Wei-Chun Kao

Structural analysis of atovaquone-inhibited cytochrome bc1 complex reveals the molecular basis of antimalarial drug action

Atovaquone, a substituted hydroxynaphthoquinone, is a potent antimalarial drug that acts by inhibiting the parasites mitochondrial cytochrome bc1 complex (cyt bc1). Mutations in cyt bc1 confer atovaquone resistance. Here we describe the X-ray structure of mitochondrial cyt bc1 from Saccharomyces cerevisiae with atovaquone bound in the catalytic Qo site, at 3.0-Å resolution. A polarized H-bond to His181 of the Rieske protein in cyt bc1 traps the ionized hydroxyl group of the drug. Side chains of highly conserved cytochrome b residues establish multiple non-polar interactions with the napththoquinone group, whereas less-conserved residues are in contact with atovaquones cyclohexyl-chlorophenyl tail. Our structural analysis reveals the molecular basis of atovaquones broad target spectrum, species-specific efficacies and acquired resistances, and may aid drug development to control the spread of resistant parasites.

The Molecular Evolution of the Qo Motif

Quinol oxidation in the catalytic quinol oxidation site (Qo site) of cytochrome (cyt) bc1 complexes is the key step of the Q cycle mechanism, which laid the ground for Mitchell’s chemiosmotic theory of energy conversion. Bifurcated electron transfer upon quinol oxidation enables proton uptake and release on opposite membrane sides, thus generating a proton gradient that fuels ATP synthesis in cellular respiration and photosynthesis. The Qo site architecture formed by cyt b and Rieske iron–sulfur protein (ISP) impedes harmful bypass reactions. Catalytic importance is assigned to four residues of cyt b formerly described as PEWY motif in the context of mitochondrial complexes, which we now denominate Qo motif as comprehensive evolutionary sequence analysis of cyt b shows substantial natural variance of the motif with phylogenetically specific patterns. In particular, the Qo motif is identified as PEWY in mitochondria, α- and ε-Proteobacteria, Aquificae, Chlorobi, Cyanobacteria, and chloroplasts. PDWY is present in Gram-positive bacteria, Deinococcus–Thermus and haloarchaea, and PVWY in β- and γ-Proteobacteria. PPWF only exists in Archaea. Distinct patterns for acidophilic organisms indicate environment-specific adaptations. Importantly, the presence of PDWY and PEWY is correlated with the redox potential of Rieske ISP and quinone species. We propose that during evolution from low to high potential electron-transfer systems in the emerging oxygenic atmosphere, cyt bc1 complexes with PEWY as Qo motif prevailed to efficiently use high potential ubiquinone as substrate, whereas cyt b with PDWY operate best with low potential Rieske ISP and menaquinone, with the latter being the likely composition of the ancestral cyt bc1 complex.

The obligate respiratory supercomplex from Actinobacteria

Actinobacteria are closely linked to human life as industrial producers of bioactive molecules and as human pathogens. Respiratory cytochrome bcc complex and cytochrome aa3 oxidase are key components of their aerobic energy metabolism. They form a supercomplex in the actinobacterial species Corynebacterium glutamicum. With comprehensive bioinformatics and phylogenetic analysis we show that genes for cyt bcc-aa3 supercomplex are characteristic for Actinobacteria (Actinobacteria and Acidimicrobiia, except the anaerobic orders Actinomycetales and Bifidobacteriales). An obligatory supercomplex is likely, due to the lack of genes encoding alternative electron transfer partners such as mono-heme cyt c. Instead, subunit QcrC of bcc complex, here classified as short di-heme cyt c, will provide the exclusive electron transfer link between the complexes as in C. glutamicum. Purified to high homogeneity, the C. glutamicum bcc-aa3 supercomplex contained all subunits and cofactors as analyzed by SDS-PAGE, BN-PAGE, absorption and EPR spectroscopy. Highly uniform supercomplex particles in electron microscopy analysis support a distinct structural composition. The supercomplex possesses a dimeric stoichiometry with a ratio of a-type, b-type and c-type hemes close to 1:1:1. Redox titrations revealed a low potential bcc complex (Em(ISP)=+160mV, Em(bL)=-291mV, Em(bH)=-163mV, Em(cc)=+100mV) fined-tuned for oxidation of menaquinol and a mixed potential aa3 oxidase (Em(CuA)=+150mV, Em(a/a3)=+143/+317mV) mediating between low and high redox potential to accomplish dioxygen reduction. The generated molecular model supports a stable assembled supercomplex with defined architecture which permits energetically efficient coupling of menaquinol oxidation and dioxygen reduction in one supramolecular entity.

Rapid Electron Transfer within the III-IV Supercomplex in Corynebacterium glutamicum

Complex III in C. glutamicum has an unusual di-heme cyt. c1 and it co-purifies with complex IV in a supercomplex. Here, we investigated the kinetics of electron transfer within this supercomplex and in the cyt. aa3 alone (cyt. bc1 was removed genetically). In the reaction of the reduced cyt. aa3 with O2, we identified the same sequence of events as with other A-type oxidases. However, even though this reaction is associated with proton uptake, no pH dependence was observed in the kinetics. For the cyt. bc1-cyt. aa3 supercomplex, we observed that electrons from the c-hemes were transferred to CuA with time constants 0.1–1 ms. The b-hemes were oxidized with a time constant of 6.5 ms, indicating that this electron transfer is rate-limiting for the overall quinol oxidation/O2 reduction activity (~210 e−/s). Furthermore, electron transfer from externally added cyt. c to cyt. aa3 was significantly faster upon removal of cyt. bc1 from the supercomplex, suggesting that one of the c-hemes occupies a position near CuA. In conclusion, isolation of the III-IV-supercomplex allowed us to investigate the kinetics of electron transfer from the b-hemes, via the di-heme cyt. c1 and heme a to the heme a3-CuB catalytic site of cyt. aa3.

Structural analysis of mitochondrial cytochrome bc1 complex with atovaquone bound reveals the molecular basis of antimalarial drug action

Results We determined the 3.0-A resolution X-ray structure of mitochondrial cyt bc1 from Saccharomyces cerevisiae with atovaquone bound in the catalytic Qo site [1]. The drug, which has a pKa of 6.9, forms a polarized H-bond between its ionized hydroxyl group and His181 of the Rieske protein subunit. Multiple non-polar interactions with side chains of cytochrome b residues stabilize hydroxynaphthoquinone and chlorophenyl-cyclohexyl groups. The cytochrome b sequence analysis showed that the majority of the interacting residues are conserved, so that atovaquone binding to the yeast cyt bc1 is likely to resemble the binding to the complexes of the target organisms. Conclusion The binding mode provides detailed insights in the molecular basis of broad target spectrum, species-specific efficacies and acquired resistances. This may aid drug development to control the spread of drug-resistant parasites.

Generation of Recombinant Antibody Fragments for Membrane Protein Crystallization

Membrane proteins are challenging targets for crystallization and structure determination by X-ray crystallography. Hurdles can be overcome by antibody-mediated crystallization. More than 25 unique structures of membrane protein:antibody complexes have already been determined. In the majority of cases, hybridoma-derived antibody fragments either in Fab or Fv fragment format were employed for these complexes. We will briefly introduce the background and current status of the strategy and describe in detail the current protocols of well-established methods for the immunization, the selection, and the characterization of antibodies, as well as the cloning, the production, and the purification of recombinant antibodies useful for structural analysis of membrane proteins.

Pneumocystis Cytochrome b Mutants Associated With Atovaquone Prophylaxis Failure as the Cause of Pneumocystis Infection Outbreak Among Heart Transplant Recipients

Background Although trimethoprim-sulfamethoxazole is the more efficient drug for prophylactic and curative treatment of pneumocystosis, atovaquone is considered a second-line prophylactic treatment in immunocompromised patients. Variations in atovaquone absorption and mutant fungi selection after atovaquone exposure have been associated with atovaquone prophylactic failure. We report here a Pneumocystis jirovecii cytochrome b (cyt b) mutation (A144V) associated with such prophylactic failure during a pneumocystosis outbreak among heart transplant recipients. Methods Analyses of clinical data, serum drug dosage, and molecular modeling of the P. jirovecii Rieske-cyt b complex were performed to investigate these prophylactic failures. Results The cyt b A144V mutation was detected in all infected, heart transplant recipient patients exposed to atovaquone prophylaxis but in none of 11 other immunocompromised, infected control patients not treated with atovaquone. Serum atovaquone concentrations associated with these prophylactic failures were similar than those found in noninfected exposed control patients under a similar prophylactic regimen. Computational modeling of the P. jirovecii Rieske-cyt b complex and in silico mutagenesis indicated that the cyt b A144V mutation might alter the volume of the atovaquone-binding pocket, which could decrease atovaquone binding. Conclusions These data suggest that the cyt b A144V mutation confers diminished sensitivity to atovaquone, resulting in spread of Pneumocystis pneumonia among heart transplant recipients submitted to atovaquone prophylaxis. Potential selection and interhuman transmission of resistant P. jirovecii strain during atovaquone prophylactic treatment has to be considered and could limit its extended large-scale use in immucompromised patients.

Unanticipated functional diversity among the TatA-type components of the Tat protein translocase

Twin-arginine translocation (Tat) systems transport folded proteins that harbor a conserved arginine pair in their signal peptides. They assemble from hexahelical TatC-type and single-spanning TatA-type proteins. Many Tat systems comprise two functionally diverse, TatA-type proteins, denominated TatA and TatB. Some bacteria in addition express TatE, which thus far has been characterized as a functional surrogate of TatA. For the Tat system of Escherichia coli we demonstrate here that different from TatA but rather like TatB, TatE contacts a Tat signal peptide independently of the proton-motive force and restricts the premature processing of a Tat signal peptide. Furthermore, TatE embarks at the transmembrane helix five of TatC where it becomes so closely spaced to TatB that both proteins can be covalently linked by a zero-space cross-linker. Our results suggest that in addition to TatB and TatC, TatE is a further component of the Tat substrate receptor complex. Consistent with TatE being an autonomous TatAB-type protein, a bioinformatics analysis revealed a relatively broad distribution of the tatE gene in bacterial phyla and highlighted unique protein sequence features of TatE orthologs.