The N-terminal signature motif on the transporter MCT1 is critical for CD147-mediated trafficking.

The human Solute Carrier (SLC) family member, monocarboxylate transporter 1 (MCT1), transports lactic and pyruvic acid across biological membranes to regulate cellular pH and metabolism. Proper trafficking of MCT1 from the endoplasmic reticulum to the plasma membrane hinges on its interactions with the membrane-bound chaperone protein, CD147. Here, using AlphaFold2 modeling and copurification, we show how a conserved signature motif located in the flexible N-terminus of MCT1 is a crucial region of interaction between MCT1 and the C-terminus of CD147. Mutations to this motif-namely, the thymic cancer linked G19C and the highly conserved W20A-destabilize the MCT1-CD147 complex and lead to a loss of proper membrane localization and cellular substrate flux. Notably, the monomeric stability of MCT1 remains unaffected in mutants, thus supporting the role of CD147 in mediating the trafficking of the heterocomplex. Using the auxiliary chaperone, GP70, we demonstrated that W20A-MCT1 can be trafficked to the plasma membrane, while G19C-MCT1 remains internalized. Overall, our findings underscore the critical role of the MCT1 transmembrane one signature motif for engaging CD147 and identify altered chaperone binding mechanisms between the CD147 and GP70 glycoprotein chaperones.

Cryo-EM structure of the tetra-phosphorylated R-domain in Ycf1 reveals key interactions for transport regulation.

Many ATP-binding cassette (ABC) transporters are regulated by phosphorylation on long and disordered loops which present a challenge to visualize with structural methods. We have trapped an activated state of the regulatory domain (R-domain) of Yeast Cadmium Factor 1 (Ycf1) by enzymatically enriching the phosphorylated state. A 3.2 Å cryo-EM structure reveals an R-domain structure with four phosphorylated residues and a position for the entire R-domain. The structure reveals key R-domain interactions including a bridging interaction between NBD1 and NBD2 as well as an interaction with the R-insertion, another regulatory region. We systematically probe these interactions with a linker substitution strategy along the R-domain and find a close match with these interactions and survival under Ycf1-dependent growth conditions. We propose a model where four overlapping phosphorylation sites bridge several regions of Ycf1 to engage in a transport-competent state.

Structural basis for autoinhibition by the dephosphorylated regulatory domain of Ycf1.

Yeast Cadmium Factor 1 (Ycf1) sequesters glutathione and glutathione-heavy metal conjugates into yeast vacuoles as a cellular detoxification mechanism. Ycf1 belongs to the C subfamily of ATP Binding Cassette (ABC) transporters characterized by long flexible linkers, notably the regulatory domain (R-domain). R-domain phosphorylation is necessary for activity, whereas dephosphorylation induces autoinhibition through an undefined mechanism. Because of its transient and dynamic nature, no structure of the dephosphorylated Ycf1 exists, limiting understanding of this R-domain regulation. Here, we capture the dephosphorylated Ycf1 using cryo-EM and show that the unphosphorylated R-domain indeed forms an ordered structure with an unexpected hairpin topology bound within the Ycf1 substrate cavity. This architecture and binding mode resemble that of a viral peptide inhibitor of an ABC transporter and the secreted bacterial WXG peptide toxins. We further reveal the subset of phosphorylation sites within the hairpin turn that drive the reorganization of the R-domain conformation, suggesting a mechanism for Ycf1 activation by phosphorylation-dependent release of R-domain mediated autoinhibition.

Structural Basis for Oxidized Glutathione Recognition by the Yeast Cadmium Factor 1.

Transporters from the ABCC family have an essential role in detoxifying electrophilic compounds including metals, drugs, and lipids, often through conjugation with glutathione complexes. The Yeast Cadmium Factor 1 (Ycf1) transports glutathione alone as well as glutathione conjugated to toxic heavy metals including Cd2+, Hg2+, and As3+. To understand the complicated selectivity and promiscuity of heavy metal substrate binding, we determined the cryo-EM structure of Ycf1 bound to the substrate, oxidized glutathione. We systematically tested binding determinants with cellular survival assays against cadmium to determine how the substrate site accommodates different-sized metal complexes. We identify a "flex-pocket" for substrate binding that binds glutathione complexes asymmetrically and flexes to accommodate different size complexes.

Vaccines to Prevent Coccidioidomycosis: A Gene-Deletion Mutant of Coccidioides Posadasii as a Viable Candidate for Human Trials.

Coccidioidomycosis is an endemic fungal infection that is reported in up to 20,000 persons per year and has an economic impact close to $1.5 billion. Natural infection virtually always confers protection from future exposure, and this suggests that a preventative vaccine strategy is likely to succeed. We here review progress toward that objective. There has been ongoing research to discover a coccidioidal vaccine over the past seven decades, including one phase III clinical trial, but for reasons of either efficacy or feasibility, a safe and effective vaccine has not yet been developed. This review first summarizes the past research to develop a coccidioidal vaccine. It then details the evidence that supports a live, gene-deletion vaccine candidate as suitable for further development as both a veterinary and a human clinical product. Finally, a plausible vaccine development plan is described which would be applicable to this vaccine candidate and also useful to other future candidates. The public health and economic impact of coccidioidomycosis fully justifies a public private partnership for vaccine development, and the development of a vaccine for this orphan disease will likely require some degree of public funding.

The structural basis for regulation of the glutathione transporter Ycf1 by regulatory domain phosphorylation.

Yeast Cadmium Factor 1 (Ycf1) sequesters heavy metals and glutathione into the vacuole to counter cell stress. Ycf1 belongs to the ATP binding cassette C-subfamily (ABCC) of transporters, many of which are regulated by phosphorylation on intrinsically-disordered domains. The regulatory mechanism of phosphorylation is still poorly understood. Here, we report two cryo-EM structures of Ycf1 at 3.4 Å and 4.0 Å resolution in inward-facing open conformations that capture previously unobserved ordered states of the intrinsically disordered regulatory domain (R-domain). R-domain phosphorylation is clearly evident and induces a topology promoting electrostatic and hydrophobic interactions with Nucleotide Binding Domain 1 (NBD1) and the Lasso motif. These interactions stay constant between the structures and are related by rigid body movements of the NBD1/R-domain complex. Biochemical data further show R-domain phosphorylation reorganizes the Ycf1 architecture and is required for maximal ATPase activity. Together, we provide insights into how R-domains control ABCC transporter activity.

Asymmetric drug binding in an ATP-loaded inward-facing state of an ABC transporter.

Substrate efflux by ATP-binding cassette (ABC) transporters, which play a major role in multidrug resistance, entails the ATP-powered interconversion between transporter intermediates. Despite recent progress in structure elucidation, a number of intermediates have yet to be visualized and mechanistically interpreted. Here, we combine cryogenic-electron microscopy (cryo-EM), double electron-electron resonance spectroscopy and molecular dynamics simulations to profile a previously unobserved intermediate of BmrCD, a heterodimeric multidrug ABC exporter from Bacillus subtilis. In our cryo-EM structure, ATP-bound BmrCD adopts an inward-facing architecture featuring two molecules of the substrate Hoechst-33342 in a striking asymmetric head-to-tail arrangement. Deletion of the extracellular domain capping the substrate-binding chamber or mutation of Hoechst-coordinating residues abrogates cooperative stimulation of ATP hydrolysis. Together, our findings support a mechanistic role for symmetry mismatch between the nucleotide binding and the transmembrane domains in the conformational cycle of ABC transporters and is of notable importance for rational design of molecules for targeted ABC transporter inhibition.

A Conserved Motif in Intracellular Loop 1 Stabilizes the Outward-Facing Conformation of TmrAB.

The ATP binding cassette (ABC) family of transporters moves small molecules (lipids, sugars, peptides, drugs, nutrients) across membranes in nearly all organisms. Transport activity requires conformational switching between inward-facing and outward-facing states driven by ATP-dependent dimerization of two nucleotide binding domains (NBDs). The mechanism that connects ATP binding and hydrolysis in the NBDs to conformational changes in a substrate binding site in the transmembrane domains (TMDs) is currently an outstanding question. Here we use sequence coevolution analyses together with biochemical characterization to investigate the role of a highly conserved region in intracellular loop 1 we define as the GRD motif in coordinating domain rearrangements in the heterodimeric peptide exporter from Thermus thermophilus, TmrAB. Mutations in the GRD motif alter ATPase activity as well as transport. Disulfide crosslinking, evolutionary trace, and evolutionary coupling analysis reveal that these effects are likely due to the destabilization of a network in which the GRD motif in TmrA bridges residues of the Q-loop, X-loop, and ABC motif in the NBDs to residues in the TmrAB peptide substrate binding site, thus providing an avenue for conformational coupling. We further find that disruption of this network in TmrA versus TmrB has different functional consequences, hinting at an intrinsic asymmetry in heterodimeric ABC transporters extending beyond that of the NBDs. These results support a mechanism in which the GRD motifs help coordinate a transition to an outward open conformation, and each half of the transporter likely plays a different role in the conformational cycle of TmrAB.

The role of novel COQ8B mutations in glomerulopathy and related kidney defects.

BACKGROUND AND OBJECTIVES: Glomerulopathies affect kidney glomeruli and can lead to end-stage renal disease if untreated. Clinical and experimental evidence have identified numerous (>20) genetic mutations in the mitochondrial coenzyme Q8B protein (COQ8B) primarily associated with nephrotic syndrome. Yet, little else is understood about COQ8B activity in renal pathogenesis and its role in mitochondrial dysfunction. We identified additional novel COQ8B mutations in a glomerulopathy patient and aimed to define the potential structural and functional defects of COQ8B mutations. DESIGN, SETTING, PARTICIPANTS, AND MEASUREMENTS: Whole exome sequencing was performed on a Hispanic female presenting with proteinuria. Novel mutations in the COQ8B gene were identified. The effects of mutation on protein function, mitochondrial morphology, and disease progression were investigated by histopathology, transmission electron microscopy, homology modeling, and in silico structural analysis. RESULTS: We have characterized the pathophysiology of novel COQ8B mutations, compound heterozygous for two alterations c.1037T>G (p.I346S), and c.1560G>A (p.W520X), in the progression of proteinuria in a Hispanic female. Histopathology revealed defects in podocyte structure and mitochondrial morphology. In silico and computation analyses highlight possible structural origins of COQ8B dysfunction in the presence of mutations. CONCLUSIONS: Novel mutations in COQ8B present promising biomarkers for the early detection and therapeutic targeting of mitochondrial glomerulopathy. Insights from structural modeling suggest roles of mutation-dependent alterations in COQ8B allosteric regulation, protein folding, or stability in renal pathogenesis.

New crystal forms of the integral membrane Escherichia coli quinol:fumarate reductase suggest that ligands control domain movement.

Quinol:fumarate reductase (QFR) is an integral membrane protein and a member of the respiratory Complex II superfamily. Although the structure of Escherichia coli QFR was first reported almost twenty years ago, many open questions of catalysis remain. Here we report two new crystal forms of QFR, one grown from the lipidic cubic phase and one grown from dodecyl maltoside micelles. QFR crystals grown from the lipid cubic phase processed as P1, merged to 7.5 Šresolution, and exhibited crystal packing similar to previous crystal forms. Crystals grown from dodecyl maltoside micelles processed as P21, merged to 3.35 Šresolution, and displayed a unique crystal packing. This latter crystal form provides the first view of the E. coli QFR active site without a dicarboxylate ligand. Instead, an unidentified anion binds at a shifted position. In one of the molecules in the asymmetric unit, this is accompanied by rotation of the capping domain of the catalytic subunit. In the other molecule, this is associated with loss of interpretable electron density for this same capping domain. Analysis of the structure suggests that the ligand adjusts the position of the capping domain.

Crystal structure and mechanistic basis of a functional homolog of the antigen transporter TAP.

ABC transporters form one of the largest protein superfamilies in all domains of life, catalyzing the movement of diverse substrates across membranes. In this key position, ABC transporters can mediate multidrug resistance in cancer therapy and their dysfunction is linked to various diseases. Here, we describe the 2.7-Å X-ray structure of heterodimeric Thermus thermophilus multidrug resistance proteins A and B (TmrAB), which not only shares structural homology with the antigen translocation complex TAP, but is also able to restore antigen processing in human TAP-deficient cells. TmrAB exhibits a broad peptide specificity and can concentrate substrates several thousandfold, using only one single active ATP-binding site. In our structure, TmrAB adopts an asymmetric inward-facing state, and we show that the C-terminal helices, arranged in a zipper-like fashion, play a crucial role in guiding the conformational changes associated with substrate transport. In conclusion, TmrAB can be regarded as a model system for asymmetric ABC exporters in general, and for TAP in particular.

Subnanometre-resolution electron cryomicroscopy structure of a heterodimeric ABC exporter.

ATP-binding cassette (ABC) transporters translocate substrates across cell membranes, using energy harnessed from ATP binding and hydrolysis at their nucleotide-binding domains. ABC exporters are present both in prokaryotes and eukaryotes, with examples implicated in multidrug resistance of pathogens and cancer cells, as well as in many human diseases. TmrAB is a heterodimeric ABC exporter from the thermophilic Gram-negative eubacterium Thermus thermophilus; it is homologous to various multidrug transporters and contains one degenerate site with a non-catalytic residue next to the Walker B motif. Here we report a subnanometre-resolution structure of detergent-solubilized TmrAB in a nucleotide-free, inward-facing conformation by single-particle electron cryomicroscopy. The reconstructions clearly resolve characteristic features of ABC transporters, including helices in the transmembrane domain and nucleotide-binding domains. A cavity in the transmembrane domain is accessible laterally from the cytoplasmic side of the membrane as well as from the cytoplasm, indicating that the transporter lies in an inward-facing open conformation. The two nucleotide-binding domains remain in contact via their carboxy-terminal helices. Furthermore, comparison between our structure and the crystal structures of other ABC transporters suggests a possible trajectory of conformational changes that involves a sliding and rotating motion between the two nucleotide-binding domains during the transition from the inward-facing to outward-facing conformations.

General qPCR and Plate Reader Methods for Rapid Optimization of Membrane Protein Purification and Crystallization Using Thermostability Assays.

This unit describes rapid and generally applicable methods to identify conditions that stabilize membrane proteins using temperature-based denaturation measurements as a proxy for target time-dependent stability. Recent developments with thiol-reactive dyes sensitive to the unmasking of cysteine residues upon protein unfolding have allowed for routine application of thermostability assays to systematically evaluate the stability of membrane protein preparations after various purification procedures. Test conditions can include different lipid cocktails, lipid-detergent micelles, pH, salts, osmolytes, and potential active-site ligands. Identification and use of conditions that stabilize the structure have proven successful in enabling the structure determination of numerous families of membrane proteins that otherwise were intractable.

Architecture of a single membrane spanning cytochrome P450 suggests constraints that orient the catalytic domain relative to a bilayer.

Bitopic integral membrane proteins with a single transmembrane helix play diverse roles in catalysis, cell signaling, and morphogenesis. Complete monospanning protein structures are needed to show how interaction between the transmembrane helix and catalytic domain might influence association with the membrane and function. We report crystal structures of full-length Saccharomyces cerevisiae lanosterol 14α-demethylase, a membrane monospanning cytochrome P450 of the CYP51 family that catalyzes the first postcyclization step in ergosterol biosynthesis and is inhibited by triazole drugs. The structures reveal a well-ordered N-terminal amphipathic helix preceding a putative transmembrane helix that would constrain the catalytic domain orientation to lie partly in the lipid bilayer. The structures locate the substrate lanosterol, identify putative substrate and product channels, and reveal constrained interactions with triazole antifungal drugs that are important for drug design and understanding drug resistance.

Plasticity of the quinone-binding site of the complex II homolog quinol:fumarate reductase.

Respiratory processes often use quinone oxidoreduction to generate a transmembrane proton gradient, making the 2H(+)/2e(-) quinone chemistry important for ATP synthesis. There are a variety of quinones used as electron carriers between bioenergetic proteins, and some respiratory proteins can functionally interact with more than one quinone type. In the case of complex II homologs, which couple quinone chemistry to the interconversion of succinate and fumarate, the redox potentials of the biologically available ubiquinone and menaquinone aid in driving the chemical reaction in one direction. In the complex II homolog quinol:fumarate reductase, it has been demonstrated that menaquinol oxidation requires at least one proton shuttle, but many of the remaining mechanistic details of menaquinol oxidation are not fully understood, and little is known about ubiquinone reduction. In the current study, structural and computational studies suggest that the sequential removal of the two menaquinol protons may be accompanied by a rotation of the naphthoquinone ring to optimize the interaction with a second proton shuttling pathway. However, kinetic measurements of site-specific mutations of quinol:fumarate reductase variants show that ubiquinone reduction does not use the same pathway. Computational docking of ubiquinone followed by mutagenesis instead suggested redundant proton shuttles lining the ubiquinone-binding site or from direct transfer from solvent. These data show that the quinone-binding site provides an environment that allows multiple amino acid residues to participate in quinone oxidoreduction. This suggests that the quinone-binding site in complex II is inherently plastic and can robustly interact with different types of quinones.

A structural model for binding of the serine-rich repeat adhesin GspB to host carbohydrate receptors.

GspB is a serine-rich repeat (SRR) adhesin of Streptococcus gordonii that mediates binding of this organism to human platelets via its interaction with sialyl-T antigen on the receptor GPIbα. This interaction appears to be a major virulence determinant in the pathogenesis of infective endocarditis. To address the mechanism by which GspB recognizes its carbohydrate ligand, we determined the high-resolution x-ray crystal structure of the GspB binding region (GspB(BR)), both alone and in complex with a disaccharide precursor to sialyl-T antigen. Analysis of the GspB(BR) structure revealed that it is comprised of three independently folded subdomains or modules: 1) an Ig-fold resembling a CnaA domain from prokaryotic pathogens; 2) a second Ig-fold resembling the binding region of mammalian Siglecs; 3) a subdomain of unique fold. The disaccharide was found to bind in a pocket within the Siglec subdomain, but at a site distinct from that observed in mammalian Siglecs. Confirming the biological relevance of this binding pocket, we produced three isogenic variants of S. gordonii, each containing a single point mutation of a residue lining this binding pocket. These variants have reduced binding to carbohydrates of GPIbα. Further examination of purified GspB(BR)-R484E showed reduced binding to sialyl-T antigen while S. gordonii harboring this mutation did not efficiently bind platelets and showed a significant reduction in virulence, as measured by an animal model of endocarditis. Analysis of other SRR proteins revealed that the predicted binding regions of these adhesins also had a modular organization, with those known to bind carbohydrate receptors having modules homologous to the Siglec and Unique subdomains of GspB(BR). This suggests that the binding specificity of the SRR family of adhesins is determined by the type and organization of discrete modules within the binding domains, which may affect the tropism of organisms for different tissues.

Geometric restraint drives on- and off-pathway catalysis by the Escherichia coli menaquinol:fumarate reductase.

Complex II superfamily members catalyze the kinetically difficult interconversion of succinate and fumarate. Due to the relative simplicity of complex II substrates and their similarity to other biologically abundant small molecules, substrate specificity presents a challenge in this system. In order to identify determinants for on-pathway catalysis, off-pathway catalysis, and enzyme inhibition, crystal structures of Escherichia coli menaquinol:fumarate reductase (QFR), a complex II superfamily member, were determined bound to the substrate, fumarate, and the inhibitors oxaloacetate, glutarate, and 3-nitropropionate. Optical difference spectroscopy and computational modeling support a model where QFR twists the dicarboxylate, activating it for catalysis. Orientation of the C2-C3 double bond of activated fumarate parallel to the C(4a)-N5 bond of FAD allows orbital overlap between the substrate and the cofactor, priming the substrate for nucleophilic attack. Off-pathway catalysis, such as the conversion of malate to oxaloacetate or the activation of the toxin 3-nitropropionate may occur when inhibitors bind with a similarly activated bond in the same position. Conversely, inhibitors that do not orient an activatable bond in this manner, such as glutarate and citrate, are excluded from catalysis and act as inhibitors of substrate binding. These results support a model where electronic interactions via geometric constraint and orbital steering underlie catalysis by QFR.

A threonine on the active site loop controls transition state formation in Escherichia coli respiratory complex II.

In Escherichia coli, the complex II superfamily members succinate:ubiquinone oxidoreductase (SQR) and quinol:fumarate reductase (QFR) participate in aerobic and anaerobic respiration, respectively. Complex II enzymes catalyze succinate and fumarate interconversion at the interface of two domains of the soluble flavoprotein subunit, the FAD binding domain and the capping domain. An 11-amino acid loop in the capping domain (Thr-A234 to Thr-A244 in quinol:fumarate reductase) begins at the interdomain hinge and covers the active site. Amino acids of this loop interact with both the substrate and a proton shuttle, potentially coordinating substrate binding and the proton shuttle protonation state. To assess the loop's role in catalysis, two threonine residues were mutated to alanine: QFR Thr-A244 (act-T; Thr-A254 in SQR), which hydrogen-bonds to the substrate at the active site, and QFR Thr-A234 (hinge-T; Thr-A244 in SQR), which is located at the hinge and hydrogen-bonds the proton shuttle. Both mutations impair catalysis and decrease substrate binding. The crystal structure of the hinge-T mutation reveals a reorientation between the FAD-binding and capping domains that accompanies proton shuttle alteration. Taken together, hydrogen bonding from act-T to substrate may coordinate with interdomain motions to twist the double bond of fumarate and introduce the strain important for attaining the transition state.

Succinate as Donor; Fumarate as Acceptor.

Succinate and fumarate are four-carbon dicarboxylates that differ in the identity of their central bond (single or double). The oxidoreduction of these small molecules plays a central role in both aerobic and anaerobic respiration. During aerobic respiration, succinate is oxidized, donating two reducing equivalents, while in anaerobic respiration, fumarate is reduced, accepting two reducing equivalents. Two related integral membrane Complex II superfamily members catalyze these reactions, succinate:ubiquinone oxidoreductase (SQR) and fumarate:menaquinol oxidoreductase (QFR). The structure, function, and regulation of these integral-membrane enzymes are summarized here. The overall architecture of these Complex II enzymes has been found to consist of four subunits: two integral membrane subunits, and a soluble domain consisting of an iron-sulfur protein subunit, and a flavoprotein subunit. This architecture provides a scaffold that houses one active site in the membrane and another in the soluble milieu, making a linear electron transfer chain that facilities shuttling of reducing equivalents between the two active sites. A combination of kinetic measurements, mutagenesis, electron paramagnetic resonance spectroscopy, UV/Vis spectroscopy, and x-ray crystallography have suggested mechanisms for succinate:fumarate interconversion, electron transfer, and quinone:quinol interconversion. Of particular interest are the structural details that control directionality and make SQR and QFR primed for preferential catalysis each in different favored directions.