VRC01 gp120 complex

The crystal structure of VRC01 and VRC01-like antibodies are studied to define with characteristics are important in neutralizing HIV-1. (cite!)

Introduction

HIV-1 has a high level of antigenic and genetic diversity. HIV-1 has also evolved mechanisms to evade the humoral immune response. These aspects of HIV-1 have made it difficult to develop a vaccine. After several years of infection, 10 to 25% of HIV-1 infected individuals develop neutralizing antibodies. Some antibodies target the transmembrane gp41 molecules of the HIV-1 viral spike, however most target the surface protein gp120. (Wu) VRC01 and VRC01-like antibodies bind to gp120 and are able to neutralize about 90% of HIV-1 isolates. Structural analysis has shown which characteristics of antibodies are essential to its binding with gp120. (Kwong) Discovery of the structure of these antibodies can help develop an effective HIV-1 vaccine.

HIV-1 Neutralization

HIV-1 enters its host by binding viral gp120, a surface glycoprotein of HIV, to the host cell’s CD4 receptor. This interaction induces conformational changes in gp120. (Wu) This conformational change results in the exposure of a binding site for the co-receptor, usually CCR5 OR CXCR4. (Li) The conformational changes also result in the formation of a pre-hairpin intermediate conformation in which gp41, a transmembrane glycoprotein of HIV, rearranges its molecules so that its N-terminal peptides form a trimer of helices that present a fusion peptide to the target cell. Once fusion occurs between the fusion peptide and the target cell membrane, HIV is able to enter and infect the target cell. (Tran) VRC01 binds to CD4’s binding site on gp120, preventing the CD4 receptor from binding to HIV and infecting the cell. (Wu).

spinqualitylabelsall models VRC01 in complex with viral gp120, 3NGB Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image   Print3D Structural Features DNA-binding. ToxT belongs to a family of transcriptional regulators headed by and known as AraC. The AraC family is characterized by a 100 amino acid region of sequence similarity that forms a DNA-binding domain with two helix-turn-helix motifs (one on either side of the black linker). This DNA binding domain is composed of seven alpha helices. HTH1 is composed of alpha helices five and six, while HTH2 is composed of alpha helices eight and nine. The two HTH regions are linked by the very polar alpha helix seven(shown in black). The overall domain is located at the C-terminus. Assuming ToxT is similar in mechanism to other AraC proteins, helix six from HTH1 and helix nine from HTH2 become aligned with the help of helix seven. Helix seven is positioned to attach to the N terminal binding pocket(the polar linking region) to allow binding to major consecutive grooves of target DNA (specific promoters for virulence genes).[1]. The conformation of helix seven is dependent on the ligand bound. Ligand-binding. A nine-stranded beta sheet sandwich or "jelly-roll" with three other alpha helices (overall making up the N-terminus) contain a binding pocket. This is made from several residues from the N-terminus (Y12, Y20, F22, L25, I27, K31, F33, L61, F69, L71, V81, and V83), and a few from the C-terminus (I226, K230, M259, V261, Y266, and M269). This pocket contains a sixteen-carbon fatty acid positioned in a conformation such that its negatively charged carboxylate group forms salt bridges between K31 of the N-terminal domain, and K230 from the C-terminal domain. The pocket is highly hydrophobic, and has a known volume of 780.9 Angstroms. This pocket contains a ligand: cis-palmitoleate which appears to have a negative effect on virulence when present in vitro. The cis-palmitoleate forms salt bridges with residues K31 and K230 (for detail, see Figure 1B of: [2]). This unsaturated fatty acid, like other UFAs,[3] tend to inhibit genes under the control of ToxT. Specifically, the cis-palmitoleate (PAM) appears to change ToxT's conformation, and thus lower its ability to bind DNA and form dimers. The presence of UFAs is associated with being in the lumen of the intestine during the bacterial infection. PAM brings K31 and K230 together from either end of the protein, and essentially closes off ToxT. K230 is at the end of helix seven, and binding to K31 causes helix six to be pulled into an unfavorable conformation that deters DNA binding. In lower concentration of fatty acids, ie: after penetrating the intestine's mucus, PAM is in lower concentration. At this point, charge-charge repulsion between K31 and K230 leads to a destabilization of the closed conformation of ToxT. This repulsion prompts the opening of the N and C terminal domains. The freedom of helices six and seven to find a favorable configuration allows DNA binding to occur. MSA 1xtc Dimerization. Though the structure shown is a monomer with two overall domains (N-terminal and C-terminal), ToxT tends to form a dimer. The preferred state of ToxT varies between promoters, but binding to the ctx promoter to generate cholera toxin appears to be possible only in the dimer form.ToxT binds to thirteen base pair sequences (can be single, direct, or inverted repeats) called toxboxes in order to activate their respective promoters.[4] Ligand In this resolved structure, cis-palmitoleate[5] is shown, which can be bound in the beta sheet barrel (as discussed above). This unsaturated fatty acid reduces virulence expression in Vibrio cholerae.

Further Study

Conclusive results about what activates ToxT itself has not yet been found. The varying activity of ToxT dependent on the presence of cis-palmitoleate or other unsaturated fatty acids represents a detailed method of effective pathogenicity in humans, but may not be a reasonable target for drug treatment. By restricting transcription (and thus translation and protein production) of virulence genes until the bacterium is determined to be in a favorable location for infection, Vibrio cholerae avoids wasting energy producing virulence factors that will just be cleared by the intestine. This is a specific mechanism to ensure that the bacterium also injects CT and TCP where they will do the most damage, perpetuating the infection. Despite the lack of information about what activates ToxT itself, it is understood that the transcription of ctxA and tcpA by vibrio cholerae is sharply reduced in the presence of oleic, linoleic acid, and arachidonic acid, all of which are components of bile. Therefore, one may hypothesize that it may be possible to use the structure of a UFA or SFA to design a small molecule inhibitor of ToxT which may be used to treat or prevent cholera.

Evolution

Vibrio cholerae is a highly diverse species in which some strains are completely harmless, whereas other strains have the capacity to cause global cholera pandemics. It has been discovered that in each epidemic and pandemic strain, there is a chromosomal pathogenicity island (PAI) that is not present in the nonpathogenic strains. The region containing two ToxR-regulated genes (aldA and tagA) is composed of 13kb of previously unidentified DNA. This region is part of a PAI that contains ToxT and a gene cluster a critical colonization factor and TCP. The PAI is 39.5 kb long, contains putative integrase and transposase genes, and inserts near a 10Sa RNA gene. One may infer that the PAI could have originated from a bacteriophage. This PAI was also found in two non-O1/non-O139 (which are both pandemic) sero type strains. Therefore, one may hypothesize that the PAI could be transferred within other strains of Vibrio cholerae.

Centroid RNA 1xtc

RNA Structure

Here we have the centroid structure of the mRNA of ToxT. This mRNA is shown in its most stable conformation, with the less stable, higher energy regions in red. The lighter colored regions are more stable and lower in energy.

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