Synthetic Antibodies Inhibit Bcl-2-associated X Protein (BAX) through Blockade of the N-terminal Activation Site.

The BCL-2 protein family plays a critical role in regulating cellular commitment to mitochondrial apoptosis. Pro-apoptotic Bcl-2-associated X protein (BAX) is an executioner protein of the BCL-2 family that represents the gateway to mitochondrial apoptosis. Following cellular stresses that induce apoptosis, cytosolic BAX is activated and translocates to the mitochondria, where it inserts into the mitochondrial outer membrane to form a toxic pore. How the BAX activation pathway proceeds and how this may be inhibited is not yet completely understood. Here we describe synthetic antibody fragments (Fabs) as structural and biochemical probes to investigate the potential mechanisms of BAX regulation. These synthetic Fabs bind with high affinity to BAX and inhibit its activation by the BH3-only protein tBID (truncated Bcl2 interacting protein) in assays using liposomal membranes. Inhibition of BAX by a representative Fab, 3G11, prevented mitochondrial translocation of BAX and BAX-mediated cytochrome c release. Using NMR and hydrogen-deuterium exchange mass spectrometry, we showed that 3G11 forms a stoichiometric and stable complex without inducing a significant conformational change on monomeric and inactive BAX. We identified that the Fab-binding site on BAX involves residues of helices α1/α6 and the α1-α2 loop. Therefore, the inhibitory binding surface of 3G11 overlaps with the N-terminal activation site of BAX, suggesting a novel mechanism of BAX inhibition through direct binding to the BAX N-terminal activation site. The synthetic Fabs reported here reveal, as probes, novel mechanistic insights into BAX inhibition and provide a blueprint for developing inhibitors of BAX activation.

The role of E3 in pH protection during alphavirus assembly and exit.

Alphaviruses are small enveloped viruses whose surface is covered by spikes composed of trimers of E2/E1 glycoprotein heterodimers. During virus entry, the E2/E1 dimer dissociates within the acidic endosomal environment, freeing the E1 protein to mediate fusion of the viral and endosome membranes. E2 is synthesized as a precursor, p62, which is cleaved by furin in the late secretory pathway to produce mature E2 and a small peripheral glycoprotein, E3. The immature p62/E1 dimer is acid resistant, but since p62 is cleaved before exit from the acidic secretory pathway, low pH-dependent binding of E3 to the spike complex is believed to prevent premature fusion. Based on analysis of the structure of the Chikungunya virus E3/E2/E1 complex, we hypothesized that interactions of E3 residues Y47 and Y48 with E2 are important in this binding. We then directly tested the in vivo role of E3 in pH protection by alanine substitutions of E3 Y47 and Y48 (Y47/48A) in Semliki Forest virus. The mutant was nonviable and was blocked in E1 transport to the plasma membrane and virus production. Although the Y47/48A mutant initially formed the p62/E1 heterodimer, the dimer dissociated during transport through the secretory pathway. Neutralization of the pH in the secretory pathway successfully rescued dimer association, E1 transport, and infectious particle production. Further mutagenesis identified the critical contact as the cation-π interaction of E3 Y47 with E2. Thus, E3 mediates pH protection of E1 during virus biogenesis via interactions strongly dependent on Y47 at the E3-E2 interface.

Analysis of the conformation and function of the Plasmodium falciparum merozoite proteins MTRAP and PTRAMP.

Thrombospondin repeat (TSR)-like domains are structures involved with cell adhesion. Plasmodium falciparum proteins containing TSR domains play crucial roles in parasite development. In particular, the preerythrocytic P. falciparum circumsporozoite protein is involved in hepatocyte invasion. The importance of these domains in two other malaria proteins, the merozoite-specific thrombospondin-related anonymous protein (MTRAP) and the thrombospondin-related apical membrane protein (PTRAMP), were assessed using near-full-length recombinant proteins composed of the extracellular domains produced in Escherichia coli. MTRAP is thought to be released from invasive organelles identified as micronemes during merozoite invasion to mediate motility and host cell invasion through an interaction with aldolase, an actin binding protein involved in the moving junction. PTRAMP function remains unknown. In this study, the conformation of recombinant MTRAP (rMTRAP) appeared to be a highly extended protein (2 nm by 33 nm, width by length, respectively), whereas rPTRAMP had a less extended structure. Using an erythrocyte binding assay, rMTRAP but not rPTRAMP bound human erythrocytes; rMTRAP binding was mediated through the TSR domain. MTRAP- and in general PTRAMP-specific antibodies failed to inhibit P. falciparum development in vitro. Altogether, MTRAP is a highly extended bifunctional protein that binds to an erythrocyte receptor and the merozoite motor.

Structure of the Plasmodium falciparum circumsporozoite protein, a leading malaria vaccine candidate.

The Plasmodium falciparum circumsporozoite protein (CSP) is critical for sporozoite function and invasion of hepatocytes. Given its critical nature, a phase III human CSP malaria vaccine trial is ongoing. The CSP is composed of three regions as follows: an N terminus that binds heparin sulfate proteoglycans, a four amino acid repeat region (NANP), and a C terminus that contains a thrombospondin-like type I repeat (TSR) domain. Despite the importance of CSP, little is known about its structure. Therefore, recombinant forms of CSP were produced by expression in both Escherichia coli (Ec) and then refolded (EcCSP) or in the methylotrophic yeast Pichia pastoris (PpCSP) for structural analyses. To analyze the TSR domain of recombinant CSP, conformation-dependent monoclonal antibodies that recognized unfixed P. falciparum sporozoites and inhibited sporozoite invasion of HepG2 cells in vitro were identified. These monoclonal antibodies recognized all recombinant CSPs, indicating the recombinant CSPs contain a properly folded TSR domain structure. Characterization of both EcCSP and PpCSP by dynamic light scattering and velocity sedimentation demonstrated that both forms of CSP appeared as highly extended proteins (R(h) 4.2 and 4.58 nm, respectively). Furthermore, high resolution atomic force microscopy revealed flexible, rod-like structures with a ribbon-like appearance. Using this information, we modeled the NANP repeat and TSR domain of CSP. Consistent with the biochemical and biophysical results, the repeat region formed a rod-like structure about 21-25 nm in length and 1.5 nm in width. Thus native CSP appears as a glycosylphosphatidylinositol-anchored, flexible rod-like protein on the sporozoite surface.