Christos Gatsogiannis

The AAA-ATPase VCP/p97 unfolds ubiquitylated proteins in key signaling and cellular homeostasis pathways, yet the mechanisms by which Ufd1-Npl4 and FAF1 adapters handle ubiquitin chains to coordinate substrate loading and processing remain unclear. Our cryo-EM study of p97-Ufd1-Npl4 and FAF1 engaging a substrate reveal interactions that drive substrate processing. While Npl4 is capped by distal ubiquitins, and the initiator ubiquitin can be traced from the Npl4 groove to the ATPase channel at near-atomic resolution, we find that the Npl4 tower adaptively responds to the configurational changes in the ATPase motor’s spiral staircase. A helix in FAF1 together with the Ufd1-UT3 domain engage proximal ubiquitins while providing the necessary structural plasticity to accommodate this motion and deliver ubiquitins to Npl4’s groove. Our study reveals the intricate, dynamic interplay between p97, Ufd1-Npl4, and FAF1 in efficient processing of ubiquitylated substrates.

Nonribosomal peptide synthetases (NRPSs) assemble structurally complex natural products but remain challenging targets for high-resolution structural analysis because of their conformational heterogeneity and dynamic catalytic cycles. SacC/SfmC is an unusual single-module NRPS that diverges from canonical peptide-forming assembly lines and instead catalyzes two sequential Pictet–Spengler reactions between a tyrosine-derived unit and a peptidyl aldehyde generated through thioester reduction. This cascade forms two C–C bonds, stereoselectively installs multiple stereogenic centers, and constructs the pentacyclic scaffold of bis-tetrahydroisoquinoline antitumor alkaloids including safracin B and the clinically important ecteinascidins. Here, we establish a state-resolved cryo-electron microscopy (cryo-EM) strategy for structural interrogation of the monomeric noncanonical NRPS SacC and report its thioesterification-state structure at 2.76 Å resolution. Stabilization of this transient catalytic state was achieved by combining the cognate MbtH-like partner protein with a tailored covalent adenosine vinylsulfonamide probe that mimics the aminoacyl-adenylate intermediate and traps the phosphopantetheine-tethered species within the adenylation domain. The structure directly visualizes a carrier protein-tethered intermediate and reveals the spatial organization of the Pictet–Spengler, adenylation, peptidyl carrier protein, and reduction domains, providing the first structural view of a Pictet–Spengler cyclization domain embedded within an NRPS assembly line. In parallel, nonreducible ester probes corresponding to downstream PCP-bound intermediates were developed to enable structural interrogation of additional catalytic states along the SacC reaction trajectory. These results demonstrate that rational chemical probe design enables state-resolved cryo-EM analysis of highly dynamic, single-module NRPSs and establishes a general strategy for capturing catalytic trajectories and enabling the engineering of noncanonical NRPS reactivity.

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. Key Points: - First apo cryo-EM structure of full-length FXIa reveals the unliganded active architecture and exposes the cryptic A3-domain FIX-binding exosite. - Three distinct apo-FXIa conformations and MD simulations reveal catalytic-domain flexibility during the transition from zymogen-like FXI to active FXIa.

Members of the nucleobase ascorbate transporter (NAT) family (SLC23) are elevator-type transporters that are responsible for the uptake of nucleobases and ascorbate. In fungi, NAT members are also responsible for the specific uptake of antifungal nucleobase analogues, such as oxypurinol, allopurinol, or 8-azaguanine. Here, we report nearly full-length cryo-EM structures of UapA, a high-affinity purine transporter from the model fungus Aspergillus nidulans , in inward-facing apo- and substrate-loaded conformations at 2.06 to 3.5 Å in detergent and lipid nanodiscs. The high-resolution structures reveal the role of water molecules and lipids in substrate binding, specificity, transporter dimerization, and activity. Notably, the N-tail of UapA is found to be structured, interacting with both the core and scaffold domains, which in combination with functional data suggests a dual role in trafficking and transport dynamics. Overall, our study provides unprecedented structural and functional insights into an elevator-type fungal transporter, which may well contribute to the exploitation of NAT transporters as specific gateways for targeted pharmacological antifungal approaches.

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 life ^2,3 . 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 sites ^6,8 . Although numerous details regarding pyruvate oxidation steps have been elucidated ⁹ , 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 assemblies ¹⁰ . Our findings provide structural context for the textbook “link reaction” ¹¹ , building on decades of biochemical knowledge, are transferable to functional aspects of other α-ketoacid dehydrogenase complexes, and, ultimately, expand our understanding of primary metabolism as a whole. Abstract Figure:

The evolution of C4 photosynthesis required extensive modification of ancestral enzymes enabling the development of an efficient carbon concentrating mechanism. A key example is NADP-malic enzyme (NADP-ME), which, in maize and sorghum—members of the same C4 lineage—underwent gene duplication and neofunctionalization, resulting in 2 plastidic isoforms with distinct oligomeric states: a tetrameric C4-specific isoform and a dimeric housekeeping (nonC4) isoform. In this study, we resolve the structural basis of this oligomeric divergence using X-ray crystallography, cryo-electron microscopy, and molecular modeling combined with targeted biochemical analysis. Our findings demonstrate that the N-terminal region of nonC4-NADP-ME is involved in its oligomeric organization, whereas a suite of adaptive substitutions at the dimer interface drives the transition to the stable tetramer characteristic of the C4 isoform. Moreover, the C-terminal region stabilizes the oligomeric states of C4- and nonC4-NADP-ME through specific interactions with adaptive residues. We propose that tetramerization mitigates aggregation at the high expression levels demanded by the C4 cycle and likely creates a scaffold for the emergence of regulatory properties. Collectively, the data show that remodeling of terminal domains and inter-subunit interfaces rewires the quaternary architecture of the enzymes, illustrating how subtle structural changes can drive the evolution of complex innovations such as C4 photosynthesis.

The voltage-dependent anion channel (VDAC) is the main gateway for metabolites across the mitochondrial outer membrane. VDAC oligomers are connected to apoptosis induced by various stimuli. However, the mechanistic and structural basis of apoptosis induction by VDAC remains poorly understood. Here, using cryo-EM and NMR we show that VDAC1 oligomerization or confinement in small lipid nanodiscs triggers the exposure of its N-terminal α-helix (VDAC1-N) which becomes available for partner protein binding. NMR and X-ray crystallography data show that VDAC1-N forms a complex with the BH3 binding groove of the anti-apoptotic Bcl2 protein BclxL. Biochemical assays demonstrate that VDAC1-N exhibits a pro-apoptotic function by promoting pore formation of the executor Bcl2 protein Bak via neutralization of BclxL. This mechanism is reminiscent of BH3-only sensitizer Bcl2 proteins that are efficient inducers of Bax/Bak-mediated mitochondrial outer membrane permeabilization and ultimately apoptosis. The VDAC pathway most likely responds to mitochondrial stress or damage.

EMPIAR, the Electron Microscopy Public Image Archive centered at EMBL-EBI, is a public resource for raw electron microscopy images related to EMDB, contains micrographs, particle sets and tilt-series.