Structure of complex III with bound antimalarial agent CK-2-68 provides insights into selective inhibition of Plasmodium cytochrome bc1 complexes.

Among the various components of the protozoan Plasmodium mitochondrial respiratory chain, only Complex III is a validated cellular target for antimalarial drugs. The compound CK-2-68 was developed to specifically target the alternate NADH dehydrogenase of the malaria parasite respiratory chain, but the true target for its antimalarial activity has been controversial. Here, we report the cryo-EM structure of mammalian mitochondrial Complex III bound with CK-2-68 and examine the structure-function relationships of the inhibitor's selective action on Plasmodium. We show that CK-2-68 binds specifically to the quinol oxidation site of Complex III, arresting the motion of the iron-sulfur protein subunit, which suggests an inhibition mechanism similar to that of Pf-type Complex III inhibitors such as atovaquone, stigmatellin, and UHDBT. Our results shed light on the mechanisms of observed resistance conferred by mutations, elucidate the molecular basis of the wide therapeutic window of CK-2-68 for selective action of Plasmodium vs. host cytochrome bc1, and provide guidance for future development of antimalarials targeting Complex III.

Crystal structure of bacterial cytochrome bc1 in complex with azoxystrobin reveals a conformational switch of the Rieske iron-sulfur protein subunit.

Cytochrome bc1 complexes (cyt bc1), also known as complex III in mitochondria, are components of the cellular respiratory chain and of the photosynthetic apparatus of non-oxygenic photosynthetic bacteria. They catalyze electron transfer (ET) from ubiquinol to cytochrome c and concomitantly translocate protons across the membrane, contributing to the cross-membrane potential essential for a myriad of cellular activities. This ET-coupled proton translocation reaction requires a gating mechanism that ensures bifurcated electron flow. Here, we report the observation of the Rieske iron-sulfur protein (ISP) in a mobile state, as revealed by the crystal structure of cyt bc1 from the photosynthetic bacterium Rhodobacter sphaeroides in complex with the fungicide azoxystrobin. Unlike cyt bc1 inhibitors stigmatellin and famoxadone that immobilize the ISP, azoxystrobin causes the ISP-ED to separate from the cyt b subunit and to remain in a mobile state. Analysis of anomalous scattering signals from the iron-sulfur cluster of the ISP suggests the existence of a trajectory for electron delivery. This work supports and solidifies the hypothesis that the bimodal conformation switch of the ISP provides a gating mechanism for bifurcated ET, which is essential to the Q-cycle mechanism of cyt bc1 function.

Structures of the Multidrug Transporter P-glycoprotein Reveal Asymmetric ATP Binding and the Mechanism of Polyspecificity.

P-glycoprotein (P-gp) is a polyspecific ATP-dependent transporter linked to multidrug resistance in cancer; it plays important roles in determining the pharmacokinetics of many drugs. Understanding the structural basis of P-gp, substrate polyspecificity has been hampered by its intrinsic flexibility, which is facilitated by a 75-residue linker that connects the two halves of P-gp. Here we constructed a mutant murine P-gp with a shortened linker to facilitate structural determination. Despite dramatic reduction in rhodamine 123 and calcein-AM transport, the linker-shortened mutant P-gp possesses basal ATPase activity and binds ATP only in its N-terminal nucleotide-binding domain. Nine independently determined structures of wild type, the linker mutant, and a methylated P-gp at up to 3.3 Å resolution display significant movements of individual transmembrane domain helices, which correlated with the opening and closing motion of the two halves of P-gp. The open-and-close motion alters the surface topology of P-gp within the drug-binding pocket, providing a mechanistic explanation for the polyspecificity of P-gp in substrate interactions.

Hydrogen Bonding to the Substrate Is Not Required for Rieske Iron-Sulfur Protein Docking to the Quinol Oxidation Site of Complex III.

Complex III or the cytochrome (cyt) bc1 complex constitutes an integral part of the respiratory chain of most aerobic organisms and of the photosynthetic apparatus of anoxygenic purple bacteria. The function of cyt bc1 is to couple the reaction of electron transfer from ubiquinol to cytochrome c to proton pumping across the membrane. Mechanistically, the electron transfer reaction requires docking of its Rieske iron-sulfur protein (ISP) subunit to the quinol oxidation site (QP) of the complex. Formation of an H-bond between the ISP and the bound substrate was proposed to mediate the docking. Here we show that the binding of oxazolidinedione-type inhibitors famoxadone, jg144, and fenamidone induces docking of the ISP to the QP site in the absence of the H-bond formation both in mitochondrial and bacterial cyt bc1 complexes, demonstrating that ISP docking is independent of the proposed direct ISP-inhibitor interaction. The binding of oxazolidinedione-type inhibitors to cyt bc1 of different species reveals a toxophore that appears to interact optimally with residues in the QP site. The effect of modifications or additions to the toxophore on the binding to cyt bc1 from different species could not be predicted from structure-based sequence alignments, as demonstrated by the altered binding mode of famoxadone to bacterial cyt bc1.

Cryo-EM Analysis of the Conformational Landscape of Human P-glycoprotein (ABCB1) During its Catalytic Cycle.

The multidrug transporter P-glycoprotein (P-gp, ABCB1) is an ATP-dependent pump that mediates the efflux of structurally diverse drugs and xenobiotics across cell membranes, affecting drug pharmacokinetics and contributing to the development of multidrug resistance. Structural information about the conformational changes in human P-gp during the ATP hydrolysis cycle has not been directly demonstrated, although mechanistic information has been inferred from biochemical and biophysical studies conducted with P-gp and its orthologs, or from structures of other ATP-binding cassette transporters. Using single-particle cryo-electron microscopy, we report the surprising discovery that, in the absence of the transport substrate and nucleotides, human P-gp can exist in both open [nucleotide binding domains (NBDs) apart; inward-facing] and closed (NBDs close; outward-facing) conformations. We also probe conformational states of human P-gp during the catalytic cycle, and demonstrate that, following ATP hydrolysis, P-gp transitions through a complete closed conformation to a complete open conformation in the presence of ADP.

Structural basis for the specific recognition of dual receptors by the homopolymeric pH 6 antigen (Psa) fimbriae of Yersinia pestis.

The pH 6 antigen (Psa) of Yersinia pestis consists of fimbriae that bind to two receptors: ß1-linked galactosyl residues in glycosphingolipids and the phosphocholine group in phospholipids. Despite the ubiquitous presence of either moiety on the surface of many mammalian cells, Y. pestis appears to prefer interacting with certain types of human cells, such as macrophages and alveolar epithelial cells of the lung. The molecular mechanism of this apparent selectivity is not clear. Site-directed mutagenesis of the consensus choline-binding motif in the sequence of PsaA, the subunit of the Psa fimbrial homopolymer, identified residues that abolish galactosylceramide binding, phosphatidylcholine binding, or both. The crystal structure of PsaA in complex with both galactose and phosphocholine reveals separate receptor binding sites that share a common structural motif, thus suggesting a potential interaction between the two sites. Mutagenesis of this shared structural motif identified Tyr126, which is part of the choline-binding consensus sequence but is found in direct contact with the galactose in the structure of PsaA, important for both receptor binding. Thus, this structure depicts a fimbrial subunit that forms a polymeric adhesin with a unique arrangement of dual receptor binding sites. These findings move the field forward by providing insights into unique types of multiple receptor-ligand interactions and should steer research into the synthesis of dual receptor inhibitor molecules to slow down the rapid progression of plague.

Tight conformational coupling between the domains of the enterotoxigenic Escherichia coli fimbrial adhesin CfaE regulates binding state transition.

CfaE, the tip adhesin of enterotoxigenic Escherichia coli colonization factor antigen I fimbriae, initiates binding of this enteropathogen to the small intestine. It comprises stacked ß-sandwich adhesin (AD) and pilin (PD) domains, with the putative receptor-binding pocket at one pole and an equatorial interdomain interface. CfaE binding to erythrocytes is enhanced by application of moderate shear stress. A G168D replacement along the AD facing the CfaE interdomain region was previously shown to decrease the dependence on shear by increasing binding at lower shear forces. To elucidate the structural basis for this functional change, we studied the properties of CfaE G168D (with a self-complemented donor strand) and solved its crystal structure at 2.6 Å resolution. Compared with native CfaE, CfaE G168D showed a downward shift in peak erythrocyte binding under shear stress and greater binding under static conditions. The thermal melting transition of CfaE G168D occurred 10 °C below that of CfaE. Compared with CfaE, the atomic structure of CfaE G168D revealed a 36% reduction in the buried surface area at the interdomain interface. Despite the location of this single modification in the AD, CfaE G168D exhibited structural derangements only in the adjoining PD compared with CfaE. In molecular dynamics simulations, the G168D mutation was associated with weakened interdomain interactions under tensile force. Taken together, these findings indicate that the AD and PD of CfaE are conformationally tightly coupled and support the hypothesis that opening of the interface plays a critical modulatory role in the allosteric activation of CfaE.

Structural analysis of cytochrome bc1 complexes: implications to the mechanism of function.

The cytochrome bc1 complex (bc1) is the mid-segment of the cellular respiratory chain of mitochondria and many aerobic prokaryotic organisms; it is also part of the photosynthetic apparatus of non-oxygenic purple bacteria. The bc1 complex catalyzes the reaction of transferring electrons from the low potential substrate ubiquinol to high potential cytochrome c. Concomitantly, bc1 translocates protons across the membrane, contributing to the proton-motive force essential for a variety of cellular activities such as ATP synthesis. Structural investigations of bc1 have been exceedingly successful, yielding atomic resolution structures of bc1 from various organisms and trapped in different reaction intermediates. These structures have confirmed and unified results of decades of experiments and have contributed to our understanding of the mechanism of bc1 functions as well as its inactivation by respiratory inhibitors. This article is part of a Special Issue entitled: Respiratory complex III and related bc complexes.

A novel ATP-dependent conformation in p97 N-D1 fragment revealed by crystal structures of disease-related mutants.

Mutations in p97, a major cytosolic AAA (ATPases associated with a variety of cellular activities) chaperone, cause inclusion body myopathy associated with Paget's disease of the bone and frontotemporal dementia (IBMPFD). IBMPFD mutants have single amino-acid substitutions at the interface between the N-terminal domain (N-domain) and the adjacent AAA domain (D1), resulting in a reduced affinity for ADP. The structures of p97 N-D1 fragments bearing IBMPFD mutations adopt an atypical N-domain conformation in the presence of Mg(2+).ATPgammaS, which is reversible by ADP, showing for the first time the nucleotide-dependent conformational change of the N-domain. The transition from the ADP- to the ATPgammaS-bound state is accompanied by a loop-to-helix conversion in the N-D1 linker and by an apparent re-ordering in the N-terminal region of p97. X-ray scattering experiments suggest that wild-type p97 subunits undergo a similar nucleotide-dependent N-domain conformational change. We propose that IBMPFD mutations alter the timing of the transition between nucleotide states by destabilizing the ADP-bound form and consequently interfere with the interactions between the N-domains and their substrates.

Mitochondrial Cytochrome bc1 Complex as Validated Drug Target: A Structural Perspective.

Mitochondrial respiratory chain Complex III, also known as cytochrome bc1 complex or cyt bc1, is a validated target not only for antibiotics but also for pesticides and anti-parasitic drugs. Although significant progress has been made in understanding the mechanisms of cyt bc1 function and inhibition by using various natural and synthetic compounds, important issues remain in overcoming drug resistance in agriculture and in evading cytotoxicity in medicine. In this review, we look at these issues from a structural perspective. After a brief description of the essential and common structural features, we point out the differences among various cyt bc1 complexes of different organisms, whose structures have been determined to atomic resolution. We use a few examples of cyt bc1 structures determined via bound inhibitors to illustrate both conformational changes observed and implications to the Q-cycle mechanism of cyt bc1 function. These structures not only offer views of atomic interactions between cyt bc1 complexes and inhibitors, but they also provide explanations for drug resistance when structural details are coupled to sequence changes. Examples are provided for exploiting structural differences in evolutionarily conserved enzymes to develop antifungal drugs for selectivity enhancement, which offer a unique perspective on differential interactions that can be exploited to overcome cytotoxicity in treating human infections.

Point mutations in Arf1 reveal cooperative effects of the N-terminal extension and myristate for GTPase-activating protein catalytic activity.

The ADP-ribosylation factors (Arfs) constitute a family of small GTPases within the Ras superfamily, with a distinguishing structural feature of a hypervariable N-terminal extension of the G domain modified with myristate. Arf proteins, including Arf1, have roles in membrane trafficking and cytoskeletal dynamics. While screening for Arf1:small molecule co-crystals, we serendipitously solved the crystal structure of the non-myristoylated engineered mutation [L8K]Arf1 in complex with a GDP analogue. Like wild-type (WT) non-myristoylated Arf1•GDP, we observed that [L8K]Arf1 exhibited an N-terminal helix that occludes the hydrophobic cavity that is occupied by the myristoyl group in the GDP-bound state of the native protein. However, the helices were offset from one another due to the L8K mutation, with a significant change in position of the hinge region connecting the N-terminus to the G domain. Hypothesizing that the observed effects on behavior of the N-terminus affects interaction with regulatory proteins, we mutated two hydrophobic residues to examine the role of the N-terminal extension for interaction with guanine nucleotide exchange factors (GEFs) and GTPase Activating Proteins (GAPs. Different than previous studies, all mutations were examined in the context of myristoylated Arf. Mutations had little or no effect on spontaneous or GEF-catalyzed guanine nucleotide exchange but did affect interaction with GAPs. [F13A]myrArf1 was less than 1/2500, 1/1500, and 1/200 efficient as substrate for the GAPs ASAP1, ARAP1 and AGAP1; however, [L8A/F13A]myrArf1 was similar to WT myrArf1. Using molecular dynamics simulations, the effect of the mutations on forming alpha helices adjacent to a membrane surface was examined, yet no differences were detected. The results indicate that lipid modifications of GTPases and consequent anchoring to a membrane influences protein function beyond simple membrane localization. Hypothetical mechanisms are discussed.

Recognition of mesothelin by the therapeutic antibody MORAb-009: structural and mechanistic insights.

Mesothelin is a tumor differentiation antigen that is highly expressed in many epithelial cancers, with limited expression in normal human tissues. Binding of mesothelin on normal mesothelial cells lining the pleura or peritoneum to the tumor-associated cancer antigen 125 (CA-125) can lead to heterotypic cell adhesion and tumor metastasis within the pleural and peritoneal cavities. This binding can be prevented by MORAb-009, a humanized monoclonal antibody against mesothelin currently under clinical trials. We show here that MORAb-009 recognizes a non-linear epitope that is contained in the first 64-residue fragment of the mesothelin. We further demonstrate that the recognition is independent of glycosylation state of the protein but sensitive to the loss of a disulfide bond linking residues Cys-7 and Cys-31. The crystal structure of the complex between the mesothelin N-terminal fragment and Fab of MORAb-009 at 2.6 Å resolution reveals an epitope encompassing multiple secondary structural elements of the mesothelin, including residues from helix α1, the loops linking helices α1 and α2, and between helices α4 and α5. The mesothelin fragment has a compact, right-handed superhelix structure consisting of five short helices and connecting loops. A residue essential for complex formation has been identified as Phe-22, which projects its side chain into a hydrophobic niche formed on the antibody recognition surface upon antigen-antibody contact. The overlapping binding footprints of both the monoclonal antibody and the cancer antigen CA-125 explains the therapeutic effect and provides a basis for further antibody improvement.

Generation, characterization and crystallization of a cytochrome c(1)-subunit IV fused cytochrome bc(1) complex from Rhodobacter sphaeroides.

Cytochrome bc(1) complex catalyzes the reaction of electron transfer from ubiquinol to cytochrome c (or cytochrome c(2)) and couples this reaction to proton translocation across the membrane. Crystallization of the Rhodobacter sphaeroides bc(1) complex resulted in crystals containing only three core subunits. To mitigate the problem of subunit IV being dissociated from the three-subunit core complex during crystallization, we recently engineered an R. sphaeroides mutant in which the N-terminus of subunit IV was fused to the C-terminus of cytochrome c(1) with a 14-glycine linker between the two fusing subunits, and a 6-histidine tag at the C-terminus of subunit IV (c(1)-14Gly-IV-6His). The purified fusion mutant complex shows higher electron transfer activity, more structural stability, and less superoxide generation as compared to the wild-type enzyme. Preliminary crystallization attempts with this mutant complex yielded crystals containing four subunits and diffracting X-rays to 5.5Å resolution.

Synthesis and Antiphytopathogenic Activity of Novel Oxazolidine-2,4-diones Bearing Phenoxypyridine Moiety.

In the present study, we conducted optimization of pyramoxadone and synthesized a series of novel oxazolidinediones. Antifungal assays showed that these compounds exhibited moderate to excellent antifungal activity against various pathogens. Further SAR analysis revealed that the introduction of substituents to the benzene ring of the phenoxy group or the inclusion of bulky groups, such as tert-butyl, on the aniline moiety, had a detrimental effect on the activity. However, the inclusion of fluorine atoms in the aniline moiety significantly enhanced the antifungal efficacy. Notably, compound 2-4 displayed significantly higher activity compared to both pyramoxadone and famoxadone against R. solani, B. cinerea, S. sclerotiorum, and P. oryzae, where it demonstrated EC50 values of 1.78, 2.47, 2.33, and 2.23 µg/mL, respectively. Furthermore, compound 2-4 exhibited potent protective and curative effects against the tomato gray mold in vivo. A mechanistic investigation revealed that compound 2-4 significantly impacted the mycelial morphology, inhibited spore germination, and impeded mycelial respiration, ultimately leading to the inhibition of pathogenic fungus growth. These findings indicate that compound 2-4 has the potential to serve as a cyt bc1 inhibitor and should be further investigated for development.

Structures of Cancer Antigen Mesothelin and Its Complexes with Therapeutic Antibodies.

The tumor-associated antigen mesothelin is expressed at high levels on the cell surface of many human cancers, while its expression in normal tissues is limited. The binding of mesothelin to the tumor-associated cancer antigen 125 (CA-125) can lead to heterotypic cell adhesion and tumor metastasis within the pleural and peritoneal cavities. Immunotherapeutic strategies targeting mesothelin are being intensively investigated. Here, we report the crystal structures of mesothelin that reveal a compact, right-handed solenoid consisting of 24 short helices and connecting loops. These helices form a nine-layered spiral coil that resembles ARM/HEAT family proteins. Glycan attachments have been identified in the structure for all three predicted N-glycosylation sites and confirmed with samples from cell culture and patient ascites. The structures of full-length mesothelin and its complex with the Fab of MORAb-009 reveal the interaction of the antibody with the complete epitope, which has not been reported previously. The N-terminal half of mesothelin is conformationally rigid, suitable for eliciting specific antibodies, whereas its C-terminal portion is more flexible. The structure of the C-terminal shedding-resistant fragment of mesothelin complexed with a mAb 15B6 displays an extended linear epitope and helps explain the protection afforded by the antibody for the shedding sites. Significance: The structures of full-length mesothelin and its complexes with antibodies reported here are the first to be determined experimentally, providing atomic models for structural organization of this protein and its interactions with antibodies. It offers insights into the function of mesothelin and guidance for further development of therapeutic antibodies.

Characterization of crystals of an antibody-recognition fragment of the cancer differentiation antigen mesothelin in complex with the therapeutic antibody MORAb-009.

The mesothelin-specific monoclonal antibody MORAb-009 is capable of blocking the binding of mesothelin to CA-125 and displays promising anticancer potential. It is currently undergoing clinical trials. In order to understand the basis of the interaction between MORAb-009 and mesothelin at atomic resolution, both the Fab fragment of MORAb-009 and the complex between the Fab and an N-terminal fragment of mesothelin (residues 7-64) were crystallized. The crystals of the Fab diffracted X-rays to 1.75 Šresolution and had the symmetry of space group P4(1)2(1)2, with unit-cell parameters a = b = 140.6, c = 282.0 Å. The crystals of the mesothelin-Fab complex diffracted to 2.6 Šresolution and belonged to the hexagonal space group P6(4), with unit-cell parameters a = b = 146.2, c = 80.9 Å. Structural analyses of these molecules are in progress.

Crystallization and preliminary X-ray diffraction analysis of PsaA, the adhesive pilin subunit that forms the pH 6 antigen on the surface of Yersinia pestis.

Yersinia pestis has been responsible for a number of high-mortality epidemics throughout human history. Like all other bacterial infections, the pathogenesis of Y. pestis begins with the attachment of bacteria to the surface of host cells. At least five surface proteins from Y. pestis have been shown to interact with host cells. Psa, the pH 6 antigen, is one of them and is deployed on the surface of bacteria as thin flexible fibrils that are the result of the polymerization of a single PsaA pilin subunit. Here, the crystallization of recombinant donor-strand complemented PsaA by the hanging-drop vapor-diffusion method is reported. X-ray diffraction data sets were collected to 1.9 Šresolution from a native crystal and to 1.5 Šresolution from a bromide-derivatized crystal. These crystals displayed the symmetry of the orthorhombic space group P222(1), with unit-cell parameters a = 26.3, b = 54.6, c = 102.1 Å. Initial phases were derived from single isomorphous replacement with anomalous scattering experiments, resulting in an electron-density map that showed a single molecule in the crystallographic asymmetric unit. Sequence assignment was aided by residues binding to bromide ions of the heavy-atom derivative.

Structural basis and mode of action for two broadly neutralizing antibodies against SARS-CoV-2 emerging variants of concern.

Emerging variants of concern for the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can transmit more efficiently and partially evade protective immune responses, thus necessitating continued refinement of antibody therapies and immunogen design. Here, we elucidate the structural basis and mode of action for two potent SARS-CoV-2 spike (S)-neutralizing monoclonal antibodies, CV3-1 and CV3-25, which remain effective against emerging variants of concern in vitro and in vivo. CV3-1 binds to the (485-GFN-487) loop within the receptor-binding domain (RBD) in the "RBD-up" position and triggers potent shedding of the S1 subunit. In contrast, CV3-25 inhibits membrane fusion by binding to an epitope in the stem helix region of the S2 subunit that is highly conserved among ß-coronaviruses. Thus, vaccine immunogen designs that incorporate the conserved regions in the RBD and stem helix region are candidates to elicit pan-coronavirus protective immune responses.

Toward understanding the mechanism of action of the yeast multidrug resistance transporter Pdr5p: a molecular modeling study.

Pleotropic drug resistant protein 5 (Pdr5p) is a plasma membrane ATP-binding cassette (ABC) transporter and the major drug efflux pump in Saccharomyces cerevisiae. The Pdr5p family of fungal transporters possesses a number of structural features significantly different from other modeled or crystallized ABC transporters, which include a reverse topology, an atypical ATP-binding site, a very low sequence similarity in the transmembrane section and long linkers between domains. These features present a considerable hurdle in molecular modeling studies of these important transporters. Here, we report the creation of an atomic model of Pdr5p based on a combination of homology modeling and ab initio methods, incorporating information from consensus transmembrane segment prediction, residue lipophilicity, and sequence entropy. Reported mutations in the transmembrane substrate-binding pocket that altered drug-resistance were used to validate the model, and one mutation that changed the communication pattern between transmembrane and nucleotide-binding domains was used in model improvement. The predictive power of the model was demonstrated experimentally by the increased sensitivity of yeast mutants to clotrimazole having alanine substitutions for Thr1213 and Gln1253, which are predicted to be in the substrate-binding pocket, without reducing the amount of Pdr5p in the plasma membrane. The quality and reliability of our model are discussed in the context of various approaches used for modeling different parts of the structure.

Structural Basis and Mode of Action for Two Broadly Neutralizing Antibodies Against SARS-CoV-2 Emerging Variants of Concern.

Emerging variants of concern for the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can transmit more efficiently and partially evade protective immune responses, thus necessitating continued refinement of antibody therapies and immunogen design. Here we elucidate the structural basis and mode of action for two potent SARS-CoV-2 Spike (S) neutralizing monoclonal antibodies CV3-1 and CV3-25 that remained effective against emerging variants of concern in vitro and in vivo. CV3-1 bound to the (485-GFN-487) loop within the receptor-binding domain (RBD) in the "RBD-up" position and triggered potent shedding of the S1 subunit. In contrast, CV3-25 inhibited membrane fusion by binding to an epitope in the stem helix region of the S2 subunit that is highly conserved among ß-coronaviruses. Thus, vaccine immunogen designs that incorporate the conserved regions in RBD and stem helix region are candidates to elicit pan-coronavirus protective immune responses.