Eleftherios Zarkadas

Azole resistance in Candida species is often caused by the overexpression of Cdr1. Despite its clinical relevance, the structural basis for its ATP-driven efflux pump function remains elusive. We present four high-resolution cryo-EM structures of Candida glabrata Cdr1 obtained in the absence and presence of ATP-Mg²⁺, itraconazole, and vanadate, capturing distinct nucleotide- and substrate-occupancy states. Additional low-population cryo-EM conformers were unveiled by 3D variability analysis, offering a detailed view of structural transitions linked with ATP-hydrolysis. The motion shows a 4 Å piston-like retraction of the C-helix from the γ-phosphate/vanadate of the hydrolyzed ATP. This causes the nearby transmembrane helix-1 (TMH-1) to open the drug-binding site via lateral displacement and unwinding of the inner-leaflet region of TMH-2. A reverse ‘squeeze-and-push’ motion of TMH-2 possibly drives substrate extrusion. High-resolution structures also reveal how itraconazole adapts its shape to fit into the drug-binding site. Our findings provide a dynamic structural framework for Cdr1-mediated azole resistance and the conserved chemo-mechanical cycle of ABC proteins, including non-membranous members.

Integrins promote immunity, embryonic development, wound healing, and hemostasis, and are activated by ‘bent/closed’ to ‘extended/open’ conformational changes. Integrin αIIbβ3, being crucial for platelet activation and aggregation, is a therapeutic target for bleeding disorders and thrombosis. Human Platelet Antigen-1a (HPA-1a) on β3 is recognized by pregnancy-associated maternal alloantibodies, potentially causing fetal/neonatal alloimmune thrombocytopenia (FNAIT) and even intracranial hemorrhage or perinatal death. We report the structure of an anti-HPA-1a antibody fragment (Fab 26.4) in complex with integrin αIIbβ3 at high resolution by cryo–electron microscopy. Fab 26.4 binding locks αIIbβ3 in the inactive, bent/closed conformation, is incompatible with integrin extension, and inhibits αIIbβ3-dependent fibrinogen binding and platelet aggregation. Thus, anti-HPA-1a antibodies directly impair integrin activation by preventing required conformational changes. These insights will improve FNAIT diagnostics and treatment, and spark the development of novel allosteric inhibitors against β3 integrins for future therapeutic applications.

The pathogenic yeast Candida glabrata is intrinsically resistant to azole antifungals through the overexpression of the multidrug transporter Cdr1. CgCdr1 detoxifies the yeast by expelling azoles out of the cell, thereby decreasing their intracellular concentration. Tacrolimus (FK506), one of the most widely used immunosuppressant medications used world-wide, has been identified as a broad-spectrum inhibitor of Cdr1 homologs in several Candida species. However, its mechanism of action remains unknown. We solved the cryoEM structure of CgCdr1 in complex with FK506, with or without ATP. The structure revealed that FK506 binds within the drug-binding site of CgCdr1, occupying the space occupied by Itraconazole. The hydrophobic face of FK506 stacks against the TMD1 and forms hydrogen bonds with TMD2, stabilizing a different conformation from the one adopted in FK-binding-proteins. FK506 binding triggered structural rearrangements bringing the nucleotide-binding-domains closer to the trans-membrane-domains, while stabilizing the inward-facing conformation. While ATP can still bind to the catalytic nucleotide-binding site, FK506 prevents the conformational transition required for ATP hydrolysis, thereby effectively blocking azole transport. Inter-particle variability analysis (3DVA) revealed significant conformational flexibility of FK506 within the binding pocket, with minimal transporter mobility. It allowed to visualize the conformational space occupied by the inhibitor within its binding-pocket, serving as a useful tool for inhibitor rational design. Overall, these findings demonstrate that FK506’s inhibition extends beyond competitive binding, involving allosteric modulation of the ATPase cycle. Significance statement: The pathogenic yeast Candida glabrata exhibits intrinsic resistance to azole antifungals via the multidrug transporter Cdr1, which expels azoles from the cell. Tacrolimus (FK506), a widely used immunosuppressant, inhibits Cdr1 homologs across Candida species, yet its mechanism remained unknown. Here, we resolved the cryoEM structure of Cg Cdr1 in complex with FK506, revealing that FK506 binds to the drug-binding site, like itraconazole. Its hydrophobic face interacts with TMD1, while hydrogen bonds form with TMD2. FK506 stabilizes the inward-facing conformation, preventing ATP hydrolysis despite ATP binding, thereby blocking azole transport. Variability analysis highlighted FK506’s conformational flexibility within the pocket, offering insights for rational inhibitor design. These findings demonstrate that FK506’s inhibition involves both competitive binding and allosteric modulation of the ATPase cycle.

Integrins promote immunity, embryonic development, wound healing, and hemostasis, and are activated by ‘bent/closed’ to ‘extended/open’ conformational changes. Integrin αIIbβ3, being crucial for platelet activation and aggregation, is a therapeutic target for bleeding disorders and thrombosis. Human Platelet Antigen-1a (HPA-1a) on β3 is recognized by pregnancy-associated maternal alloantibodies, potentially causing fetal/neonatal alloimmune thrombocytopenia (FNAIT) and even intracranial hemorrhage or perinatal death. However, severe disease determinants are largely unknown. We report the first structure of an anti-HPA-1a antibody fragment (Fab 26.4) in complex with integrin αIIbβ3 at high resolution by cryo-electron microscopy. Fab 26.4 binding traps αIIbβ3 in the inactive, bent/closed conformation, is incompatible with integrin extension, and inhibits αIIbβ3-dependent fibrinogen binding and platelet aggregation. Thus, anti-HPA-1a antibodies directly impair integrin activation by preventing required conformational changes. These insights will improve FNAIT diagnostics and treatment, and spark the development of novel allosteric inhibitors against β3 integrins for future therapeutic applications.

Communication at synapses is facilitated by postsynaptic receptors, which convert a chemical signal into an electrical response. For ligand-gated ion channels, agonist binding triggers rapid transitions through intermediate states leading to a transient open-pore conformation, with these transitions shaping the postsynaptic response. In this work, we determine structures of the muscle-type nicotinic acetylcholine receptor in unliganded, mono-liganded, and di-liganded states. Agonist binding to a single site stabilizes a closed structure where an entire principal agonist-binding subunit transitions to an active-like conformation, whereas the other unoccupied principal subunit remains inactive, albeit poised for activation. Uniting this intermediate structure with single-channel recordings informs a sequential activation mechanism where asynchronous subunit transitions prime the receptor for activation—a finding with implications for an entire superfamily of pentameric ligand-gated ion channels.

Azole resistance in Candida species is often caused by the overexpression of Cdr1. Despite its clinical relevance, the structural basis for its ATP-driven efflux pump function remains elusive. We present four high-resolution cryo-EM structures for Candida glabrata Cdr1 under active turnover conditions in the absence and presence of ATP-Mg²⁺, itraconazole, and vanadate. Additional transient cryo-EM structures were unveiled by 3D variability analysis offering a detailed view of the step-by-step transitions triggered by ATP-hydrolysis. The motion cascade starts with a 4 Å piston-like retraction of the C-helix from the γ-phosphate/vanadate of the hydrolyzed ATP. This causes the nearby transmembrane helix-1 (TMH-1) to open the drug-binding site via lateral displacement and unwinding of the inner-leaflet region of TMH-2. A reverse ‘squeeze-and-push’ motion of TMH-2 possibly drives substrate extrusion. High resolution structures also reveal how itraconazole adapts its shape to fit into the drug-binding site. Our findings provide a dynamic structural framework for Cdr1-mediated azole resistance and the conserved chemo-mechanical cycle of ABC proteins, including non-membranous members.

Multidrug ABC transporters harness the energy of ATP binding and hydrolysis to translocate substrates out of the cell and detoxify them. While this involves a well-accepted alternating access mechanism, molecular details of this interplay are still elusive. Rhodamine6G binding on a catalytic inactive mutant of the homodimeric multidrug ABC transporter BmrA triggers a cooperative binding of ATP on the two identical nucleotide-binding-sites, otherwise michaelian. Here, we investigate this asymmetric behavior via a structural-enzymology approach, solving cryoEM structures of BmrA at defined ATP ratios, highlighting the plasticity of BmrA as it undergoes the transition from inward to outward facing conformations. Analysis of continuous heterogeneity within cryoEM data and structural dynamics, reveals that Rhodamine6G narrows the conformational spectrum explored by the nucleotide-binding domains. We observe the same behavior for the other drug Hœchst33342. Following on these findings, the effect of drug-binding showed an ATPase stimulation and a maximal transport activity of the wild-type protein at the concentration-range where the cooperative transition occurs. Altogether, these findings provide a description of the influence of drug binding on the ATP-binding sites through a change in conformational dynamics.

Influenza A viruses are responsible for human seasonal epidemics and severe animal pandemics with a risk of zoonotic transmission to humans. The viral segmented RNA genome is encapsidated by nucleoproteins (NP) and attached to the heterotrimeric polymerase, forming the viral ribonucleoproteins (vRNPs). Flexible helical vRNPs are central for viral transcription and replication. In this study, we present an advanced biological tool, the antiparallel helical RNP-like complex, assembled from recombinant N-terminally truncated NP and short synthetic RNA. The 3.0 Å cryo-electron microscopy structure details for the first time the whole RNA pathway across NP as well as NP-NP interactions that drive the antiparallel helical assembly accommodating major and minor grooves. Our findings show that the surface of the protein can harbour several conformations of the RNA, confirming that the number of nucleobases that binds to NP is not fixed, but ranges probably between 20 and 24. Taking all together, our data provide details to further understand the genome encapsidation and explain the inherent flexibility of influenza A virus vRNPs.

Multidrug ABC transporters harness the energy of ATP binding and hydrolysis to change conformation and thereby translocate substrates out of the cell to detoxify them. While this general access mechanism scheme is well accepted, molecular details of this interplay is still elusive. Rhodamine6G binding on a catalytic mutant of the homodimeric multidrug ABC transporter BmrA triggers a cooperative binding of ATP on the two identical nucleotide-binding-sites, otherwise Michaelian. We investigated this asymmetric behavior via a structural-enzymology approach, solving cryoEM structure of BmrA at defined ATP ratio along the enzymatic transition, highlighting the plasticity of BmrA as it undergoes the transition from inward to outward facing conformations. Analysis of continuous heterogeneity within cryoEM data and structural dynamics, revealed that Rhodamine6G narrows the conformational spectrum explored by the nucleotide-binding-domains, describing the allosteric effect of drug binding that optimizes the ATP-dependent conversion of the transporter to the outward-facing state. Following on these findings, the effect of drug-binding showed an ATPase stimulation and a maximal transport activity of the wild-type protein at the concentration-range where the allosteric transition occurs. Drug diffusion rate is the likely rate-limiting step of the reaction, while drug transport and ATPase activities are in effect uncoupled.