Tea Pavkov-Keller

Structural predictions have reached unprecedented accuracy. They leverage sequence‐specific data to capture all potential interactions a sequence has evolved to fulfill. AlphaFold derives information from three sources: learned parameters capturing intrinsic amino acid secondary structure and environment propensity; models of related proteins providing structural templates; and aligned sequences encoding profiles and concerted evolutionary changes of residues involved in contacts. However, function demands dynamic changes; hence not all possible interactions can coexist simultaneously. Comprehensive information entails contradictions, which resolved in favor of the better‐informed structure will silence less stable states and associations. Here, we introduce a method using all three channels to include prior knowledge: site‐specific variants, predefined alignments and templates. Selecting information relevant to a particular state delimits the functional context of a prediction. Our program VAIRO allows us to rescue asymmetric and weaker interactions to complete the view of molecular assemblies in the architecture of a bacterial surface layer, and reveals otherwise inaccessible dynamic states in a pneumococcal multimeric membrane protein complex. VAIRO is distributed via the python package index (PyPI) ( https://pypi.org/project/vairo ) and the code is also available on Github ( https://github.com/arcimboldo-team/vairo ).

Type IV secretion systems (T4SS) are found in both monoderm and diderm bacteria. The broad-host-range conjugative plasmid pIP501 from Enterococcus faecalis harbors a T4SS encoding 15 tra genes responsible for the spread of antimicrobial resistance genes among diverse G+ pathogens. Eight Tra proteins (TraB, TraC_B3, TraF, TraH_B8, TraI, TraK, TraL_B6, and TraM_B8) are postulated to form the mating pair formation (MPF) complex representing the central DNA translocation pore. One of these proteins is TraF, a 52.8 kDa transmembrane protein which lacks any homologs in other well described T4SSs. In this study, TraF was proven to be an essential conjugative transfer protein. The TraF pulldown co-eluted all Tra proteins except TraG_B1 and TraN. Bacterial-two-hybrid assay showed a strong interaction between TraF and TraM_B8. We present a 1.25 Å resolution crystal structure of the N-terminal domain of TraF, which adopts a pseudokinase fold. AlphaFold predictions of full-length TraF with membrane mimetics show a transmembrane protein with two distinct soluble domains. FoldSeek revealed a strong similarity to YukC (EssB), a transmembrane pseudokinase from type VII secretion system (T7SS). YukC was shown to function as an interaction hub by mediating contacts between its pseudokinase domain and other T7SS proteins as part of the central membrane core complex. We postulate that TraF might play an important role in T4SS complex formation.

Background: Allergic sensitization to cats and other furry animals is a major cause of asthma and allergic rhinitis in more than 200 million people worldwide. According to the frequency of IgE recognition, allergen‐specific IgE levels, and allergenic activity, Fel d 4 is a major allergen in the cat ( Felis domesticus ). The lipocalin allergen Fel d 4 is highly homologous to dog (Can f 6) and major horse (Equ c 1) allergens. Accordingly, IgE cross‐reactivity to these allergens contributes to polysensitization and allergic responses upon exposure to different animals. Methods: Fel d 4 was recombinantly produced in two systems, E. coli and Expi293F mammalian cells. Recombinant forms were characterized by circular dichroism and mass spectrometry. The Fel d 4 3D structure was determined using X‐ray crystallography. Immunoreactivity, epitope analyses, and cross‐reactive properties were assessed by ELISA and basophil release assays using allergic patients’ sera. Results: We reveal the rFel d 4 crystal structures and demonstrate that mammalian cells produce an N‐glycosylated recombinant Fel d 4 allergen. The C‐terminal regions of Fel d 4, Can f 6, and Equ c 1 constitute conformational IgE ‐epitope‐containing areas responsible for cross‐reactivity. Conclusion: Uncovering the IgE ‐binding sites of Fel d 4 and cross‐reactive allergens contributes to future rational design of active and passive allergen‐specific treatment forms.

Transportin 3 (TNPO3) is a nuclear import receptor known for its broad substrate specificity, often recognizing arginine-serine (SR/RS) repeat-rich nuclear localization signals (NLS) in SRSF proteins. While serine phosphorylation or glutamate presence has been associated with these NLSs, recent proteomic studies identified TNPO3 cargoes lacking SR/RS repeats. One such example is the cold-inducible RNA-binding protein (CIRBP), which contains a non-classical RSY-NLS. Using X-ray crystallography, here we investigate the TNPO3-CIRBP interaction and find that tyrosines within the RSY-NLS play a key role in binding, independent of phosphorylation. Surprisingly, serine and tyrosine phosphorylation in CIRBP’s NLS inhibits TNPO3 binding, suggesting a regulatory mechanism for nuclear import. Our study reveals a non-conventional nuclear import mechanism mediated by TNPO3, which may extend to other known or yet undiscovered TNPO3 cargoes.

Symmetry in nature often emerges from self-assembly processes and serves a wide range of functions. Cell surface layers (S-layers) form symmetrical lattices on many bacterial and archaeal cells, playing essential roles such as facilitating cell adhesion, evading the immune system, and protecting against environmental stress. However, the experimental structural characterization of these S-layers is challenging due to their self-assembly properties and high sequence variability. In this study, we introduce the SymProFold pipeline, which utilizes the high accuracy of AlphaFold-Multimer predictions to derive symmetrical assemblies from protein sequences, specifically focusing on two-dimensional S-layer arrays and spherical viral capsids. The pipeline tests all known symmetry operations observed in these systems (p1, p2, p3, p4, and p6) and identifies the most likely symmetry for the assembly. The predicted models were validated using available experimental data at the cellular level, and additional crystal structures were obtained to confirm the symmetry and interfaces of several SymProFold assemblies. Overall, the SymProFold pipeline enables the determination of symmetric protein assemblies linked to critical functions, thereby opening possibilities for exploring functionalities and designing targeted applications in diverse fields such as nanotechnology, biotechnology, medicine, and materials and environmental sciences.

Flexible Substrataufnahme aus Nährstoffreservoirs ist für Mikroorganismen entscheidend, um sich in kompetitiven natürlichen Umgebungen durchzusetzen. Zur Aufnahme von Glucose aus verschiedenen Glucosid‐ und Disaccharid‐Substraten haben viele frei‐ und symbiotisch lebende Bakterien, neben der Hydrolyse, einen nicht‐hydrolytischen Stoffwechselweg entwickelt. Dieser besteht aus vier biochemischen Schritten und wird von einem distinkten Genlocus (glycoside utilization locus, GUL) codiert. Mechanistisch integriert der Stoffwechselweg die Oxidation und Reduktion am Glucosyl/Glucose‐C3 mit der eliminativen Spaltung der glycosidischen Bindung und der Addition von Wasser an das Eliminationsprodukt in zwei aufeinanderfolgenden, von Lyasen katalysierten Reaktionen. In dieser Studie zeigen wir, basierend auf der Untersuchung von Enzymen des Pflanzenpathogens Agrobacterium tumefaciens, ein konserviertes Aktivitätszentrum in beiden Lyasen, welches um ein zentrales Mn²⁺ Metallion angeordnet ist. Weiters identifizieren wir die strukturellen Voraussetzungen für die spezifische Katalyse der 3‐Ketoglucosid Eliminierung zu 2‐Hydroxy‐3‐ketoglycal und der Hydratisierung des Glycal zu 3‐Ketoglucose. Die Erweiterung unserer Suche nach GUL‐codierten putativen Lyasen auf das menschliche darmkommensale Bakterium Bacteroides thetaiotaomicron führte uns zur Entdeckung eines Ca²⁺‐koordinierten Aktivitätszentrums in einem Glycosidase‐ähnlichen Protein. Das Enzym katalysiert die eliminative Spaltung von 3‐Ketoglucosiden entgegengesetzter (α) anomerer Konfiguration im Vergleich zu den von der A. tumefaciens Lyase präferierten Substraten (β). Strukturelle und biochemische Vergleiche zeigen die molekular‐mechanistische Basis der Stereokomplementarität der beiden 3‐Ketoglucosid Lyasen. Unsere Ergebnisse identifizieren ein grundlegendes Set von GUL‐codierten Lyasen für den Glucosidmetabolismus und erlauben es, der Gendiversität von GULs in der Domäne von Bakterien physiologische Bedeutung zuzuschreiben.

The flexible acquisition of substrates from nutrient pools is critical for microbes to prevail in competitive environments. To acquire glucose from diverse glycoside and disaccharide substrates, many free‐living and symbiotic bacteria have developed, alongside hydrolysis, a non‐hydrolytic pathway comprised of four biochemical steps and conferred from a single glycoside utilization gene locus (GUL). Mechanistically, this pathway integrates within the framework of oxidation and reduction at the glucosyl/glucose C3, the eliminative cleavage of the glycosidic bond and the addition of water in two consecutive lyase‐catalyzed reactions. Here, based on study of enzymes from the phytopathogen Agrobacterium tumefaciens, we reveal a conserved Mn²⁺ metallocenter active site in both lyases and identify the structural requirements for specific catalysis to elimination of 3‐keto‐glucosides and water addition to the resulting 2‐hydroxy‐3‐keto‐glycal product, yielding 3‐keto‐glucose. Extending our search of GUL‐encoded putative lyases to the human gut commensal Bacteroides thetaiotaomicron, we discover a Ca²⁺ metallocenter active site in a putative glycoside hydrolase‐like protein and demonstrate its catalytic function in the eliminative cleavage of 3‐keto‐glucosides of opposite (α) anomeric configuration as preferred by the A. tumefaciens enzyme (β). Structural and biochemical comparisons reveal the molecular‐mechanistic origin of 3‐keto‐glucoside lyase stereo‐complementarity. Our findings identify a basic set of GUL‐encoded lyases for glucoside metabolism and assign physiological significance to GUL genetic diversity in the bacterial domain of life.

S-layers are crystalline arrays found on bacterial and archaeal cells. Lactobacillus is a diverse family of bacteria known especially for potential gut health benefits. This study focuses on the S-layer proteins from Lactobacillus acidophilus and Lactobacillus amylovorus common in the mammalian gut. Atomic resolution structures of Lactobacillus S-layer proteins SlpA and SlpX exhibit domain swapping, and the obtained assembly model of the main S-layer protein SlpA aligns well with prior electron microscopy and mutagenesis data. The S-layer’s pore size suggests a protective role, with charged areas aiding adhesion. A highly similar domain organization and interaction network are observed across the Lactobacillus genus. Interaction studies revealed conserved binding areas specific for attachment to teichoic acids. The structure of the SlpA S-layer and the suggested incorporation of SlpX as well as its interaction with teichoic acids lay the foundation for deciphering its role in immune responses and for developing effective treatments for a variety of infectious and bacteria-mediated inflammation processes, opening opportunities for targeted engineering of the S-layer or lactobacilli bacteria in general.