Molecular basis of F-actin regulation and sarcomere assembly via myotilin.

Sarcomeres, the basic contractile units of striated muscle cells, contain arrays of thin (actin) and thick (myosin) filaments that slide past each other during contraction. The Ig-like domain-containing protein myotilin provides structural integrity to Z-discs-the boundaries between adjacent sarcomeres. Myotilin binds to Z-disc components, including F-actin and α-actinin-2, but the molecular mechanism of binding and implications of these interactions on Z-disc integrity are still elusive. To illuminate them, we used a combination of small-angle X-ray scattering, cross-linking mass spectrometry, and biochemical and molecular biophysics approaches. We discovered that myotilin displays conformational ensembles in solution. We generated a structural model of the F-actin:myotilin complex that revealed how myotilin interacts with and stabilizes F-actin via its Ig-like domains and flanking regions. Mutant myotilin designed with impaired F-actin binding showed increased dynamics in cells. Structural analyses and competition assays uncovered that myotilin displaces tropomyosin from F-actin. Our findings suggest a novel role of myotilin as a co-organizer of Z-disc assembly and advance our mechanistic understanding of myotilin's structural role in Z-discs.

Small conformational changes in IgG1 detected as acidic charge variants by cation exchange chromatography.

Fully characterizing the post-translational modifications present in charge variants of therapeutic monoclonal antibodies (mAbs), particularly acidic variants, is challenging and remains an open area of investigation. In this study, to test the possibility that chromatographically separated acidic fractions of therapeutic mAbs contain conformational variants, we undertook a mAb refolding approach using as a case study an IgG1 that contains many unidentified acidic peaks with few post-translational modifications, and examined whether different acidic peak fractions could be generated corresponding to these variants. The IgG1 drug substance was denatured by guanidine hydrochloride, without a reducing agent present, and gradually refolded by stepwise dialysis against arginine hydrochloride used as an aggregation suppressor. Each acidic chromatographic peak originally contained in the IgG1 drug substance was markedly increased by this stepwise refolding process, indicating that these acidic variants are conformational variants. However, no conformational changes were detected by small-angle X-ray scattering experiments for the whole IgG1, indicating that the conformational changes are minor. Chromatographic, thermal and fluorescence analyses suggested that the conformational changes are a localized denaturation effect centred around the aromatic amino acid regions. This study provides new insights into the characterization of acidic variants that are currently not fully understood.

Molecular mechanism of leukocidin GH-integrin CD11b/CD18 recognition and species specificity.

Host-pathogen interactions are central to understanding microbial pathogenesis. The staphylococcal pore-forming cytotoxins hijack important immune molecules but little is known about the underlying molecular mechanisms of cytotoxin-receptor interaction and host specificity. Here we report the structures of a staphylococcal pore-forming cytotoxin, leukocidin GH (LukGH), in complex with its receptor (the α-I domain of complement receptor 3, CD11b-I), both for the human and murine homologs. We observe 2 binding interfaces, on the LukG and the LukH protomers, and show that human CD11b-I induces LukGH oligomerization in solution. LukGH binds murine CD11b-I weakly and is inactive toward murine neutrophils. Using a LukGH variant engineered to bind mouse CD11b-I, we demonstrate that cytolytic activity does not only require binding but also receptor-dependent oligomerization. Our studies provide an unprecedented insight into bicomponent leukocidin-host receptor interaction, enabling the development of antitoxin approaches and improved animal models to explore these approaches.

Multi-channel in situ dynamic light scattering instrumentation enhancing biological small-angle X-ray scattering experiments at the PETRA III beamline P12.

Small-angle X-ray scattering (SAXS) analysis of biomolecules is increasingly common with a constantly high demand for comprehensive and efficient sample quality control prior to SAXS experiments. As monodisperse sample suspensions are desirable for SAXS experiments, latest dynamic light scattering (DLS) techniques are most suited to obtain non-invasive and rapid information about the particle size distribution of molecules in solution. A multi-receiver four-channel DLS system was designed and adapted at the BioSAXS endstation of the EMBL beamline P12 at PETRA III (DESY, Hamburg, Germany). The system allows the collection of DLS data within round-shaped sample capillaries used at beamline P12. Data obtained provide information about the hydrodynamic radius of biological particles in solution and dispersity of the solution. DLS data can be collected directly prior to and during an X-ray exposure. To match the short X-ray exposure times of around 1 s for 20 exposures at P12, the DLS data collection periods that have been used up to now of 20 s or commonly more were substantially reduced, using a novel multi-channel approach collecting DLS data sets in the SAXS sample capillary at four different neighbouring sample volume positions in parallel. The setup allows online scoring of sample solutions applied for SAXS experiments, supports SAXS data evaluation and for example indicates local inhomogeneities in a sample solution in a time-efficient manner. Biological macromolecules with different molecular weights were applied to test the system and obtain information about the performance. All measured hydrodynamic radii are in good agreement with DLS results obtained by employing a standard cuvette instrument. Moreover, applying the new multi-channel DLS setup, a reliable radius determination of sample solutions in flow, at flow rates normally used for size-exclusion chromatography-SAXS experiments, and at higher flow rates, was verified as well. This study also shows and confirms that the newly designed sample compartment with attached DLS instrumentation does not disturb SAXS measurements.

Sample and Buffer Preparation for SAXS.

In this book chapter, a practical approach for conducting small angle X-ray scattering (SAXS) experiments is given. Our aim is to guide SAXS users through a three-step process of planning, preparing and performing a basic SAXS measurement. The minimal requirements necessary to prepare samples are described specifically for protein and other macromolecular samples in solution. We address the very important aspects in terms of sample characterization using additional techniques as well as the essential role of accurately subtracting background scattering contributions. At the end of the chapter some advice is given for trouble-shooting problems that may occur during the course of the SAXS measurements. Automated pipelines for data processing are described which are useful in allowing users to evaluate the quality of the data 'on the spot' and consequently react to events such as radiation damage, the presence of unwanted sample aggregates or miss-matched buffers.

Structural Basis for Antigen Recognition by Transglutaminase 2-specific Autoantibodies in Celiac Disease.

Antibodies to the autoantigen transglutaminase 2 (TG2) are a hallmark of celiac disease. We have studied the interaction between TG2 and an anti-TG2 antibody (679-14-E06) derived from a single gut IgA plasma cell of a celiac disease patient. The antibody recognizes one of four identified epitopes targeted by antibodies of plasma cells of the disease lesion. The binding interface was identified by small angle x-ray scattering, ab initio and rigid body modeling using the known crystal structure of TG2 and the crystal structure of the antibody Fab fragment, which was solved at 2.4 Å resolution. The result was confirmed by testing binding of the antibody to TG2 mutants by ELISA and surface plasmon resonance. TG2 residues Arg-116 and His-134 were identified to be critical for binding of 679-14-E06 as well as other epitope 1 antibodies. In contrast, antibodies directed toward the two other main epitopes (epitopes 2 and 3) were not affected by these mutations. Molecular dynamics simulations suggest interactions of 679-14-E06 with the N-terminal domain of TG2 via the CDR2 and CDR3 loops of the heavy chain and the CDR2 loop of the light chain. In addition there were contacts of the framework 3 region of the heavy chain with the catalytic domain of TG2. The results provide an explanation for the biased usage of certain heavy and light chain gene segments by epitope 1-specific antibodies in celiac disease.

Limiting radiation damage for high-brilliance biological solution scattering: practical experience at the EMBL P12 beamline PETRAIII.

Radiation damage is the general curse of structural biologists who use synchrotron small-angle X-ray scattering (SAXS) to investigate biological macromolecules in solution. The EMBL-P12 biological SAXS beamline located at the PETRAIII storage ring (DESY, Hamburg, Germany) caters to an extensive user community who integrate SAXS into their diverse structural biology programs. The high brilliance of the beamline [5.1 × 10(12) photons s(-1), 10 keV, 500 (H) µm × 250 (V) µm beam size at the sample position], combined with automated sample handling and data acquisition protocols, enable the high-throughput structural characterization of macromolecules in solution. However, considering the often-significant resources users invest to prepare samples, it is crucial that simple and effective protocols are in place to limit the effects of radiation damage once it has been detected. Here various practical approaches are evaluated that users can implement to limit radiation damage at the P12 beamline to maximize the chances of collecting quality data from radiation sensitive samples.

Structural and functional insights into tRNA recognition by human tRNA guanine transglycosylase.

Eukaryotic tRNA guanine transglycosylase (TGT) is an RNA-modifying enzyme which catalyzes the base exchange of the genetically encoded guanine 34 of tRNAsAsp,Asn,His,Tyr for queuine, a hypermodified 7-deazaguanine derivative. Eukaryotic TGT is a heterodimer comprised of a catalytic and a non-catalytic subunit. While binding of the tRNA anticodon loop to the active site is structurally well understood, the contribution of the non-catalytic subunit to tRNA binding remained enigmatic, as no complex structure with a complete tRNA was available. Here, we report a cryo-EM structure of eukaryotic TGT in complex with a complete tRNA, revealing the crucial role of the non-catalytic subunit in tRNA binding. We decipher the functional significance of these additional tRNA-binding sites, analyze solution state conformation, flexibility, and disorder of apo TGT, and examine conformational transitions upon tRNA binding.

On the Influence of Fabrication Methods and Materials for mRNA-LNP Production: From Size and Morphology to Internal Structure and mRNA Delivery Performance In Vitro and In Vivo.

Lipid nanoparticle (LNP) remains the most advanced platform for messenger RNA (mRNA) delivery. To date, mRNA LNPs synthesis is mostly performed by mixing lipids and mRNA with microfluidics. In this study, a cost-effective microfluidic setup for synthesizing mRNA LNPs is developed. It allows to fine-tune the LNPs characteristics without compromising LNP properties. It is compared with a commercial device (NanoAssemblr) and ethanol injection and the influence of manufacturing conditions on the performance of mRNA LNPs is investigated. LNPs prepared by ethanol injection exhibit broader size distributions and more inhomogeneous internal structure (e.g., bleb-like substructures), while other LNPs show uniform structure with dense cores. Small angel X-ray scattering (SAXS) data indicate a tighter interaction between mRNA and lipids within LNPs synthesized by custom device, compared to LNPs produced by NanoAssemblr. Interestingly, the better transfection efficiency of polysarcosine (pSar)-modified LNPs correlates with a higher surface roughness than that of PEGylated ones. The manufacturing approach, however, shows modest influence on mRNA expression in vivo. In summary, the home-developed cost-effective microfluidic device can synthesize LNPs and represents a potent alternative to NanoAssemblr. The preparation methods show notable effect on LNPs' structure but a minor influence on mRNA delivery in vitro and in vivo.

Reconstruction of quaternary structure from X-ray scattering by equilibrium mixtures of biological macromolecules.

A recent renaissance in small-angle X-ray scattering (SAXS) made this technique a major tool for the low-resolution structural characterization of biological macromolecules in solution. The major limitation of existing methods for reconstructing 3D models from SAXS is imposed by the requirement of solute monodispersity. We present a novel approach that couples low-resolution 3D SAXS reconstruction with composition analysis of mixtures. The approach is applicable to polydisperse and difficult to purify systems, including weakly associated oligomers and transient complexes. Ab initio shape analysis is possible for symmetric homo-oligomers, whereas rigid body modeling is applied also to dissociating complexes when atomic structures of the individual subunits are available. In both approaches, the sample is considered as an equilibrium mixture of intact complexes/oligomers with their dissociation products or free subunits. The algorithms provide the 3D low-resolution model (for ab initio modeling, also the shape of the monomer) and the volume fractions of the bound and free state(s). The simultaneous fitting of multiple scattering data sets collected under different conditions allows one to restrain the modeling further. The possibilities of the approach are illustrated in simulated and experimental SAXS data from protein oligomers and multisubunit complexes including nucleoproteins. Using this approach, new structural insights are provided in the association behavior and conformations of estrogen-related receptors ERRα and ERRγ. The possibility of 3D modeling from the scattering by mixtures significantly widens the range of applicability of SAXS and opens novel avenues in the analysis of oligomeric mixtures and assembly/dissociation processes.

Structural and functional analysis of a plant nucleolar RNA chaperone-like protein.

Ribosome biogenesis is a key process in all eukaryotic cells that requires hundreds of ribosome biogenesis factors (RBFs), which are essential to build the mature ribosomes consisting of proteins and rRNAs. The processing of the required rRNAs has been studied extensively in yeast and mammals, but in plants much is still unknown. In this study, we focused on a RBF from A. thaliana that we named NUCLEOLAR RNA CHAPERONE-LIKE 1 (NURC1). NURC1 was localized in the nucleolus of plant cell nuclei, and other plant RBF candidates shared the same localization. SEC-SAXS experiments revealed that NURC1 has an elongated and flexible structure. In addition, SEC-MALLS experiments confirmed that NURC1 was present in its monomeric form with a molecular weight of around 28 kDa. RNA binding was assessed by performing microscale thermophoresis with the Arabidopsis internal transcribed spacer 2 (ITS2) of the polycistronic pre-rRNA precursor, which contains the 5.8S, 18S, and 25S rRNA. NURC1 showed binding activity to the ITS2 with a dissociation constant of 228 nM and exhibited RNA chaperone-like activity. Our data suggested that NURC1 may have a function in pre-rRNA processing and thus ribosome biogenesis.

Quantitative size-resolved characterization of mRNA nanoparticles by in-line coupling of asymmetrical-flow field-flow fractionation with small angle X-ray scattering.

We present a generically applicable approach to determine an extensive set of size-dependent critical quality attributes inside nanoparticulate pharmaceutical products. By coupling asymmetrical-flow field-flow fractionation (AF4) measurements directly in-line with solution small angle X-ray scattering (SAXS), vital information such as (i) quantitative, absolute size distribution profiles, (ii) drug loading, (iii) size-dependent internal structures, and (iv) quantitative information on free drug is obtained. Here the validity of the method was demonstrated by characterizing complex mRNA-based lipid nanoparticle products. The approach is particularly applicable to particles in the size range of 100 nm and below, which is highly relevant for pharmaceutical products-both biologics and nanoparticles. The method can be applied as well in other fields, including structural biology and environmental sciences.

Form factor determination of biological molecules with X-ray free electron laser small-angle scattering (XFEL-SAS).

Free-electron lasers (FEL) are revolutionizing X-ray-based structural biology methods. While protein crystallography is already routinely performed at FELs, Small Angle X-ray Scattering (SAXS) studies of biological macromolecules are not as prevalent. SAXS allows the study of the shape and overall structure of proteins and nucleic acids in solution, in a quasi-native environment. In solution, chemical and biophysical parameters that have an influence on the structure and dynamics of molecules can be varied and their effect on conformational changes can be monitored in time-resolved XFEL and SAXS experiments. We report here the collection of scattering form factors of proteins in solution using FEL X-rays. The form factors correspond to the scattering signal of the protein ensemble alone; the scattering contributions from the solvent and the instrument are separately measured and accurately subtracted. The experiment was done using a liquid jet for sample delivery. These results pave the way for time-resolved studies and measurements from dilute samples, capitalizing on the intense and short FEL X-ray pulses.

Advanced sample environments and sample requirements for biological SAXS.

In this chapter, we discuss the various sample delivery systems that are available at most biological SAXS beamlines. The focus is laid on the EMBL Biosaxs beamline P12 at the Petra 3 storage ring on the DESY site in Hamburg, Germany. The minimal requirements necessary to prepare samples are described specifically for macromolecular samples in solution and the background is given on how the physical properties of the scattering process itself determine the sample requirements. We provide a number of exemplary applications as well as guidelines for selecting and performing the right data collection strategy depending on the scientific question at hand. This chapter is aimed at novices to the SAXS technique with biochemical background as well as more experienced users setting out to employ more advanced SAXS set-ups.

EFAMIX, a tool to decompose inline chromatography SAXS data from partially overlapping components.

Small-angle X-ray scattering (SAXS) is an established technique for structural analysis of biological macromolecules in solution. During the last decade, inline chromatography setups coupling SAXS with size exclusion (SEC-SAXS) or ion exchange (IEC-SAXS) have become popular in the community. These setups allow one to separate individual components in the sample and to record SAXS data from isolated fractions, which is extremely important for subsequent data interpretation, analysis, and structural modeling. However, in case of partially overlapping elution peaks, inline chromatography SAXS may still yield scattering profiles from mixtures of components. The deconvolution of these scattering data into the individual fractions is nontrivial and potentially ambiguous. We describe a cross-platform computer program, EFAMIX, for restoring the scattering and concentration profiles of the components based on the evolving factor analysis (EFA). The efficiency of the program is demonstrated in a number of simulated and experimental SEC-SAXS data sets. Sensitivity and limitations of the method are explored, and its applicability to IEC-SAXS data is discussed. EFAMIX requires minimal user intervention and is available to academic users through the program package ATSAS as from release 3.1.

Chaperoning of the histone octamer by the acidic domain of DNA repair factor APLF.

Nucleosome assembly requires the coordinated deposition of histone complexes H3-H4 and H2A-H2B to form a histone octamer on DNA. In the current paradigm, specific histone chaperones guide the deposition of first H3-H4 and then H2A-H2B. Here, we show that the acidic domain of DNA repair factor APLF (APLFAD) can assemble the histone octamer in a single step and deposit it on DNA to form nucleosomes. The crystal structure of the APLFAD-histone octamer complex shows that APLFAD tethers the histones in their nucleosomal conformation. Mutations of key aromatic anchor residues in APLFAD affect chaperone activity in vitro and in cells. Together, we propose that chaperoning of the histone octamer is a mechanism for histone chaperone function at sites where chromatin is temporarily disrupted.

A round-robin approach provides a detailed assessment of biomolecular small-angle scattering data reproducibility and yields consensus curves for benchmarking.

Through an expansive international effort that involved data collection on 12 small-angle X-ray scattering (SAXS) and four small-angle neutron scattering (SANS) instruments, 171 SAXS and 76 SANS measurements for five proteins (ribonuclease A, lysozyme, xylanase, urate oxidase and xylose isomerase) were acquired. From these data, the solvent-subtracted protein scattering profiles were shown to be reproducible, with the caveat that an additive constant adjustment was required to account for small errors in solvent subtraction. Further, the major features of the obtained consensus SAXS data over the q measurement range 0-1 Å-1 are consistent with theoretical prediction. The inherently lower statistical precision for SANS limited the reliably measured q-range to <0.5 Å-1, but within the limits of experimental uncertainties the major features of the consensus SANS data were also consistent with prediction for all five proteins measured in H2O and in D2O. Thus, a foundation set of consensus SAS profiles has been obtained for benchmarking scattering-profile prediction from atomic coordinates. Additionally, two sets of SAXS data measured at different facilities to q > 2.2 Å-1 showed good mutual agreement, affirming that this region has interpretable features for structural modelling. SAS measurements with inline size-exclusion chromatography (SEC) proved to be generally superior for eliminating sample heterogeneity, but with unavoidable sample dilution during column elution, while batch SAS data collected at higher concentrations and for longer times provided superior statistical precision. Careful merging of data measured using inline SEC and batch modes, or low- and high-concentration data from batch measurements, was successful in eliminating small amounts of aggregate or interparticle interference from the scattering while providing improved statistical precision overall for the benchmarking data set.

The USR domain of USF1 mediates NF-Y interactions and cooperative DNA binding.

The trimeric CCAAT-binding NF-Y is a "pioneer" Transcription Factor -TF- known to cooperate with neighboring TFs to regulate gene expression. Genome-wide analyses detected a precise stereo-alignment -10/12 bp- of CCAAT with E-box elements and corresponding colocalization of NF-Y with basic-Helix-Loop-Helix (bHLH) TFs. We dissected here NF-Y interactions with USF1 and MAX. USF1, but not MAX, cooperates in DNA binding with NF-Y. NF-Y and USF1 synergize to activate target promoters. Reconstruction of complexes by structural means shows independent DNA binding of MAX, whereas USF1 has extended contacts with NF-Y, involving the USR, a USF-specific amino acid sequence stretch required for trans-activation. The USR is an intrinsically disordered domain and adopts different conformations based on E-box-CCAAT distances. Deletion of the USR abolishes cooperative DNA binding with NF-Y. Our data indicate that the functionality of certain unstructured domains involves adapting to small variation in stereo-alignments of the multimeric TFs sites.

The allosteric modulation of complement C5 by knob domain peptides.

Bovines have evolved a subset of antibodies with ultra-long heavy chain complementarity determining regions that harbour cysteine-rich knob domains. To produce high-affinity peptides, we previously isolated autonomous 3-6 kDa knob domains from bovine antibodies. Here, we show that binding of four knob domain peptides elicits a range of effects on the clinically validated drug target complement C5. Allosteric mechanisms predominated, with one peptide selectively inhibiting C5 cleavage by the alternative pathway C5 convertase, revealing a targetable mechanistic difference between the classical and alternative pathway C5 convertases. Taking a hybrid biophysical approach, we present C5-knob domain co-crystal structures and, by solution methods, observed allosteric effects propagating >50 Å from the binding sites. This study expands the therapeutic scope of C5, presents new inhibitors, and introduces knob domains as new, low molecular weight antibody fragments, with therapeutic potential.

Antibodies are proteins produced by the immune system that can selectively bind to other molecules and modify their behaviour. Cows are highly equipped at fighting-off disease-causing microbes due to the unique shape of some of their antibodies. Unlike other jawed vertebrates, cows' antibodies contain an ultra-long loop region that contains a 'knob domain' which sticks out from the rest of the antibody. Recent research has shown that when detached, the knob domain behaves like an antibody fragment, and can independently bind to a range of different proteins. Antibody fragments are commonly developed in the laboratory to target proteins associated with certain diseases, such as arthritis and cancer. But it was unclear whether the knob domains from cows' antibodies could also have therapeutic potential. To investigate this, Macpherson et al. studied how knob domains attach to complement C5, a protein in the inflammatory pathway which is a drug target for various diseases, including severe COVID-19. The experiments identified various knob domains that bind to complement C5 and inhibits its activity by altering its structure or movement. Further tests studying the structure of these interactions, led to the discovery of a common mechanism by which inhibitors can modify the behaviour of this inflammatory protein. Complement C5 is involved in numerous molecular pathways in the immune system, which means many of the drugs developed to inhibit its activity can also leave patients vulnerable to infection. However, one of the knob domains identified by Macpherson et al. was found to reduce the activity of complement C5 in some pathways, whilst leaving other pathways intact. This could potentially reduce the risk of bacterial infections which sometimes arise following treatment with these types of inhibitors. These findings highlight a new approach for developing drug inhibitors for complement C5. Furthermore, the ability of knob domains to bind to multiple sites of complement C5 suggests that this fragment could be used to target proteins associated with other diseases.

Structures of three MORN repeat proteins and a re-evaluation of the proposed lipid-binding properties of MORN repeats.

MORN (Membrane Occupation and Recognition Nexus) repeat proteins have a wide taxonomic distribution, being found in both prokaryotes and eukaryotes. Despite this ubiquity, they remain poorly characterised at both a structural and a functional level compared to other common repeats. In functional terms, they are often assumed to be lipid-binding modules that mediate membrane targeting. We addressed this putative activity by focusing on a protein composed solely of MORN repeats-Trypanosoma brucei MORN1. Surprisingly, no evidence for binding to membranes or lipid vesicles by TbMORN1 could be obtained either in vivo or in vitro. Conversely, TbMORN1 did interact with individual phospholipids. High- and low-resolution structures of the MORN1 protein from Trypanosoma brucei and homologous proteins from the parasites Toxoplasma gondii and Plasmodium falciparum were obtained using a combination of macromolecular crystallography, small-angle X-ray scattering, and electron microscopy. This enabled a first structure-based definition of the MORN repeat itself. Furthermore, all three structures dimerised via their C-termini in an antiparallel configuration. The dimers could form extended or V-shaped quaternary structures depending on the presence of specific interface residues. This work provides a new perspective on MORN repeats, showing that they are protein-protein interaction modules capable of mediating both dimerisation and oligomerisation.