Biology subjects

Gatsogiannis, C.

Publications and source records attributed to Gatsogiannis, C..

At least 19 recordsLinked to original sources

bioRxiv2026

Cryo-EM structures of apo human Factor XIa reveal catalytic-domain flexibility and exposure of the Factor IX-binding site

Factor XI (FXI) is a key coagulation protease of the intrinsic pathway of blood coagulation and an emerging antithrombotic target. However, the structural transition from zymogen to active Factor XIa (FXIa) has remained poorly understood. Using cryo-EM, we demonstrate that FXI activation results in a global reorganization of the homodimer, extending beyond the activation loop to include a significant reorientation of the catalytic domain (CD) relative to the apple-domain (AD) platform. The CD displays pronounced conformational heterogeneity; we identify three distinct conformers, suggesting that FXIa exists as a dynamic ensemble rather than a single rigid state. MD analysis indicates that activation disrupts the inter-CD allosteric communication present in the zymogen, thereby facilitating this flexibility. CD plasticity allows for the dynamic exposure of the A3 exosite, facilitating the binding of Factor IX. Comparison with plasma kallikrein (PKa) suggests that such structural flexibility may be a shared feature of apple-domain-containing contact-system proteases. Our results reveal that FXIa functions as a dynamic ensemble, providing a structural framework for understanding substrate recognition and identifying novel, non-catalytic sites for the development of specific FXIa inhibitors.

biochemistry↗

bioRxiv2026

A nested shell structure coordinates enzyme communication in pyruvate oxidation

The pyruvate dehydrogenase complex (PDHc)1 links glycolysis to the Krebs cycle by catalyzing the oxidative decarboxylation of pyruvate to acetyl-CoA and CO2, a process essential for life2,3. PDHc is formed by structural proteins (E3-binding protein, E3BP)4-7, enzymatic subunits (E1, E2, E3)4,6, and mobile lipoyl domains (LDs), the latter shuttling intermediates across active sites6,8. Although numerous details regarding pyruvate oxidation steps have been elucidated9, the precise organization of the entire PDHc remains unknown due to its large size and dynamic heterogeneity. Here, we employ in silico, in vitro, and in situ methods to propose a multi-scale model of PDHc that includes approximately one million atoms and to visualize multiple conformational states. This model reveals a [~]40-50 nm nested shell structure, formed by flexible linkers that spatially coordinate the E1 and E3 enzyme complexes around the E2-E3BP core scaffold. This structure acts as a molecular sieve, selectively guiding lipoyl arms while maintaining enzyme positioning with sub-nm precision. During catalysis, the nested shell structure expands and adopts a mechanically reinforced state comparable in magnitude to viral assemblies10. Our findings provide structural context for the textbook "link reaction"11, building on decades of biochemical knowledge, are transferable to functional aspects of other -ketoacid dehydrogenase complexes, and, ultimately, expand our understanding of primary metabolism as a whole. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=195 SRC="FIGDIR/small/724543v1_ufig1.gif" ALT="Figure 1"> View larger version (70K): org.highwire.dtl.DTLVardef@1615612org.highwire.dtl.DTLVardef@159bc8dorg.highwire.dtl.DTLVardef@69f8c1org.highwire.dtl.DTLVardef@14a83a3_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2025

Structural basis of apoptosis induction by the mitochondrial voltage dependent anion channel

The voltage-dependent anion channel (VDAC) is the main gateway for metabolites across the mitochondrial outer membrane1. In addition, VDAC oligomers have been associated with apoptosis at mitochondrial stress conditions2. However, the mechanistic and structural basis of VDACs capability to induce apoptosis pathways remains poorly understood. Here, we show with biochemical and structural methods that VDAC1 oligomerization triggers the dissociation of its N-terminal -helix (VDAC1-N) from the channel interior. We used advanced lipid nanodiscs as a tool to selectively trap VDAC1 in its canonical helix-inserted and helix-exposed state to facilitate a structural characterization of both conformations by cryo-electron microscopy. The results show that slight changes in the shape and dynamics of the VDAC1 {beta}-barrel suffice to release the N-terminal helix to the channel exterior. This conformational switch addresses the long-standing question how VDAC1 can regulate partner protein binding. To confirm this hypothesis, we performed interaction studies between VDAC1 in both conformational states and the anti-apoptotic partner protein BclxL using nuclear magnetic resonance spectroscopy and could detect binding only for the helix-exposed state. These insights enabled the X-ray structure determination of the BclxL-VDAC1-N complex at high resolution and provided atomistic details on the VDAC1-N binding mode at the BH3-groove in BclxL. Further biochemical assays showed that VDAC1-N promotes pore formation of the pro-apoptotic Bcl2 protein Bak by neutralizing BclxLs inhibitory activity. These findings suggest that stress-induced oligomerization of VDAC can trigger the exposure of its N-terminal -helix leading to the neutralization of anti-apoptotic Bcl2 proteins. This mode-of-action is reminiscent of BH3-only sensitizer Bcl2 proteins3 that are efficient inducers of Bax/Bak-mediated mitochondrial outer membrane permeabilization and ultimately apoptosis.

biophysics↗

bioRxiv2025

From dimer to tetramer: the evolutionary trajectory of C4 photosynthetic-NADP-ME oligomeric state in Poaceae

The C4 carbon concentrating mechanism relies on specialized enzymes that have evolved unique expression patterns and biochemical properties distinct to their ancestral housekeeping forms. In maize and sorghum, the evolution of C4-NADP-malic enzyme (C4-NADP-ME) involved gene duplication and neofunctionalization, leading to the emergence of two plastidic isoforms: C4-NADP-ME and nonC4-NADP-ME, each with distinct kinetic and structural features. While C4-NADP-ME functions primarily as a tetramer, nonC4-NADP-ME exists in an equilibrium between dimeric and tetrameric forms, favoring the dimer in solution. This study shows which evolutionary changes in amino acid sequences influence the structure and function of these isoforms. By integrating X-ray crystallography, cryo-electron microscopy, computational molecular modeling and targeted biochemical analysis of mutant and truncated protein variants, we identify crucial roles for the N- and C-terminal regions and specific amino acid residues in governing isoform oligomerization. Our results reveal that the N-terminal region is essential for stabilizing the dimeric form of nonC4-NADP-ME, whereas specific adaptive substitutions and interactions with the C-terminal region enhance the stability of the tetrameric state characteristic of the C4-adapted isoform. We propose that differences in the N-terminal domain between the C4 and nonC4 isoforms reflect distinct selective pressures, which have driven their evolutionary divergence to fulfill specialized cellular functions.

evolutionary biology↗

bioRxiv2024

Structure and mechanism of the RalGAP tumor suppressor complex

The RalGAP (GTPase activating protein) complexes are negative regulators of the Ral GTPases and thus crucial components that counteract (oncogenic) Ras signaling. However, no structural information on the architecture of this tumor suppressor complex is available hampering a mechanistic understanding of its functionality. Here, we present a cryo-EM structure of RalGAP that reveals an extended 58 nm tetrameric architecture comprising two heterodimers of the RalGAP and RalGAP{beta} subunits. We show that the catalytic domain of RalGAP requires stabilization by a unique domain of RalGAP{beta}, providing the molecular basis for why RalGAP complexes are obligatory heterodimers. Formation of RalGAP tetramers is not required for activity in vitro, but essential for function of the complex in vivo. Structural analysis of RalGAP subunit variants reported in cancer patients suggests effects on complex formation and thus functional relevance in tumor development, emphasizing the significance of the obtained structural information for medical research.

biochemistry↗

bioRxiv2024

High-resolution structures of the UapA purine transporter reveal unprecedented aspects of the elevator-type transport mechanism.

UapA is an extensively studied elevator-type purine transporter from the model fungus Aspergillus nidulans. Determination of a 3.6[A] inward-facing crystal structure lacking the cytoplasmic N-and C-tails, molecular dynamics (MD), and functional studies have led to speculative models of its transport mechanism and determination of substrate specificity. Here, we report full-length cryo-EM structures of UapA in new inward-facing apo- and substrate-loaded conformations at 2.05-3.5 [A] in detergent and lipid nanodiscs. The structures reveal in an unprecedented level of detail the role of water molecules and lipids in substrate binding, specificity, dimerization, and activity, rationalizing accumulated functional data. Unexpectedly, the N-tail is structured and interacts with both the core and scaffold domains. This finding, combined with mutational and functional studies and MD, points out how N-tail interactions couple proper subcellular trafficking and transport activity by wrapping UapA in a conformation necessary for ER-exit and but also critical for elevator-type conformational changes associated with substrate translocation once UapA has integrated into the plasma membrane. Our study provides detailed insights into important aspects of the elevator-type transport mechanism and opens novel issues on how the evolution of extended cytosolic tails in eukaryotic transporters, apparently needed for subcellular trafficking, might have been integrated into the transport mechanism.

biophysics↗

bioRxiv2024

Structural basis for bispecific antibody design: arrangement of domain linkage produces activity enhancement

A bispecific antibody (BsAb) is a protein genetically engineered from two different antibodies, allowing simultaneous binding to two kinds of antigen to bring them into close proximity. BsAbs have been developed as anti-cancer drugs that accumulate lymphocytes onto cancer cells by bridging antigens present on each. Ex3 is a bispecific diabody composed of the two fused variable regions (Fvs) of an anti-epidermal growth factor receptor (EGFR) antibody and an anti-CD3 antibody with potent cancer cytotoxic activity. In Ex3, the LH-type, in which the variable regions of the light chain (VLs) are located at the N-terminus of those of the heavy chain (VHs), exerted 1000-fold greater anticancer activity than the HL-type, in which the VHs are located at the N-terminus of the VLs. This effect (termed activity enhancement), in which the activity is greatly enhanced by domain rearrangement, has been reported not only for Ex3 but also for several other BsAbs. However, the molecular details of this activity enhancement have yet to be elucidated. In this study, we determined the cryo-EM structures of Ex3 LH- and HL-types in complex with CD3 and EGFR. Structural comparison of the LH- and HL-types showed that rearrangement of the domain linkage produces drastic structural differences in the overall shape of these complexes, and dynamics attributed to the flexibility between the two Fvs. These findings provide valuable insights into the molecular mechanism for the activity enhancement of BsAbs. This study will be a stepping stone towards establishing a design foundation for BsAb development.

biochemistry↗

bioRxiv2024

Molecular mechanism of α-latrotoxin action

The potent neurotoxic venom of the black widow spider contains a cocktail of seven phylum-specific latrotoxins (LTXs), but only one, -LTX, targets vertebrates. This 130 kDa toxin binds to receptors at presynaptic nerve terminals and triggers a massive release of neurotransmitters. It is widely accepted that LTXs tetramerize and insert into the presynaptic membrane, thereby forming Ca2+-conductive pores, but the underlying mechanism remains poorly understood. LTXs are homologous and consist of an N-terminal region with three distinct domains, along with a C-terminal domain containing up to 22 consecutive ankyrin repeats. Here we report the first high resolution structures of the vertebrate-specific -LTX tetramer in its prepore and pore state. Our structures, in combination with AlphaFold2-based structural modeling and molecular dynamics simulations, reveal dramatic conformational changes in the N-terminal region of the complex. Four distinct helical bundles synchronously rearrange to progressively form a highly stable 15 nm cation-impermeable coiled-coil stalk. This stalk, in turn, positions an N-terminal pair of helices within the membrane, thereby enabling the assembly of a cation-permeable channel. Taken together, these data unveil a unique mechanism for membrane insertion and channel formation, characteristic of the LTX family, and provide the necessary framework for advancing novel therapeutics and biotechnological applications.

biochemistry↗

bioRxiv2022

Structure of the peroxisomal Pex1/Pex6 ATPase complex bound to a substrate

The double-ring AAA+ ATPase Pex1/Pex6 is required for peroxisomal receptor recycling and is essential for peroxisome formation. Pex1/Pex6 mutations cause severe peroxisome associated developmental disorders. Despite its pathophysiological importance, mechanistic details of the heterohexamer are not yet available. Here, we report cryoEM structures of Pex1/Pex6 from Saccharomyces cerevisiae, with an endogenous protein substrate trapped in the central pore of the second ring (D2). Pairs of Pex1/Pex6(D2) subdomains engage the substrate via a staircase of pore-1 loops with distinct properties. The first ring (D1) is catalytically inactive but undergoes significant conformational changes resulting in alternate widening and narrowing of its pore. These events are fueled by ATP hydrolysis in the D2 ring and disengagement of a "twin-seam" Pex1/Pex6(D2) heterodimer from the staircase. Mechanical forces are propagated in a unique manner along Pex1/Pex6 interfaces that are not available in homo-oligomeric AAA-ATPases. Our structural analysis reveals the mechanisms of how Pex1 and Pex6 coordinate to achieve substrate translocation.

molecular biology↗

bioRxiv2022

Asymmetric horseshoe-like assembly of peroxisomal Yeast Oxalyl-CoA synthetase

Oxalyl-CoA synthetase from Saccharomyces cerevisiae is one of the most abundant peroxisomal protein in yeast and hence has become a model to study peroxisomal translocation. It contains a C-terminal Peroxisome Targeting Signal 1, which however is partly dispensable, suggesting additional receptor bindings sites. To unravel any additional features that may contribute to its capacity to be recognized as peroxisomal target, we determined its assembly and overall architecture by an integrated structural biology approach, including X-ray crystallography, single particle cryo-electron microscopy and small angle X-ray scattering. Surprisingly, it assembles into mixture of concentration-dependent dimers, tetramers and hexamers by dimer self-association. Hexameric particles form an unprecedented asymmetric horseshoe-like arrangement, which considerably differs from symmetric hexameric assembly found in many other protein structures. A single mutation within the self-association interface is sufficient to abolish any higher-level oligomerization, resulting in homogenous dimeric assembly. The small C-terminal domain of yeast Oxalyl-CoA synthetase is connected by a partly flexible hinge with the large N-terminal domain, which provides the sole basis for oligomeric assembly. Our data provide a basis to mechanistically study peroxisomal translocation of this target.

molecular biology↗

bioRxiv2021

Structure of a bacterial Rhs effector exported by the type VI secretion system

The type VI secretion system (T6SS) is a widespread protein export apparatus found in Gram-negative bacteria. The majority of T6SSs deliver toxic effector proteins into competitor bacteria. Yet, the structure, function, and activation of many of these effectors remains poorly understood. Here, we present the structures of the T6SS effector RhsA from Pseudomonas protegens and its cognate T6SS spike protein, VgrG1, at 3.3 [A] resolution. The structures reveal that the rearrangement hotspot (Rhs) repeats of RhsA assemble into a closed anticlockwise {beta}-barrel spiral similar to that found in bacterial insecticidal Tc toxins and in metazoan teneurin proteins. We find that the C-terminal toxin domain of RhsA is autoproteolytically cleaved but remains inside the Rhs cocoon where, with the exception of three ordered structural elements, most of the toxin is disordered. The N-terminal plug domain is unique to T6SS Rhs proteins and resembles a champagne cork that seals the Rhs cocoon at one end while also mediating interactions with VgrG1. Interestingly, this domain is also autoproteolytically cleaved inside the cocoon but remains associated with it. We propose that mechanical force is required to remove the cleaved part of the plug, resulting in the release of the toxin domain as it is delivered into a susceptible bacterial cell by the T6SS.

biochemistry↗

bioRxiv2021

Molecular architecture of black widow spider neurotoxins

Latrotoxins (LaTXs) are presynaptic pore-forming neurotoxins found in the venom of Latrodectus spiders. The venom contains a toxic cocktail of seven LaTXs, with one of them targeting vertebrates (-latrotoxin (-LTX)), five specialized on insects (, {beta}, {gamma}, {delta}, {varepsilon}-latroinsectotoxins (LITs), and one on crustaceans (-latrocrustatoxin (-LCT)). LaTXs bind to specific receptors on the surface of neuronal cells, inducing the release of neurotransmitters either by directly stimulating exocytosis or by forming Ca2+-conductive tetrameric pores in the membrane. Despite extensive studies in the past decades, a high-resolution structure of a LaTX is not yet available and the precise mechanism of LaTX action remains unclear. Here, we report cryoEM structures of the -LCT monomer and the {delta}-LIT dimer. The structures reveal that LaTXs are organized in four domains. A C-terminal domain of ankyrin-like repeats shields a central membrane insertion domain of six parallel -helices. Both domains are flexibly linked via an N-terminal -helical domain and a small {beta}-sheet domain. A comparison between the structures suggests that oligomerization involves major conformational changes in LaTXs with longer C-terminal domains. Based on our data we propose a cyclic mechanism of oligomerization, taking place prior membrane insertion. Both recombinant -LCT and {delta}-LIT form channels in artificial membrane bilayers, that are stabilized by Ca2+ ions and allow calcium flux at negative membrane potentials. Our comparative analysis between -LCT and {delta}-LIT provides first crucial insights towards understanding the molecular mechanism of the LaTX family.

biophysics↗

bioRxiv2020

TranSPHIRE: Automated and feedback-optimized on-the-fly processing for cryo-EM

Single particle electron cryomicroscopy (cryo-EM) requires full automation to allow high-throughput structure determination which is especially important for drug discovery research. Although several software packages exist where parts of the cryo-EM pipeline are automated, a complete solution that offers reliable, quality-optimized on-the-fly processing, resulting in a high-resolution three-dimensional reconstruction does not exist. Here we present TranSPHIRE: A software package for fully automated processing of cryo-EM data sets during data acquisition. TranSPHIRE transfers data from the microscope, automatically applies the common pre-processing steps, picks particles, performs 2D clustering, and 3D refinement parallel to image recording. Importantly, TranSPHIRE introduces a machine learning-based feedback loop to re-train its internally used picking model to adapt to any given data set live during processing. This elegant approach enables TranSPHIRE to process data more effectively, producing high-quality particle stacks. TranSPHIRE collects, and displays all microscope settings and metrics generated by its individual tools, in order to allow users to quickly evaluate data during acquisition. TranSPHIRE can run on a single work station and also includes the automated processing of filaments.

biophysics↗

bioRxiv2020

Cryo-EM structure of the fully-loaded asymmetric anthrax lethal toxin in its heptameric pre-pore state

Anthrax toxin is the major virulence factor secreted by Bacillus anthracis, causing high mortality in humans and other mammals. It consists of a membrane translocase, known as protective antigen (PA), that catalyzes the unfolding of its cytotoxic substrates lethal factor (LF) and edema factor (EF), followed by translocation into the host cell. Substrate recruitment to the heptameric PA pre-pore and subsequent translocation, however, are not well understood. Here, we report three high-resolution cryo-EM structures of the fully-loaded anthrax lethal toxin in its heptameric pre-pore state, which differ in the position and conformation of LFs. The structures reveal that three LFs interact with the heptameric PA and upon binding change their conformation to form a continuous chain of head-to-tail interactions. As a result of the underlying symmetry mismatch, one LF binding site in PA remains unoccupied. Whereas one LF directly interacts with a part of PA called -clamp, the others do not interact with this region, indicating an intermediate state between toxin assembly and translocation. Interestingly, the interaction of the N-terminal domain with the -clamp correlates with a higher flexibility in the C-terminal domain of the protein. Based on our data, we propose a model for toxin assembly, in which the order of LF binding determines which factor is translocated first.

molecular biology↗

bioRxiv2019

Towards the molecular architecture of the peroxisomal receptor docking complex

Import of yeast peroxisomal matrix proteins is initiated by cytosolic receptors, which specifically recognize and bind the respective cargo proteins. At the peroxisomal membrane, the cargo-loaded receptor interacts with the docking protein Pex14p that is tightly associated with Pex17p. Previous data suggest that this interaction triggers the formation of an import pore for further translocation of the cargo. The mechanistic principles are however unclear, mainly because structures of higher order assemblies are still lacking. Here, using an integrative approach, we provide the first structural characterization of the major components of the peroxisomal docking complex Pex14p/Pex17p, in a native bilayer environment and reveal its subunit organization. Our data show that three copies of Pex14p and a single copy of Pex17p assemble to form a 20 nm rod-like particle. The different subunits are arranged in a parallel manner, showing interactions along their complete sequences and providing receptor binding-sites on both membrane sides. The long rod facing the cytosol is mainly formed by the predicted coiled-coil domains of Pex14p and Pex17p, possibly providing the necessary structural support for the formation of the import pore. Further implications of Pex14p/Pex17p for formation of the peroxisomal translocon are discussed.

molecular biology↗

bioRxiv2019

Structure of the human BBSome core complex in the open conformation

The BBSome is a heterooctameric protein complex that plays a central role in primary cilia homeostasis. Its malfunction causes the severe ciliopathy Bardet-Biedl syndrome (BBS). The complex acts as a cargo adapter that recognizes signaling proteins such as GPCRs and links them to the intraflagellar transport machinery. The underlying mechanism is poorly understood. Here we present a high-resolution cryo-EM structure of a human heterohexameric core subcomplex of the BBSome. The structure reveals the architecture of the complex in atomic detail. It explains how the subunits interact with each other and how disease-causing mutations hamper this interaction. The complex adopts a conformation that is open for binding to membrane-associated GTPase Arl6 and a large positively charged patch likely strengthens the interaction with the membrane. A prominent negatively charged cleft at the center of the complex is likely involved in binding of positively charged signaling sequences of cargo proteins.

molecular biology↗

bioRxiv2019

Cryo-EM structure of the ClpXP protein degradation machinery

The ClpXP machinery is a two component protease complex performing targeted protein degradation in bacteria and eukaryotes. The complex consists of the AAA+ chaperone ClpX and the peptidase ClpP. The hexameric ClpX utilizes the energy of ATP binding and hydrolysis to engage, unfold and translocate substrates into the catalytic chamber of tetradecameric ClpP where they are degraded. Formation of the complex involves a symmetry mismatch, since hexameric AAA+ rings bind axially to the opposing stacked heptameric rings of the tetradecameric ClpP. Here we present the first high-resolution cryo-EM structure of ClpXP from Listeria monocytogenes. We unravel the heptamer-hexamer binding interface and provide novel insights into the ClpX-ClpP crosstalk and activation mechanism. The comparison with available crystal structures of ClpP and ClpX in different states allows us to understand important aspects of ClpXPs complex mode of action and provides a structural framework for future pharmacological applications.

molecular biology↗

bioRxiv2019

Conserved architecture of Tc toxins from human and insect pathogenic bacteria

Tc toxin complexes use a syringe-like mechanism to penetrate the membrane and translocate a toxic enzyme into the host cytosol. They are composed of three components: TcA, TcB and TcC. Until recently, low-resolution structures of TcA from different bacteria suggested that Tc toxins differ considerably in their architecture and possibly in their mechanism of action. Here, we present high-resolution structures and functional studies of five TcAs from different insect and human pathogenic bacteria. Contrary to previous expectations, their overall composition and domain organization is almost identical. The TcAs assemble as a pentamer with a central -helical channel surrounded by a shell composed of conserved -helical domains and variable {beta}-sheet domains. Essential structural features, including a conserved trefoil protein knot, are present in all five TcAs, suggesting a common mechanism of action. All TcAs form functional pores and can be combined with TcB-TcC subunits from other species resulting in chimeric holotoxins. We have identified a conserved ionic pair that stabilizes the shell, likely operating as a strong latch that only springs open after the destabilization of other regions. Our results lead to new insights into the architecture and host specificity of the Tc toxin family.

molecular biology↗