Quaternary structures of HIV Env immunogen exhibit conformational vicissitudes and interface diminution elicited by ligand binding.

The human immunodeficiency virus envelope protein is the key element mediating entry into host cells. Conformational rearrangement of Env upon binding to the host CD4 receptor and chemokine coreceptor drives membrane fusion. We elucidated the quaternary arrangement of the soluble Env trimeric immunogen o-gp140ΔV2TV1, in both its native (unliganded) and CD4-induced (liganded) states by cryoelectron microscopy and molecular modeling. The liganded conformation was elicited by binding gp140 to the synthetic CD4-mimicking miniprotein CD4m. Upon CD4m binding, an outward domain shift of the three gp120 subunits diminishes gp120-gp41 interactions, whereas a "flat open" concave trimer apex is observed consequent to gp120 tilting away from threefold axis, likely juxtaposing the fusion peptide with the host membrane. Additional features observed in the liganded conformation include rotations of individual gp120 subunits that may release gp41 for N- and C-helix refolding and also may lead to optimal exposure of the elicited coreceptor binding site. Such quaternary arrangements of gp140 lead to the metastable liganded conformation, with putative locations of exposed epitopes contributing to a description of sequential events occurring prior to membrane fusion. Our observations imply a mechanism whereby a soluble Env trimeric construct, as opposed to trimers extracted from virions, may better expose crucial epitopes such as the CD4 binding site and V3, as well as epitopes in the vicinity of gp41, subsequent to conjugation with CD4m. Structural features gleaned from our studies should aid the design of Env-based immunogens for inducement of potent broadly neutralizing antibodies against exposed conformational epitopes.

Self-assembly of heptameric nanoparticles derived from tag-functionalized phi29 connectors.

The structure of an induced macromolecular assembly was characterized and found to consist of an ordered heptameric arrangement of recombinant phi29 gp10 connector molecules. Insertion of an N-terminal Strep-II/His(6) tag to the connectors led to the spontaneous formation of large nanoparticles that were distinct from free, wild-type phi29 connectors in both size and symmetry elements. The determination of single-molecule tomograms and image-averaged reconstructions allowed for the stoichiometric and topological characterization of the ordered assemblage, revealing that the nanoparticle is composed of five equatorial connectors arranged with pseudo-5-fold rotational symmetry, capped on its ends by two polar connectors. Additionally, all seven connectors are oriented with their narrower N-terminal necks into the nanoparticle core and wider C-terminal ends out toward the nanoparticle surface, a geometric arrangement accommodated by the shape complementarity of the conical connector profiles. A significant amount of conformational heterogeneity was detected, ranging from changes in overall nanoparticle diameter, to tilting of individual connectors, to variations in connector stoichiometry. Nevertheless, a stable, heptameric nanoparticle was resolved, revealing the significant potential of guided, peptide-mediated supramolecular self-assembly. With this construct, we anticipate the further design of variable N-terminal tags to allow for the generation of nanoparticles with tailored connector stoichiometry and topological arrangements. By modifying the surface-exposed C-terminal ends with application-appropriate moieties, the consistent structure and compact nature of these nanoparticles may prove beneficial in nanotechnological and nanomedical approaches.

Adjustable ellipsoid nanoparticles assembled from re-engineered connectors of the bacteriophage phi29 DNA packaging motor.

A 24 x 30 nm ellipsoid nanoparticle containing 84 subunits or 7 dodecamers of the re-engineered core protein of the bacteriophage phi29 DNA packaging motor was constructed. Homogeneous nanoparticles were obtained with simple one-step purification. Electron microscopy and analytical ultracentrifugation were employed to elucidate the structure, shape, size, and mechanism of assembly. The formation of this structure was mediated and stabilized by N-terminal peptide extensions. Reversal of the 84-subunit ellipsoid nanoparticle to its dodecamer subunit was controlled by the cleavage of the extended N-terminal peptide with a protease. The 84 outward-oriented C-termini were conjugated with a streptavidin binding peptide which can be used for the incorporation of markers. This further extends the application of this nanoparticle to pathogen detection and disease diagnosis by signal enhancement.