truncation variants expressed using the Vertiga-IM shaker. Three of these variants were then compared to wild-type beta(2)AR using three metrics: cell surface expression, saturation ligand binding and protein immunoblot analysis of dodecylmaltoside extracted material. Second, a series of systematic Cx26 truncation variants were evaluated for expression by protein immunoblot analysis. The cumulative results for these two systems show that the Vertiga-IM instrument can be used effectively in the parallel insect cell microexpression of membrane protein variants, and that the expression of cell surface molecules as monitored with the Guava EasyCyte instrument can be used to rapidly assess the production of properly folded proteins in the baculovirus expression system. This approach expedites the in vitro evaluation of a large number of mammalian membrane protein variants.

Z. Zhang, C. Ma, O. Pornillos, X. Xiu, G. Chang, and M. H. Saier Jr (2007) “Functional characterization of the heterooligomeric EbrAB multidrug efflux transporter of Bacillus subtilis,” Biochemistry 46, 5218-5225 (Pubmed)


O. Pornillos and G. Chang (2006) “Inverted repeat domains in membrane proteins,” FEBS Lett 580, 358-362 (Pubmed)


O. Pornillos, Y.-J. Chen, A. P. Chen, and G. Chang (2005) “X-ray structure of the EmrE multidrug transporter in complex with a substrate,” Science 310, 1950-1953.  Retraction: G. Chang, C. B. Roth, C. L. Reyes, O. Pornillos, Y.-J. Chen, A. P. Chen (2006) Science 314, 1875

J. Benjamin, B. K. Ganser-Pornillos, W. F. Tivol, W. I. Sundquist, and G. J. Jensen (2005) “Three-dimensional structure of HIV-1 virus-like particles by electron cryotomography,” J Mol Biol 346, 577-588 (Pubmed)


B. K. Ganser-Pornillos, U. K. von Schwedler, K. M. Stray, C. Aiken, and W. I. Sundquist (2004) “Assembly properties of the human immunodeficiency virus type 1 CA protein,” J Virol 78, 2545-2552 (Pubmed) (Abstract) During retroviral maturation, the CA protein oligomerizes to form a closed capsid that surrounds the viral genome. We have previously identified a series of deleterious surface mutations within human immunodeficiency virus type 1 (HIV-1) CA that alter infectivity, replication, and assembly in vivo. For this study, 27 recombinant CA proteins harboring 34 different mutations were tested for the ability to assemble into helical cylinders in vitro. These cylinders are composed of CA hexamers and are structural models for the mature viral capsid. Mutations that diminished CA assembly clustered within helices 1 and 2 in the N-terminal domain of CA and within the crystallographically defined dimer interface in the CA C-terminal domain. These mutations demonstrate the importance of these regions for CA cylinder production and, by analogy, mature capsid assembly. One CA mutant (R18A) assembled into cylinders, cones, and spheres. We suggest that these capsid shapes occur because the R18A mutation alters the frequency at which pentamers are incorporated into the hexagonal lattice. The fact that a single CA protein can simultaneously form all three known retroviral capsid morphologies supports the idea that these structures are organized on similar lattices and differ only in the distribution of 12 pentamers that allow them to close. In further support of this model, we demonstrate that the considerable morphological variation seen for conical HIV-1 capsids can be recapitulated in idealized capsid models by altering the distribution of pentamers.


O. Pornillos, D. S. Higginson, K. M. Stray, R. D. Fisher, J. E. Garrus, M. Payne, H. E. Wang, S. G. Morham, and W. I. Sundquist (2003) “HIV Gag mimics the Tsg101-recruiting activity of the human Hrs protein,” J Cell Biol 162, 425-434 (PDF) (Pubmed) (Abstract) The HIV-1 Gag protein recruits the cellular factor Tsg101 to facilitate the final stages of virus budding. A conserved P(S/T)AP tetrapeptide motif within Gag (the "late domain") binds directly to the NH2-terminal ubiquitin E2 variant (UEV) domain of Tsg101. In the cell, Tsg101 is required for biogenesis of vesicles that bud into the lumen of late endosomal compartments called multivesicular bodies (MVBs). However, the mechanism by which Tsg101 is recruited from the cytoplasm onto the endosomal membrane has not been known. Now, we report that Tsg101 binds the COOH-terminal region of the endosomal protein hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs; residues 222-777). This interaction is mediated, in part, by binding of the Tsg101 UEV domain to the Hrs 348PSAP351 motif. Importantly, Hrs222-777 can recruit Tsg101 and rescue the budding of virus-like Gag particles that are missing native late domains. These observations indicate that Hrs normally functions to recruit Tsg101 to the endosomal membrane. HIV-1 Gag apparently mimics this Hrs activity, and thereby usurps Tsg101 and other components of the MVB vesicle fission machinery to facilitate viral budding.

B. K. Ganser, A. Cheng, W. I. Sundquist, and M. Yeager (2003) “Three-dimensional structure of the M-MuLV CA protein on a lipid monolayer: a general model for retroviral capsid assembly,” EMBO J 22, 2886-2892 (Pubmed) (Abstract) Although retroviruses from different genera form morphologically distinct capsids, we have proposed that all of these structures are composed of similar hexameric arrays of capsid (CA) protein subunits and that their distinct morphologies reflect different distributions of pentameric declinations that allow the structures to close. Consistent with this model, CA proteins from both HIV-1 and Rous sarcoma virus (RSV) form similar hexagonal lattices. However, recent structural studies have suggested that the Moloney murine leukemia virus (M-MuLV) CA protein may assemble differently. We now report an independent three-dimensional reconstruction of two-dimensional crystals of M-MuLV CA. This new reconstruction reveals a hexameric lattice that is similar to those formed by HIV-1 and RSV CA, supporting a generalized model for retroviral capsid assembly.

B. K. Ganser (2003) Retroviral capsid assembly, Ph.D. Thesis, University of Utah


O. Pornillos, J. E. Garrus, and W. I. Sundquist (2002) “Mechanisms of enveloped RNA virus budding,” Trends Cell Biol 12, 569-579 (Pubmed)

O. Pornillos, S. L. Alam, D. R. Davis, and W. I. Sundquist (2002) “Structure of the Tsg101 UEV domain in complex with the PTAP motif of the HIV-1 p6 protein,” Nat Struct Biol 9, 812-817 (Journal) (PDF) (Pubmed) (PDB: 1M4P) (PDB: 1M4Q) (Abstract) The structural proteins of HIV and Ebola display PTAP peptide motifs (termed 'late domains') that recruit the human protein Tsg101 to facilitate virus budding. Here we present the solution structure of the UEV (ubiquitin E2 variant) binding domain of Tsg101 in complex with a PTAP peptide that spans the late domain of HIV-1 p6Gag. The UEV domain of Tsg101 resembles E2 ubiquitin-conjugating enzymes, and the PTAP peptide binds in a bifurcated groove above the vestigial enzyme active site. Each PTAP residue makes important contacts, and the Ala9-Pro10 dipeptide binds in a deep pocket of the UEV domain that resembles the X-Pro binding pockets of SH3 and WW domains. The structure reveals the molecular basis of HIV PTAP late domain function and represents an attractive starting point for the design of novel inhibitors of virus budding.

O. Pornillos (2002) Ligand interactions of the HIV-1 Gag p6 domain, Ph.D. Thesis, University of Utah

O. Pornillos, S. L. Alam, R. L. Rich, D. G. Myszka, D. R. Davis, and W. I. Sundquist (2002) “Structure and functional interactions of the Tsg101 UEV domain,” EMBO J 21, 2397-2406 (PDF) (Pubmed) (PDB: 1KPP) (PDB: 1KPQ) (Abstract) Human Tsg101 plays key roles in HIV budding and in cellular vacuolar protein sorting (VPS). In performing these functions, Tsg101 binds both ubiquitin (Ub) and the PTAP tetrapeptide 'late domain' motif located within the viral Gag protein. These interactions are mediated by the N-terminal domain of Tsg101, which belongs to the catalytically inactive ubiquitin E2 variant (UEV) family. We now report the structure of Tsg101 UEV and chemical shift mapping of the Ub and PTAP binding sites. Tsg101 UEV resembles canonical E2 ubiquitin conjugating enzymes, but has an additional N-terminal helix, an extended β-hairpin that links strands 1 and 2, and lacks the two C-terminal helices normally found in E2 enzymes. PTAP-containing peptides bind in a hydrophobic cleft exposed by the absence of the C-terminal helices, whereas ubiquitin binds in a novel site surrounding the β-hairpin. These studies provide a structural framework for understanding how Tsg101 mediates the protein–protein interactions required for HIV budding and VPS.


J. E. Garrus, U. K. von Schwedler, O. Pornillos, S. G. Morham, K. H. Zavitz, H. E. Wang, D. A. Wettstein, K. M. Stray, M. Côté, R. L. Rich, D. G. Myszka, and W. I. Sundquist (2001) “Tsg101 and the vacuolar protein sorting pathway are essential for HIV-1 budding,” Cell 107, 55-65 (Pubmed)

Y. Jenkins, O. Pornillos, R. L. Rich, D. G. Myszka, W. I. Sundquist, and M. H. Malim (2001) “Biochemical analyses of the interactions between human immunodeficiency virus type 1 Vpr and p6Gag,” J Virol 75, 10537-10542 (PDF) (Pubmed) (Abstract) The nonstructural human immunodeficiency virus type 1 Vpr protein is packaged into progeny virions at significant levels (~200 copies/virion). Genetic analyses have demonstrated that efficient Vpr packaging is dependent upon a leucine-X-X-leucine-phenylalanine (LXXLF) motif located in the p6Gag domain of the structural Gag polyprotein. Recombinant proteins spanning full-length Vpr (Vpr1-97) or the amino-terminal 71 amino acids (Vpr1-71) formed specific complexes with recombinant p6 proteins in vitro. Complex formation required an intact LXXLF motif and exhibited an intrinsic dissociation constant of ~75 µM. Gel filtration and cross-linking analyses further revealed that Vpr1-71 self-associated in solution. Our experiments demonstrate that Vpr can bind directly and specifically to p6 and suggest that oligomerization of both Vpr and Gag may serve to increase the avidity and longevity of Vpr-Gag complexes, thereby ensuring efficient Vpr packaging.


B. K. Ganser, S. Li, V. Y. Klishko, J. T. Finch, and W. I. Sundquist (1999) “Assembly and analysis of conical models for the HIV-1 core,” Science 283, 80-83 (Pubmed) (Abstract) The genome of the human immunodeficiency virus (HIV) is packaged within an unusual conical core particle located at the center of the infectious virion. The core is composed of a complex of the NC (nucleocapsid) protein and genomic RNA, surrounded by a shell of the CA (capsid) protein. A method was developed for assembling cones in vitro using pure recombinant HIV-1 CA-NC fusion proteins and RNA templates. These synthetic cores are capped at both ends and appear similar in size and morphology to authentic viral cores. It is proposed that both viral and synthetic cores are organized on conical hexagonal lattices, which by Euler's theorem requires quantization of their cone angles. Electron microscopic analyses revealed that the cone angles of synthetic cores were indeed quantized into the five allowed angles. The viral core and most synthetic cones exhibited cone angles of approximately 19 degrees (the narrowest of the allowed angles). These observations suggest that the core of HIV is organized on the principles of a fullerene cone, in analogy to structures recently observed for elemental carbon.

D. B. Stierle, A. A. Stierle, and B. K. Ganser (1999) “Isolation of two highly methylated polyketide derivatives from a yew-associated penicillium species,” J Nat Prod 62, 1147-1150 (Pubmed)


D. B. Stierle, A. A. Stierle, and B. Ganser (1997) “New phomopsolides from a Penicillium sp,” J Nat Prod 60, 1207-1209 (Pubmed)