Competitive effects of salt and surfactant on the structure of nanoparticles in a binary system of nanoparticle and protein.

Small-angle neutron scattering (SANS) and dynamic light scattering (DLS) experiments have been carried out to study the competitive effects of NaCl and sodium dodecyl sulfate (SDS) surfactant on the evolution of the structure and interactions in a silica nanoparticle-Bovine serum albumin (BSA) protein system. The unique advantage of contrast-matching SANS has been utilized to particularly probe the structure of nanoparticles in the multi-component system. Silica nanoparticles and BSA protein both being anionic remain largely individual in the solution without significant adsorption. The non-adsorbing nature of protein is known to cause depletion attraction between nanoparticles at higher protein concentrations. The nanoparticles undergo immediate aggregation in the nanoparticle-BSA system on the addition of a small amount of salt [referred as the critical salt concentration (CSC)], much less than that required to induce aggregation in a pure nanoparticle dispersion. The salt ions screen the electrostatic repulsion between the nanoparticles, whereby the BSA-induced depletion attraction dominates the system and contributes to the nanoparticle aggregation of a mass fractal kind of morphology. Further, the addition of SDS in this system interestingly suppresses nanoparticle aggregation for salt concentrations lower than the CSC. The presence of SDS gives rise to additional electrostatic repulsion in the system by binding with the BSA protein via electrostatic and hydrophobic interactions. For salt concentrations higher than the CSC, the formation of clusters of nanoparticles is inevitable even in the presence of protein-surfactant complexes, but the mass fractal kind of branched aggregates transform to surface fractals. This has been attributed to the BSA-SDS complex induced depletion attraction along with salt-driven screening of electrostatic repulsion. Thus, the interplay of depletion and electrostatic and hydrophobic interactions has been utilized to tune the structures formed in a multicomponent silica nanoparticle-BSA-SDS/NaCl system.

Tuning of silica nanoparticle-lysozyme protein complexes in the presence of the SDS surfactant.

The structures of the complexes of anionic silica nanoparticle (size ∼ 16 nm)-lysozyme (cationic) protein, tuned by the addition of the anionic surfactant sodium dodecyl sulfate (SDS), have been investigated by dynamic light scattering (DLS) and small-angle neutron scattering (SANS). The unique advantage of contrast variation SANS has been used to probe the role of individual components in binary and ternary systems. The cationic lysozyme protein (at pH ∼ 7) adsorbs on the anionic silica nanoparticles and forms mass fractal aggregates due to the strong attractive interaction, whereas similarly charged SDS does not interact physically with silica nanoparticles. The presence of SDS, however, remarkably affects the nanoparticle-protein interactions via binding with the oppositely charged segments of lysozyme. In general, the SDS-lysozyme complexes possess a variety of structures (e.g., insoluble complexes of Ly(DS)8, crystalline structure, or micelle-like structure) depending on the surfactant-to-protein molar ratio (S/P). In the ternary system (HS40-lysozyme-SDS), lysozyme preferentially binds with SDS, instead of directly to nanoparticles. At low S/Ps (0 ≤ S/P ≤ 10), the SDS concentration is not enough to fully neutralize the charge of lysozyme, leading to the formation of cationic SDS-lysozyme complex-mediated nanoparticle aggregation. The morphology of the nanoparticle-(lysozyme-SDS) complexes is also found to be mass fractal kind where the fractal dimension increases with increasing SDS concentration. At S/P > 10, there is sufficient SDS to fully neutralize the lysozyme in the absence of competing charges from the particle but it is at S/P = 50 before all lysozyme desorbs from the particle and binds completely to the overwhelming amount of SDS, creating an oppositely charged lysozyme-SDS complex, which is repelled from the particle.

Structural basis of the zinc-induced cytoplasmic aggregation of the RNA-binding protein SFPQ.

SFPQ is a ubiquitous nuclear RNA-binding protein implicated in many aspects of RNA biogenesis. Importantly, nuclear depletion and cytoplasmic accumulation of SFPQ has been linked to neuropathological conditions such as Alzheimer's disease (AD) and amyotrophic lateral sclerosis (ALS). Here, we describe a molecular mechanism by which SFPQ is mislocalized to the cytoplasm. We report an unexpected discovery of the infinite polymerization of SFPQ that is induced by zinc binding to the protein. The crystal structure of human SFPQ in complex with zinc at 1.94 Å resolution reveals intermolecular interactions between SFPQ molecules that are mediated by zinc. As anticipated from the crystal structure, the application of zinc to primary cortical neurons induced the cytoplasmic accumulation and aggregation of SFPQ. Mutagenesis of the three zinc-coordinating histidine residues resulted in a significant reduction in the zinc-binding affinity of SFPQ in solution and the zinc-induced cytoplasmic aggregation of SFPQ in cultured neurons. Taken together, we propose that dysregulation of zinc availability and/or localization in neuronal cells may represent a mechanism for the imbalance in the nucleocytoplasmic distribution of SFPQ, which is an emerging hallmark of neurodegenerative diseases including AD and ALS.

Whaddaya Know: A Guide to Uncertainty and Subjectivity in Structural Biology.

The methods of structural biology, while powerful, are technically complex. Although the Protein Data Bank (PDB) provides a repository that allows anyone to download any structure, many users would not appreciate the caveats that should be considered when examining a structure. Here, we describe several key uncertainties associated with the application of X-ray crystallography, NMR spectroscopy, single-particle electron microscopy (SPEM), and small-angle scattering (SAS) to biological macromolecules. The take-home message is that structures are not absolute truths - they are models that fit the experimental data and therefore have uncertainty and subjectivity associated with them. These uncertainties must be appreciated - careful reading of the associated paper, and any validation report provided by the structure database, is highly recommended.

Disulfide isomerase activity of the dynamic, trimeric Proteus mirabilis ScsC protein is primed by the tandem immunoglobulin-fold domain of ScsB.

Correct disulfide bond formation is essential for proper folding of many proteins, including bacterial virulence factors. The suppressor of copper sensitivity (Scs) proteins have roles in dithiol/disulfide interchange and the bacterial response to copper stress. Encoded in a four-gene cassette (ScsABCD) present in many Gram-negative bacteria, the Scs proteins are enigmatic and poorly characterized. Here, we show that the periplasmic α-domain of the membrane protein ScsB in the Gram-negative bacterium Proteus mirabilis forms a redox relay with the soluble periplasmic protein PmScsC. We also found that the periplasmic α-domain is sufficient to activate the disulfide isomerase activity of PmScsC. The crystal structure of PmScsBα at a resolution of 1.54 Å revealed that it comprises two structurally similar immunoglobulin-like folds, one of which includes a putative redox-active site with the sequence CXXXC. We confirmed the importance of these cysteine residues for PmScsBα function, and in addition, we engineered cysteine variants that produced a stable complex between PmScsC and PmScsBα. Using small-angle X-ray and neutron scattering analyses with contrast variation, we determined a low-resolution structure of the PmScsC-PmScsBα complex. The structural model of this complex suggested that PmScsBα uses both of its immunoglobulin-like folds to interact with PmScsC and revealed that the highly dynamic PmScsC becomes ordered upon PmScsBα binding. These findings add to our understanding of the poorly characterized Scs proteins.

Soft and Hard Interactions between Polystyrene Nanoplastics and Human Serum Albumin Protein Corona.

Upon contact with biological fluids, the surface of nanoparticles is surrounded by many types of proteins, forming a so-called "protein corona". The physicochemical properties of the nanoparticle/corona complex depend predominantly on the nature of the protein corona. An understanding of the structure of the corona and the resulting complex provides insight into the structure-activity relationship. Here, we structurally evaluate the soft and hard components of the protein corona, formed from polystyrene (PS) nanoplastics and human serum albumin (HSA). Using circular dichroism spectroscopy to elucidate the structure of HSA within the complex, we establish the effect of nanoparticle size and pH on the nature of the protein corona formed- whether hard or soft. Despite the weak interaction between PS and the HSA corona, small angle neutron scattering revealed the formation of a complex structure that enhanced the intermolecular interactions between HSA proteins, PS particles, and the HS/PSA complexes. Fractal formation occurred under conditions where the interaction between PS and HSA was strong, and increasing HSA concentrations suppressed the degree of aggregation. The size of the nanoparticles directly influenced the nature of the protein corona, with larger particles favoring the formation of a soft corona, due to the decreased PS-HSA attraction.

Interfacial Structures of Droplet-Stabilized Emulsions Formed with Whey Protein Microgel Particles as Revealed by Small- and Ultra-Small-Angle Neutron Scattering.

Droplet-stabilized emulsions (DSEs) were made from oil droplets coated with whey protein microgel (WPM) particles. The WPM particles with z-average hydrodynamic diameters of 270.9 ± 4.7 and 293.8 ± 6.7 nm were obtained by heating whey proteins with 10 mM phosphate buffer, pH 5.9 (-PB) and no buffer (-NPB), respectively. The primary emulsions coated by WPM-NPB and WPM-PB particles had mass fractal dimensions of ∼2.75, as determined by small- and ultra-small-angle neutron scattering (SANS and USANS). The size of the subsequently formed DSEs (D32 ≈ 7-23 µm), which were stabilized by the primary emulsion droplets, made with either WPM-NPB (termed DSE-NPB) or WPM-PB (termed DSE-PB) was dependent on the concentration of the primary emulsion (10-60 wt %) in the aqueous phase. At the DSE-NPB interface, the adsorbed primary emulsion droplets formed a fractal network with a surface fractal dimension of about 3, indicating a rough interfacial layer. Combined SANS and USANS allowed a comprehensive understanding of the multilength scale structures from WPM particles to DSEs.

Identification of a novel tetrameric structure for human apolipoprotein-D.

Apolipoprotein-D is a 25 kDa glycosylated member of the lipocalin family that folds into an eight-stranded ß-barrel with a single adjacent α-helix. Apolipoprotein-D specifically binds a range of small hydrophobic ligands such as progesterone and arachidonic acid and has an antioxidant function that is in part due to the reduction of peroxidised lipids by methionine-93. Therefore, apolipoprotein-D plays multiple roles throughout the body and is protective in Alzheimer's disease, where apolipoprotein-D overexpression reduces the amyloid-ß burden in Alzheimer's disease mouse models. Oligomerisation is a common feature of lipocalins that can influence ligand binding. The native structure of apolipoprotein-D, however, has not been conclusively defined. Apolipoprotein-D is generally described as a monomeric protein, although it dimerises when reducing peroxidised lipids. Here, we investigated the native structure of apolipoprotein-D derived from plasma, breast cyst fluid (BCF) and cerebrospinal fluid. In plasma and cerebrospinal fluid, apolipoprotein-D was present in high-molecular weight complexes, potentially in association with lipoproteins. In contrast, apolipoprotein-D in BCF formed distinct oligomeric species. We assessed apolipoprotein-D oligomerisation using native apolipoprotein-D purified from BCF and a suite of complementary methods, including multi-angle laser light scattering, analytical ultracentrifugation and small-angle X-ray scattering. Our analyses showed that apolipoprotein-D predominantly forms a ∼95 to ∼100 kDa tetramer. Small-angle X-ray scattering analysis confirmed these findings and provided a structural model for apolipoprotein-D tetramer. These data indicate apolipoprotein-D rarely exists as a free monomer under physiological conditions and provide insights into novel native structures of apolipoprotein-D and into oligomerisation behaviour in the lipocalin family.

Mesoporous Metallic Iridium Nanosheets.

Two-dimensional (2D) metals are an emerging class of nanostructures that have attracted enormous research interest due to their unusual electronic and thermal transport properties. Adding mesopores in the plane of ultrathin 2D metals is the next big step in manipulating these structures because increasing their surface area improves the utilization of the material and the availability of active sites. Here, we report a novel synthetic strategy to prepare an unprecedented type of 2D mesoporous metallic iridium (Ir) nanosheet. Mesoporous Ir nanosheets can be synthesized with close-packed assemblies of diblock copolymer (poly-(ethylene oxide)- b-polystyrene, PEO- b-PS) micelles aligned in the 2D plane of the nanosheets. This novel synthetic route opens a new dimension of control in the synthesis of 2D metals, enabling new kinds of mesoporous architectures with abundant catalytically active sites. Because of their unique structural features, the mesoporous metallic Ir nanosheets exhibit a high electrocatalytic activity toward the oxygen evolution reaction (OER) in acidic solution as compared to commercially available catalysts.

The antigen 43 structure reveals a molecular Velcro-like mechanism of autotransporter-mediated bacterial clumping.

Aggregation and biofilm formation are critical mechanisms for bacterial resistance to host immune factors and antibiotics. Autotransporter (AT) proteins, which represent the largest group of outer-membrane and secreted proteins in Gram-negative bacteria, contribute significantly to these phenotypes. Despite their abundance and role in bacterial pathogenesis, most AT proteins have not been structurally characterized, and there is a paucity of detailed information with regard to their mode of action. Here we report the structure-function relationships of Antigen 43 (Ag43a), a prototypic self-associating AT protein from uropathogenic Escherichia coli. The functional domain of Ag43a displays a twisted L-shaped ß-helical structure firmly stabilized by a 3D hydrogen-bonded scaffold. Notably, the distinctive Ag43a L shape facilitates self-association and cell aggregation. Combining all our data, we define a molecular "Velcro-like" mechanism of AT-mediated bacterial clumping, which can be tailored to fit different bacterial lifestyles such as the formation of biofilms.

Effects of Alkali Cations and Halide Anions on the Self-Assembly of Phosphatidylcholine in Oils.

The interactions between ions and phospholipids are closely associated with the structures and functions of cell membrane. Instead of conventional aqueous systems, we systematically investigated the effects of inorganic ions on the self-assembly of lecithin, a zwitterionic phosphatidylcholine, in cyclohexane. Previous studies have shown that addition of inorganic salts with specific divalent and trivalent cations can transform lecithin organosols into organogels. In this study, we focused on the effect of monovalent alkali halides. Fourier transform infrared spectroscopy was used to demonstrate that the binding strength of the alkali cations with the phosphate of lecithin is in the order Li+ > Na+ > K+. More importantly, the cation-phosphate interaction is affected by the paired halide anions, and the effect follows the series I- > Br- > Cl-. The salts of stronger interactions with lecithin, including LiCl, LiBr, LiI, and NaI, were found to induce cylindrical micelles sufficiently long to form organogels, while others remain organosols. A mechanism based on the charge density of ions and the enthalpy change of the ion exchange between alkali halides and lecithin headgroup is provided to explain the contrasting interactions and the effectiveness of the salts to induce organogelation.

Low-resolution solution structures of Munc18:Syntaxin protein complexes indicate an open binding mode driven by the Syntaxin N-peptide.

When nerve cells communicate, vesicles from one neuron fuse with the presynaptic membrane releasing chemicals that signal to the next. Similarly, when insulin binds its receptor on adipocytes or muscle, glucose transporter-4 vesicles fuse with the cell membrane, allowing glucose to be imported. These essential processes require the interaction of SNARE proteins on vesicle and cell membranes, as well as the enigmatic protein Munc18 that binds the SNARE protein Syntaxin. Here, we show that in solution the neuronal protein Syntaxin1a interacts with Munc18-1 whether or not the Syntaxin1a N-peptide is present. Conversely, the adipocyte protein Syntaxin4 does not bind its partner Munc18c unless the N-peptide is present. Solution-scattering data for the Munc18-1:Syntaxin1a complex in the absence of the N-peptide indicates that this complex adopts the inhibitory closed binding mode, exemplified by a crystal structure of the complex. However, when the N-peptide is present, the solution-scattering data indicate both Syntaxin1a and Syntaxin4 adopt extended conformations in complexes with their respective Munc18 partners. The low-resolution solution structure of the open Munc18:Syntaxin binding mode was modeled using data from cross-linking/mass spectrometry, small-angle X-ray scattering, and small-angle neutron scattering with contrast variation, indicating significant differences in Munc18:Syntaxin interactions compared with the closed binding mode. Overall, our results indicate that the neuronal Munc18-1:Syntaxin1a proteins can adopt two alternate and functionally distinct binding modes, closed and open, depending on the presence of the N-peptide, whereas Munc18c:Syntaxin4 adopts only the open binding mode.

Vortex fluidic regulated phospholipid equilibria involving liposomes down to sub-micelle size assemblies.

Conventional channel-based microfluidic platforms have gained prominence in controlling the bottom-up formation of phospholipid based nanostructures including liposomes. However, there are challenges in the production of liposomes from rapidly scalable processes. These have been overcome using a vortex fluidic device (VFD), which is a thin film microfluidic platform rather than channel-based, affording ∼110 nm diameter liposomes. The high yielding and high throughput continuous flow process has a 45° tilted rapidly rotating glass tube with an inner hydrophobic surface. Processing is also possible in the confined mode of operation which is effective for labelling pre-VFD-prepared liposomes with fluorophore tags for subsequent mechanistic studies on the fate of liposomes under shear stress in the VFD. In situ small-angle neutron scattering (SANS) established the co-existence of liposomes ∼110 nm with small rafts, micelles, distorted micelles, or sub-micelle size assemblies of phospholipid, for increasing rotation speeds. The equilibria between these smaller entities and ∼110 nm liposomes for a specific rotational speed of the tube is consistent with the spatial arrangement and dimensionality of topological fluid flow regimes in the VFD. The prevalence for the formation of ∼110 nm diameter liposomes establishes that this is typically the most stable structure from the bottom-up self-assembly of the phospholipid and is in accord with dimensions of exosomes.

Membrane curvature protein exhibits interdomain flexibility and binds a small GTPase.

The APPL1 and APPL2 proteins (APPL (adaptor protein, phosphotyrosine interaction, pleckstrin homology (PH) domain, and leucine zipper-containing protein)) are localized to their own endosomal subcompartment and interact with a wide range of proteins and small molecules at the cell surface and in the nucleus. They play important roles in signal transduction through their ability to act as Rab effectors. (Rabs are a family of Ras GTPases involved in membrane trafficking.) Both APPL1 and APPL2 comprise an N-terminal membrane-curving BAR (Bin-amphiphysin-Rvs) domain linked to a PH domain and a C-terminal phosphotyrosine-binding domain. The structure and interactions of APPL1 are well characterized, but little is known about APPL2. Here, we report the crystal structure and low resolution solution structure of the BARPH domains of APPL2. We identify a previously undetected hinge site for rotation between the two domains and speculate that this motion may regulate APPL2 functions. We also identified Rab binding partners of APPL2 and show that these differ from those of APPL1, suggesting that APPL-Rab interaction partners have co-evolved over time. Isothermal titration calorimetry data reveal the interaction between APPL2 and Rab31 has a K(d) of 140 nM. Together with other biophysical data, we conclude the stoichiometry of the complex is 2:2.

Data analysis and modeling of small-angle neutron scattering data with contrast variation from bio-macromolecular complexes.

Small-angle neutron scattering (SANS) with contrast variation (CV) is a valuable technique in the structural biology toolchest. Accurate structural parameters-e.g., radii of gyration, volumes, dimensions, and distance distribution(s)-can be derived from the SANS-CV data to yield the shape and disposition of the individual components within stable complexes. Contrast variation is achieved through the substitution of hydrogen isotopes (1H for 2H) in molecules and solvents to alter the neutron scattering properties of each component of a complex. While SANS-CV can be used a stand-alone technique for interrogating the overall structure of biomacromolecules in solution, it also complements other methods such as small-angle X-ray scattering, crystallography, nuclear magnetic resonance, and cryo-electron microscopy. Undertaking a SANS-CV experiment is challenging, due in part to the preparation of significant quantities of monodisperse samples that may require deuterium (2H) labeling. Nevertheless, SANS-CV can be used to study a diverse range biomacromolecular complexes including protein-protein and protein-nucleic acid systems, membrane proteins, and flexible systems resistant to crystallization. This chapter describes how to approach the data analysis and modeling of SANS data, including: (1) Analysis of the forward scattering (I(0)) and calculation of theoretical estimates of contrast; (2) Analysis of the contrast dependence of the radius of gyration using the Stuhrmann plot and parallel axis theorem; (3) Calculation of composite scattering functions to evaluate the size, shape, and dispositions of individual components within a complex, and; (4) Development of real-space models to fit the SANS-CV data using volume-element bead modeling or atomistic rigid body modeling.

2023 update of template tables for reporting biomolecular structural modelling of small-angle scattering data.

In 2017, guidelines were published for reporting structural modelling of small-angle scattering (SAS) data from biomolecules in solution that exemplified best-practice documentation of experiments and analysis. Since then, there has been significant progress in SAS data and model archiving, and the IUCr journal editors announced that the IUCr biology journals will require the deposition of SAS data used in biomolecular structure solution into a public archive, as well as adherence to the 2017 reporting guidelines. In this context, the reporting template tables accompanying the 2017 publication guidelines have been reviewed with a focus on making them both easier to use and more general. With input from the SAS community via the IUCr Commission on SAS and attendees of the triennial 2022 SAS meeting (SAS2022, Campinas, Brazil), an updated reporting template table has been developed that includes standard descriptions for proteins, glycosylated proteins, DNA and RNA, with some reorganization of the data to improve readability and interpretation. In addition, a specialized template has been developed for reporting SAS contrast-variation (SAS-cv) data and models that incorporates the additional reporting requirements from the 2017 guidelines for these more complicated experiments. To demonstrate their utility, examples of reporting with these new templates are provided for a SAS study of a DNA-protein complex and a SAS-cv experiment on a protein complex. The examples demonstrate how the tabulated information promotes transparent reporting that, in combination with the recommended figures and additional information best presented in the main text, enables the reader of the work to readily draw their own conclusions regarding the quality of the data and the validity of the models presented.

Structural basis underlying the synergism of NADase and SLO during group A Streptococcus infection.

Group A Streptococcus (GAS) is a strict human pathogen possessing a unique pathogenic trait that utilizes the cooperative activity of NAD+-glycohydrolase (NADase) and Streptolysin O (SLO) to enhance its virulence. How NADase interacts with SLO to synergistically promote GAS cytotoxicity and intracellular survival is a long-standing question. Here, the structure and dynamic nature of the NADase/SLO complex are elucidated by X-ray crystallography and small-angle scattering, illustrating atomic details of the complex interface and functionally relevant conformations. Structure-guided studies reveal a salt-bridge interaction between NADase and SLO is important to cytotoxicity and resistance to phagocytic killing during GAS infection. Furthermore, the biological significance of the NADase/SLO complex in GAS virulence is demonstrated in a murine infection model. Overall, this work delivers the structure-functional relationship of the NADase/SLO complex and pinpoints the key interacting residues that are central to the coordinated actions of NADase and SLO in the pathogenesis of GAS infection.

Technical considerations for small-angle neutron scattering from biological macromolecules in solution: Cross sections, contrasts, instrument setup and measurement.

Small angle scattering affords an approach to evaluate the structure of dilute populations of macromolecules in solution where the measured scattering intensities relate to the distribution of scattering-pair distances within each macromolecule. When small angle neutron scattering (SANS) with contrast variation is employed, additional structural information can be obtained regarding the internal organization of biomacromolecule complexes and assemblies. The technique allows for the components of assemblies to be selectively 'matched in' and 'matched out' of the scattering profiles due to the different ways the isotopes of hydrogen-protium 1H, and deuterium 2H (or D)-scatter neutrons. The isotopic substitution of 1H for D in the sample enables the controlled variation of the scattering contrasts. A contrast variation experiment requires trade-offs between neutron beam intensity, q-range, wavelength and q-resolution, isotopic labelling levels, sample concentration and path-length, and measurement times. Navigating these competing aspects to find an optimal combination is a daunting task. Here we provide an overview of how to calculate the neutron scattering contrasts of dilute biological macromolecule samples prior to an experiment and how this then informs the approach to configuring SANS instruments and the measurement of a contrast variation series dataset.

The suppressor of copper sensitivity protein C from Caulobacter crescentus is a trimeric disulfide isomerase that binds copper(I) with subpicomolar affinity.

The introduction of disulfide bonds into periplasmic proteins is a critical process in many Gram-negative bacteria. The formation and regulation of protein disulfide bonds have been linked to the production of virulence factors. Understanding the different pathways involved in this process is important in the development of strategies to disarm pathogenic bacteria. The well characterized disulfide bond-forming (DSB) proteins play a key role by introducing or isomerizing disulfide bonds between cysteines in substrate proteins. Curiously, the suppressor of copper sensitivity C proteins (ScsCs), which are part of the bacterial copper-resistance response, share structural and functional similarities with DSB oxidase and isomerase proteins, including the presence of a catalytic thioredoxin domain. However, the oxidoreductase activity of ScsC varies with its oligomerization state, which depends on a poorly conserved N-terminal domain. Here, the structure and function of Caulobacter crescentus ScsC (CcScsC) have been characterized. It is shown that CcScsC binds copper in the copper(I) form with subpicomolar affinity and that its isomerase activity is comparable to that of Escherichia coli DsbC, the prototypical dimeric bacterial isomerase. It is also reported that CcScsC functionally complements trimeric Proteus mirabilis ScsC (PmScsC) in vivo, enabling the swarming of P. mirabilis in the presence of copper. Using mass photometry and small-angle X-ray scattering (SAXS) the protein is demonstrated to be trimeric in solution, like PmScsC, and not dimeric like EcDsbC. The crystal structure of CcScsC was also determined at a resolution of 2.6 Å, confirming the trimeric state and indicating that the trimerization results from interactions between the N-terminal α-helical domains of three CcScsC protomers. The SAXS data analysis suggested that the protomers are dynamic, like those of PmScsC, and are able to sample different conformations in solution.

Variation of Antigen 43 self-association modulates bacterial compacting within aggregates and biofilms.

The formation of aggregates and biofilms enhances bacterial colonisation and infection progression by affording protection from antibiotics and host immune factors. Despite these advantages there is a trade-off, whereby bacterial dissemination is reduced. As such, biofilm development needs to be controlled to suit adaptation to different environments. Here we investigate members from one of largest groups of bacterial adhesins, the autotransporters, for their critical role in the assembly of bacterial aggregates and biofilms. We describe the structural and functional characterisation of autotransporter Ag43 variants from different Escherichia coli pathotypes. We show that specific interactions between amino acids on the contacting interfaces of adjacent Ag43 proteins drives a common mode of trans-association that leads to cell clumping. Furthermore, subtle variation of these interactions alters aggregation kinetics and the degree of compacting within cell clusters. Together, our structure-function investigation reveals an underlying molecular basis for variations in the density of bacterial communities.