Through different genetic barriers, epidemics broke out in different regions of the world: especially subtype C in Africa and subtypes A and B in Europe and America (Faria et al., 2014). Subtype B is thought to have spread to Haiti from Africa in the 1960s, then spread to the United States via a single migration event at the end of the decade (Worobey et al., 2008). The first structure of HIV-1 gp120, from strain HxBc2, was in complex with an antigen-binding fragment (Fab) of the CD4-induced antibody 17b as well as two domains of the CD4 receptor (Kwong et al., 1998).
Two of the 2G12 dimers (dimer B and C) in the P6122 crystals were conformationally identical, while the third dimer (dimer A) was distinct ( Figure 4A,B ; Figure S4A,B ). The difference in relative positions of the (Fab)2 arms is consistent with flexibility between the two (Fab)2 portions of the 2G12 dimer structure, in contrast to the inflexibility of the single rigid (Fab)2 of 2G12 monomer. The span between the combining sites of the two (Fab)2 arms of 2G12 dimers, ranging from 105 to 170 Å, was similar to the combining sites of the two Fab arms of a conventional IgG, which are separated by ~150 Å (Klein) and Bjorkman, 2010).
However, the orientation of the Fc regions in the three 2G12 dimers supported the connectivity model. It was initially difficult to determine the connectivity between the (Fab)2 and Fc regions of the 2G12 dimer in the 2G12/2G12.1 structure because the Fc regions are arranged almost parallel to the (Fab)2 units was (Figure 4C). In many of the classes for 2G12 dimer and higher-order oligomers, the 2G12 (Fab)2 was detectable and the Fc less evident.
This effector function is mediated by CD16, an Fc receptor that interacts with the hinge-proximal region of the Fc CH2 domain ( Sondermann et al., 2000 ). The IgG dimer was consistent with the orientation of the Fc domains in the 2G12 and 2G12/2G12.1 structures. Equilibrium gel filtration chromatography ( Hummel and Dreyer, 1962 ) was performed to determine the binding stoichiometry of 2G12 monomer and 2G12 dimer to FcRn.
The symmetry of two of the 2G12 dimer conformations, dimer A and the dimer in the 2G12/2G12.1 peptide structure (panels A and C), was point group C2 (the two-fold axis of dimer symmetry coincided with the crystallographic twofold axis). Middle: Top-down view of a 2G12 dimer molecule with Fc regions pointing to the side. The P(r) for rituximab showed a smaller second peak, indicating the presence of more elongated domains in solution (Figure S5D) (Putnam et al., 2007).
The putative tantalum bromide sites corresponded to locations on the surface of the 2G12 dimer model. One of the most distinct structural differences between HIV-2 and HIV-1 gp120 is the extended E loop of HIV-2 gp120. Fewer potential N-linked glycans, and thus reduced glycan shielding, of the V3 and V4 loops in HIV-2 may lead to higher immunogenicity in those regions (Shi et al., 2005).
A study (Decker et al., 2005) exploring co-receptor site conservation suggested that many CD4-induced antibodies (CD4i) against HIV-1 gp120, including 21c, were able to neutralize or bind to HIV-2 gp120.
Unpublished work in progress)
Because of the labile nature of the gp120-gp41 non-covalent interaction, recombinant production of fully post-translationally processed trimers has been problematic for many years (Sanders et al., 2002). To stabilize the trimerization interface, an isoleucine-proline (IP) mutation at position 559 was introduced (Binley et al., 2000; Sanders et al., 2002) to destabilize the post-fusion configuration and thereby increase the stability of the pre-fusion configuration, which is biologically most stable. interesting since most existing structures have a post-fusion form of Env. Both structures agreed on the overall architecture of the soluble trimer and revealed key interactions between gp120 and gp41 and between gp120 subunits.
The BG505 SOSIP.664 trimer represents a new, native-like standard for studying the function of the HIV envelope spike during host infection as well as providing crucial insights into immunogenic and prophylactic design. The original BG505 SOSIP.664 constructs contained a mutation to RRRRRR (6R) instead of the original Furin cleavage site. An anti-His6 Western blot of the SOSVP 6R fractions showed equivocal results (Figure 3.4, lower panel).
Fraction A6 or A7, which corresponded to the trimeric protein for UC1 or 7312A SOSVP 6R respectively, did not appear to show cleavage due to the absence of the gp41ECTO band under reducing conditions (lanes 2 and 6). We characterized the molar mass of the entire glycoprotein using the RI signal (carbohydrate and protein contributions). Analysis of HIV-2 SOSVP Trimers by Negative Staining Electron Microscopy Negative stain EM has been used as an assay to study the overall behavior and.
This may be caused by intrinsic instability of the uncleaved trimers or unwanted responses of the proteins to staining. The rationale for the SOS mutation is to covalently link the cleaved gp120 and gp41ECTO, and the IP mutation is to destabilize the gp41-gp41 postfusion interface to promote the stability of the prefusion configuration of Env trimers. Most of the proteins had a taller trimer peak (centered around fractions A6 or A7), with a shorter multimer or aggregate peak that was slightly larger.
We purified protein for all versions of the 7312A strain except for VP WT and SOS 6R. Negative stain grids were prepared by applying ~10 µg/ml of the purified trimer to a freshly glow-discharged ultrathin carbon film-coated 400 Cu mesh grid (Ted Pella) and stained with 2% uranyl acetate. Crystal structure of the gp41 core of simian immunodeficiency virus (SIV): conserved helical interactions underlie the broad inhibitory activity of gp41 peptides.
