Tiago N. Cordeiro

Hub proteins are highly connected nodes in protein-protein interaction networks and are often intrinsically disordered proteins (IDPs) or contain intrinsically disordered regions. In Bacillus subtilis, the morphogenesis of the spore surface is orchestrated by a set of so-called morphogenetic proteins that guide the assembly of distinct layers. Formation of the inner coat is directed by SafAFL and its shorter isoform, C30. Both are expressed early in sporulation under the control of σE and localize at the interface between the developing inner coat and the underlying cortex peptidoglycan. From this site, they act as organizational hubs, recruiting client proteins essential for coat maturation. Among these is Tgl, a transglutaminase synthesized later in development following activation of σK after engulfment completion. We show that the C30 domain exhibits IDP-like features yet self-assembles into >1200 kDa complexes stabilized by disulfide bonds and that these bonds are required for subsequent proper Tgl-mediated “spotwelding” cross-linking. Small-angle X-ray scattering (SAXS) and photobleaching show that Tgl immobilizes but does not drastically alter these assemblies. These findings support a hierarchical, biphasic model for inner coat assembly: initial self-assembly and disulfide stabilization, followed by Tgl-mediated cross-linking and structural stabilization. According to this model, the forms of SafAFL/C30 that dominate the two stages recruit different client proteins in register with the course of morphogenesis.

Decoupling substrate binding from catalysis remains a central challenge in mechanistic enzymology, particularly for metalloenzymes in which metal cofactors intrinsically couple molecular recognition to electron transfer. Here, we introduce a hybrid nuclear magnetic resonance (NMR) strategy that isolates binding thermodynamics by exploiting metal-free (apo) variants of multicopper oxidases that abolish turnover while retaining structural integrity and binding competence. By combining ligand-based saturation transfer difference (STD) NMR with protein-centered methionine-selective methyl NMR, we resolve substrate-binding interactions at atomic resolution and directly interrogate how dynamic structural elements shape molecular recognition independently of catalysis. Using the metallo-oxidase McoA, its evolved laccase-like variant 2F4, and their Met-looptruncated counterparts, ligand-centered NMR reveals a conserved aromatic binding epitope for the reporter substrate ABTS across all enzyme backgrounds, while distinguishing specific contacts from transient, nonproductive encounters. Quantitative analysis shows that 2F4 binds ABTS more weakly than wild-type McoA despite exhibiting higher catalytic activity, indicating that directed evolution enhances turnover rather than binding. Methionine-selective methyl NMR identifies substrate-induced perturbations in the Met-loop and adjacent surface residues consistent with heterogeneous, multi-site binding. NMR-guided filtering of ensemble docking models supports two adjacent substrate-access cavities that accommodate multiple transient ligand orientations, validating mechanistic features previously inferred only 2 computationally. Collectively, these results define substrate recognition in multicopper oxidases as tunable, with the Met-loop modulating accessibility, promiscuity, and conformational sampling rather than dictating catalytic chemistry. By decoupling binding from catalysis and quantifying loop-modulated interactions, this hybrid NMR framework provides a generalizable strategy for dissecting substrate-recognition mechanisms in multicopper oxidases and other loop-regulated metalloenzymes.

Hub proteins are highly connected nodes in protein-protein interaction networks and are often intrinsically disordered proteins (IDPs) or contain intrinsically disordered regions. In Bacillus subtilis , the morphogenesis of the spore surface is orchestrated by a set of so-called morphogenetic proteins that guide the assembly of distinct layers. Formation of the inner coat is directed by SafA ^FL and its shorter isoform, C30. Both are expressed early in sporulation under the control of σ ^E and localize at the interface between the developing inner coat and the underlying cortex peptidoglycan. From this site, they act as organizational hubs, recruiting client proteins essential for coat maturation. Among these is Tgl, a transglutaminase synthesized later in development following activation of σ ^K after engulfment completion. We show that the C30 domain exhibits IDP-like features yet self-assembles into >1200 kDa complexes stabilized by disulfide bonds and that these bonds are required for subsequent proper Tgl-mediated “spotwelding” cross-linking. Small-angle X-ray scattering (SAXS) and photobleaching show that Tgl immobilizes but does not drastically alter these assemblies. These findings support a hierarchical, biphasic model for inner coat assembly: initial self-assembly and disulfide stabilization, followed by Tgl-mediated cross-linking and structural stabilization. According to this model, the forms of SafA ^FL /C30 that dominate the two stages recruit different client proteins in register with the course of morphogenesis.

Motivation: Intrinsically disordered proteins (IDPs) and regions (IDRs) challenge structural characterization due to their dynamic conformational ensembles and lack of stable structure. Existing computational approaches for modelling these ensembles are either computationally intensive, limited in flexibility, or inaccessible to non-experts, especially when dealing with multi-domain or multi-chain proteins. Results: We present Ensemblify, an open-source, user-friendly Python package for generating and analyzing conformational ensembles of IDPs/IDRs. Ensemblify uses a Monte Carlo algorithm coupled with neighbour-aware sampling of dihedral angles from curated or user-defined fragment libraries to explore conformational space. It directly incorporates information from AlphaFold’s confidence metrics as flexible energy restraints in PyRosetta to guide the sampling. It supports multi-chain and multi-domain proteins and can sample N-terminal, C-terminal, and inter-domain linkers while preserving folded regions. Ensemble quality can be validated and refined against experimental data such as SAXS via Bayesian/Maximum Entropy (BME) reweighting. Interactive dashboards provide in-depth structural analysis and comparison. Testing across 10 diverse proteins demonstrated Ensemblify’s accuracy, flexibility, and ability to recover experimentally observed structural features. Incorporating AlphaFold confidence metrics shows potential to improve the ensemble-data agreement. Availability: Ensemblify is freely available at https://github.com/CordeiroLab/ensemblify , along with detailed installation instructions and usage tutorials. Ensemblify can be used for scripting through its Python API or directly through the provided command-line interface (CLI). Complete documentation is available within the source-code and CLI and on Ensemblify’s official documentation page ( https://ensemblify.readthedocs.io ). Contact: tiago.gomes@itqb.unl.pt , tiago.cordeiro@itqb.unl.pt Supplementary information: Supplementary data is available online.

The SARS-CoV-2 nucleocapsid (N) protein is essential for the viral life cycle, facilitating RNA packaging, replication, and host-cell interactions. Its ability to self-assemble and undergo phase separation is critical for these functions but remains poorly understood. Using an integrated approach combining small-angle X-ray scattering (SAXS), nuclear magnetic resonance spectroscopy, computational modeling, and biophysical assays, we uncover key mechanisms underpinning N-protein’s dynamic self-assembly. We show that the N-protein’s interdomain linker (IDL) contains a conserved coiled-coil (CC) motif that drives transient interactions between protein subunits, enabling the formation of progressively larger complexes at higher concentrations. SAXS analysis and ensemble modeling reveal that the IDL exists in a concentration-dependent equilibrium between monomeric, dimeric, and trimeric states. The CC motif facilitates parallel, head-to-head oligomerization of N-protein dimers, transitioning between compact (closed) and extended (open) configurations depending on the interaction network within the IDL. This linker-driven assembly modulates phase separation, impacting the size, stability, and dynamics of biomolecular condensates. Here, we present the most comprehensive conformational landscape analysis of the N-protein to date, providing a detailed model of its self-assembly and phase separation. Our findings highlight how the structural plasticity of the IDL and CC-mediated interactions are pivotal to its roles in the SARS-CoV-2 life cycle.

The SARS-CoV-2 nucleocapsid (N) protein is essential for the viral lifecycle, facilitating RNA packaging, replication, and host-cell interactions. Its ability to self-assemble and undergo liquid-liquid phase separation (LLPS) is critical for these functions but remains poorly understood. Using an integrated approach combining small-angle X-ray scattering (SAXS), nuclear magnetic resonance (NMR) spectroscopy, computational modeling, and complementary biophysical assays, we uncover key mechanisms underpinning N-protein's dynamic self-assembly. We show that the N-protein's interdomain linker (IDL) contains a conserved coiled-coil (CC) motif that drives transient interactions between protein subunits, enabling the formation of progressively larger complexes at higher concentrations. SAXS analysis and ensemble modeling reveal that the IDL exists in a concentration-dependent equilibrium between monomeric, dimeric, and trimeric states. The CC motif facilitates parallel, head-to-head oligomerization of N-protein dimers, transitioning between compact (closed) and extended (open) configurations depending on the interaction network within the IDL. This linker-driven assembly modulates LLPS, impacting the size, stability, and dynamics of biomolecular condensates. Here, we present the most comprehensive conformational landscape analysis of the N-protein to date, providing a detailed model of its self-assembly and LLPS. Our findings highlight how the structural plasticity of the IDL and CC-mediated interactions are pivotal to its roles in the SARS-CoV-2 lifecycle.

The translocated intimin receptor (Tir) is an essential type III secretion system (T3SS) effector of attaching and effacing pathogens contributing to the global foodborne disease burden. Tir acts as a cell-surface receptor in host cells, rewiring intracellular processes by targeting multiple host proteins. We investigated the molecular basis for Tir’s binding diversity in signalling, finding that Tir is a disordered protein with host-like binding motifs. Unexpectedly, also are several other T3SS effectors. By an integrative approach, we reveal that Tir dimerises via an antiparallel OB-fold within a highly disordered N-terminal cytosolic domain. Also, it has a long disordered C-terminal cytosolic domain partially structured at host-like motifs that bind lipids. Membrane affinity depends on lipid composition and phosphorylation, highlighting a previously unrecognised host interaction impacting Tir-induced actin polymerisation and cell death. Furthermore, multi-site tyrosine phosphorylation enables Tir to engage host SH2 domains in a multivalent fuzzy complex, consistent with Tir’s scaffolding role and binding promiscuity. Our findings provide insights into the intracellular Tir domains, highlighting the ability of T3SS effectors to exploit host-like protein disorder as a strategy for host evasion.