Biology subjects

Smits, S. H. J.

Publications and source records attributed to Smits, S. H. J..

8 recordsLinked to original sources

bioRxiv2026

In vitro characterization of the baker's yeast deubiquitinase Ubp3

Ubp3 is one of about 20 deubiquitinases (DUBs) in S. cerevisiae. The current view generally assumes that Ubp3 requires its interaction partner Bre5, which is proposed to function as a positive regulator. Accordingly, the Ubp3/Bre5 complex has been implicated in a broad range of cellular processes for example trafficking between ER and Golgi, stress granule formation and selective autophagy. However, the molecular basis of this proposed Bre5-dependent activity remains unclear. To address this at a molecular level, Ubp3, Bre5, and related constructs were heterologously expressed in E. coli, purified to homogeneity, and characterized in vitro. Both proteins contain folded domains as well as extensive intrinsically disordered regions (IDRs). Despite this structural complexity, the Ubp3/Bre5 complex could be isolated following either co-expression in vivo or after in vitro assembly. Unexpectedly, complex formation with Bre5 was not required for the catalytic activity of full length Ubp3. Furthermore, even the isolated catalytic domain of Ubp3 was fully active against two distinct substrates in the absence of Bre5, demonstrating that its deubiquitinating activity is intrinsically independent of Bre5. These findings indicate that the catalytic domain alone is sufficient for substrate cleavage, whereas the extensive IDRs of Ubp3 and its cofactor Bre5 might contribute to substrate recognition or specificity. Overall, this study challenges the prevailing model of Bre5-dependent activation of Ubp3 and provides new insights into the molecular organization of the Ubp3/Bre5 system. More broadly, it highlights the importance of intrinsically disordered regions in regulating deubiquitinase function and cellular signaling networks.

biochemistry↗

bioRxiv2025

Mechanistic Insights into the Structural Asymmetry of the LanFEG Transporter NisFEG in Lantibiotic Immunity

Nisin is one of the best studied antimicrobial peptides. Still, how nisin-producing strains can protect themselves against nisins bactericidal effects is only partially understood. Located within the nisin biosynthesis operon, the heterotetrameric ABC transporter NisFEG transports nisin to the extracellular environment, granting autoimmunity to the producer strain. NisFEG belongs to the LanFEG family of ABC transporters, members of which are found in some lantibiotic-producing bacterial strains. However, their structure has not been elucidated. In this work, we constructed a full atom model of NisFEG in the ATP-bound conformation. The architecture of the complex reveals a narrow contact interface between the two transmembrane chains, with prominent lateral clefts, similar to those observed in other exporters of hydrophobic compounds. Through molecular dynamics (MD) simulations, we observed that one of the most conserved elements of the LanFEG family, the E-loop of the nucleotide-binding domain, interacts preferentially with a small intracellular helix of the NisG transmembrane chain. By combining co-solvent MD simulations and predictions of the binding mode of the terminal segment of nisin, we could identify a putative interaction surface, located predominantly on NisE. Our results suggest that nisin extrusion operates in an asymmetric manner, where contacts between the E-loop and NisG are the driving force for the conformational changes triggered by ATP hydrolysis, whereas the NisE subunit is the main mediator of interactions with the lantibiotic. This functional asymmetry could explain why the LanFEG family has evolved two distinct transmembrane chains, where each one was selected to perform a single step in an optimal way, maximizing the immunity of lantibiotic-producing bacteria.

biophysics↗

bioRxiv2025

The Paulinella chromatophore transit peptide part2 adopts a structural fold similar to the γ-glutamyl-cyclotransferase fold

The chromatophores of the cercozoan amoeba Paulinella are photosynthetic organelles that evolved from a cyanobacterial endosymbiont. Many nucleus-encoded chromatophore-targeted proteins carry unusual N-terminal targeting signals termed crTPs. The crTPs are bipartite. Whereas crTPpart1 that likely mediates trafficking through the secretory pathway is cleaved off during import, crTPpart2 remains attached to its cargo protein. The function of crTPpart2 is unknown. To contribute to unravel the functional role of crTPpart2, here we elucidated the structures of crTPpart2 from two different chromatophore-targeted proteins by X-ray crystallography at [~]2.3 [A] resolution. Interestingly, the crTPpart2 of both proteins adopts a structural fold. Both structures share a conserved structured core and a flexible N-terminal arm. The structured core resembles proteins of the {gamma}-glutamyl cyclotransferase superfamily within which crTPpart2 structures form a novel protein (sub)-family. The proposed catalytic center typical for proteins with cyclotransferase activity is not conserved in crTPpart2. A Cys pair that is conserved in crTPpart2 of many chromatophore-targeted proteins has been captured as disulfide bridge. Together, our data suggests that chromatophore-targeted proteins are imported in their folded state and that the fold adopted by crTPpart2 plays a functional role during import. The characterization of its structure and flexibility provides important steps towards elucidating this novel protein translocation mechanism.

microbiology↗

bioRxiv2024

Jasmonic acid signalling is targeted by a smut fungal Tin2-fold effector

In plants, jasmonate signaling is a hub integrating environmental cues with growth and development. Due to its role in balancing defense responses against pathogens, it is a target of effector proteins from various pathogens. Here, we characterized the fungal effector protein Tue1 from the Brassicaceae smut fungus Thecaphora thlaspeos. T. thlaspeos naturally infects Arabis hirsuta but can also colonize the non-host Arabidopsis thaliana. In planta, the fungal protein Tue1 hijacks the plant importin- dependent nuclear transporter to reach the plant nucleus. It interacts with jasmonate ZIM domain 10 (JAZ10) proteins of both A. thaliana and Ar. hirsuta. Structure-guided analysis of Tue1 suggests that it binds the Jas motif of JAZ10 indicating a role in stabilization or binding competition with proteins like MYC3 and COI1. A subset of jasmonate-responsive genes is differentially regulated during T. thlaspeos infection, proposing a link of the Tue1 function to infection. Tue1 share structural similarity to the Tin2-fold family recently described in the corn smut Ustilago maydis. Our study therefore suggests that this structural effector family is expanded across fungal pathogens, although future studies have to reveal whether targeting JAZ-repressors is a conserved mechanism or specifically acquired as an adaptation to its perennial host.

biochemistry↗

bioRxiv2024

Full-length structure of the host targeted bacterial effector Bep1 reveals a novel structural domain conserved in FIC effector proteins from Bartonella

Bacterial effector proteins translocated via a type-IV secretion system (T4SS) typically harbor a C-terminal segment required for recognition by the type-IV secretion coupling protein 1. In the -proteobacterial pathogen Bartonella, the signal is bipartite being composed of a BID (Bep intracellular delivery) domain and a positively charged C-terminal tail 2. Here, we show the crystal structure of full length Bartonella effector protein 1 (Bep1), which shows a novel FIC - OB - BAS(BID) domain arrangement conserved in the majority of Beps with the BID domain inserted into the newly discovered BAS parent domain. We propose that the BAS domain is necessary for the overall "boomerang"-like shape of Bep1 and that it plays a role during translocation through the T4SS.

microbiology↗

bioRxiv2022

The first archaeal PET-degrading enzyme belongs to the feruloyl-esterase family

Polyethylene terephthalate (PET) is a commodity polymer known to globally contaminate marine and terrestrial environments. Today, around 40 bacterial and fungal PET-active enzymes (PETases) are known, originating from four bacterial and two fungal phyla. In contrast, no archaeal enzyme has been identified to degrade PET. Here we report on the structural and biochemical characterization of PET46, an archaeal promiscuous feruloyl esterase exhibiting degradation activitiy on PET, bis-, and mono-(2-hydroxyethyl) terephthalate (BHET and MHET). The enzyme, found by a sequence-based metagenome search, was derived from a non-cultivated, deep-sea Candidatus Bathyarchaeota archaeon. Biochemical characterization demonstrated that PET46 is a promiscuous, heat-adapted hydrolase. Its crystal structure was solved at a resolution of 1.71 [A]. It shares the core alpha/beta-hydrolase fold with bacterial PETases, but contains a unique lid common in feruloyl esterases, which is involved in substrate binding. Thus, our study significantly widens the currently known diversity of PET-hydrolyzing enzymes, by demonstrating PET depolymerization by a lignin-degrading esterase.

biochemistry↗

bioRxiv2021

Biophysical and pharmacokinetic characterization of a small-molecule inhibitor of RUNX1/ETO tetramerization with anti-leukemic effects

Acute myeloid leukemia (AML) is a malignant disease of immature myeloid cells and the most prevalent acute leukemia among adults. The oncogenic homo-tetrameric fusion protein RUNX1/ETO results from the chromosomal translocation t(8;21) and is found in AML patients. The nervy homology region 2 (NHR2) domain of ETO mediates tetramerization; this oligomerization is essential for oncogenic activity. Previously, we identified the first-in-class small-molecule inhibitor of NHR2 tetramer formation, 7.44, which was shown to specifically interfere with NHR2, restore gene expression down-regulated by RUNX1/ETO, inhibit the proliferation of RUNX1/ETO-depending SKNO-1 cells, and reduce the RUNX1/ETO-related tumor growth in a mouse model. However, no biophysical and structural characterization of 7.44 binding to the NHR2 domain has been reported. Likewise, the compound has not been characterized as to physicochemical, pharmacokinetic, and toxicological properties. Here, we characterize the interaction between the NHR2 domain of RUNX1/ETO and 7.44 by biophysical assays and show that 7.44 interferes with NHR2 tetramer stability and leads to an increase in the dimer population of NHR2. The affinity of 7.44 with respect to binding to NHR2 is Klig = 3.95 {+/-} 1.28 M. By NMR spectroscopy combined with molecular dynamics simulations, we show that 7.44 binds with both heteroaromatic moieties to NHR2 and interacts with or leads to conformational changes in the N-termini of the NHR2 tetramer. Finally, we demonstrate that 7.44 has favorable physicochemical, pharmacokinetic, and toxicological properties. Together with biochemical, cellular, and in vivo assessments, the results reveal 7.44 as a lead for further optimization towards targeted therapy of t(8;21) AML.

biophysics↗

bioRxiv2021

Structure and efflux mechanism of the yeast pleiotropic drug resistance transporter Pdr5

Pdr5, a member of the extensive ABC transporter superfamily, is representative of a clinically relevant subgroup involved in pleiotropic drug resistance. Through the coupling of nucleotide hydrolysis with drug efflux, Pdr5 homologues enable pathogenic species to survive in the presence of chemically diverse antifungal agents. Our structural and functional results reveal details of an ATP-driven conformational cycle, which mechanically drives drug translocation through an amphipathic channel, and a clamping switch within a conserved linker loop that acts as a nucleotide sensor. One half of the transporter remains nearly invariant throughout the cycle, while its partner undergoes changes that are transmitted across interdomain interfaces to support a peristaltic motion of the pumped molecule. The efflux model proposed here rationalises the pleiotropic impact of Pdr5 and opens avenues for the development of effective antifungal compounds.

microbiology↗