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Glycosaminoglycans are extended linear polysaccharides present on cell surfaces and within the extracellular matrix that play crucial roles in various biological processes. Two prominent glycosaminoglycans, heparan sulfate and chondroitin sulfate, are covalently linked to proteoglycan core proteins through a common tetrasaccharide linker comprising glucuronic acid, galactose, galactose, and xylose moities. This tetrasaccharide linker is meticulously assembled step by step by four Golgi-localized glycosyltransferases. The addition of the fifth sugar moiety, either N-acetylglucosamine or N-acetylgalactosamine, initiates further chain elongation, resulting in the formation of heparan sulfate or chondroitin sulfate, respectively. Despite the fundamental significance of this step in glycosaminoglycan biosynthesis, its regulatory mechanisms have remained elusive. In this study, we detail the expression and purification of the four linker-synthesizing glycosyltransferases and their utilization in the production of fluorescent peptides carrying the native tetrasaccharide linker. We generated five tetrasaccharide peptides, mimicking the core proteins of either heparan sulfate or chondroitin sulfate proteoglycans. These peptides were readily accepted as substrates by the EXTL3 enzyme, which adds an N-acetylglucosamine moiety, thereby initiating heparan sulfate biosynthesis. Importantly, EXTL3 showed a preference towards peptides mimicking the core proteins of heparan sulfate proteoglycans over the ones from chondroitin sulfate proteoglycans. This suggests that EXTL3 could play a role in the decision-making step during glycosaminoglycan biosynthesis. The innovative strategy for chemo-enzymatic synthesis of fluorescent-labeled linker-peptides promises to be instrumental in advancing future investigations into the initial steps and the divergent step of glycosaminoglycan biosynthesis., (© The Author(s) 2024. Published by Oxford University Press.)

Invasion of brain endothelium protein A (IbeA) is a virulence factor specific to pathogenic Escherichia coli. Originally identified in the K1 strain causing neonatal meningitis, it was more recently found in avian pathogenic Escherichia coli (APEC) and adherent invasive Escherichia coli (AIEC). In these bacteria, IbeA facilitates host cell invasion and intracellular survival, in particular, under harsh conditions like oxidative stress. Furthermore, IbeA from AIEC contributes to intramacrophage survival and replication, thus enhancing the inflammatory response within the intestine. Therefore, this factor is a promising drug target for anti-AIEC strategies in the context of Crohn's disease. Despite such an important role, the biological function of IbeA remains largely unknown. In particular, its exact nature and cellular localization, i.e., membrane-bound invasin versus cytosolic factor, are still of debate. Here, we developed an efficient protocol for recombinant expression of IbeA under native conditions and demonstrated that IbeA from AIEC is a soluble, homodimeric flavoprotein. Using mass spectrometry and tryptophan fluorescence measurements, we further showed that IbeA preferentially binds flavin adenine dinucleotide (FAD), with an affinity in the one-hundred nanomolar range and optimal binding under reducing conditions. 3D-modeling with AlphaFold revealed that IbeA shares strong structural homology with FAD-dependent oxidoreductases. Finally, we used ligand docking, mutational analyses, and molecular dynamics simulations to identify the FAD binding pocket within IbeA and characterize possible conformational changes occurring upon ligand binding. Overall, we suggest that the role of IbeA in the survival of AIEC within host cells, notably macrophages, is linked to modulation of redox processes., (© 2023 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

The synthesis of fatty acids from acetyl-coenzyme A (AcCoA) is deregulated in diverse pathologies, including cancer. Here, we report that fatty acid accumulation is negatively regulated by nucleoside diphosphate kinases 1 and 2 (NME1/2), housekeeping enzymes involved in nucleotide homeostasis that were recently found to bind CoA. We show that NME1 additionally binds AcCoA and that ligand recognition involves a unique binding mode dependent on the CoA/AcCoA 3' phosphate. We report that Nme2 knockout mice fed a high-fat diet (HFD) exhibit excessive triglyceride synthesis and liver steatosis. In liver cells, NME2 mediates a gene transcriptional response to HFD leading to the repression of fatty acid accumulation and activation of a protective gene expression program via targeted histone acetylation. Our findings implicate NME1/2 in the epigenetic regulation of a protective liver response to HFD and suggest a potential role in controlling AcCoA usage between the competing paths of histone acetylation and fatty acid synthesis.

RIPK2 is an essential adaptor for NOD signalling and its kinase domain is a drug target for NOD-related diseases, such as inflammatory bowel disease. However, recent work indicates that the phosphorylation activity of RIPK2 is dispensable for signalling and that inhibitors of both RIPK2 activity and RIPK2 ubiquitination prevent the essential interaction between RIPK2 and the BIR2 domain of XIAP, the key RIPK2 ubiquitin E3 ligase. Moreover, XIAP BIR2 antagonists also block this interaction. To reveal the molecular mechanisms involved, we combined native mass spectrometry, NMR, and cryo-electron microscopy to determine the structure of the RIPK2 kinase BIR2 domain complex and validated the interface with in cellulo assays. The structure shows that BIR2 binds across the RIPK2 kinase antiparallel dimer and provides an explanation for both inhibitory mechanisms. It also highlights why phosphorylation of the kinase activation loop is dispensable for signalling while revealing the structural role of RIPK2-K209 residue in the RIPK2-XIAP BIR2 interaction. Our results clarify the features of the RIPK2 conformation essential for its role as a scaffold protein for ubiquitination., (© 2023 Lethier et al.)

Meiotic recombination is initiated by the formation of DNA double-strand breaks (DSBs), essential for fertility and genetic diversity. In the mouse, DSBs are formed by the catalytic TOPOVIL complex consisting of SPO11 and TOPOVIBL. To preserve genome integrity, the activity of the TOPOVIL complex is finely controlled by several meiotic factors including REC114, MEI4, and IHO1, but the underlying mechanism is poorly understood. Here, we report that mouse REC114 forms homodimers, that it associates with MEI4 as a 2:1 heterotrimer that further dimerizes, and that IHO1 forms coiled-coil-based tetramers. Using AlphaFold2 modeling combined with biochemical characterization, we uncovered the molecular details of these assemblies. Finally, we show that IHO1 directly interacts with the PH domain of REC114 by recognizing the same surface as TOPOVIBL and another meiotic factor ANKRD31. These results provide strong evidence for the existence of a ternary IHO1-REC114-MEI4 complex and suggest that REC114 could act as a potential regulatory platform mediating mutually exclusive interactions with several partners., (© 2023 The Authors. Published under the terms of the CC BY NC ND 4.0 license.)

Most bacteriophages present a tail allowing host recognition, cell wall perforation, and viral DNA channeling from the capsid to the infected bacterium cytoplasm. The majority of tailed phages bear a long flexible tail ( Siphoviridae ) at the tip of which receptor binding proteins (RBPs) specifically interact with their host, triggering infection. In siphophage T5, the unique RBP is located at the extremity of a central fiber. We present the structures of T5 tail tip, determined by cryo-electron microscopy before and after interaction with its E. coli receptor, FhuA, reconstituted into nanodisc. These structures bring out the important conformational changes undergone by T5 tail tip upon infection, which include bending of T5 central fiber on the side of the tail tip, tail anchoring to the membrane, tail tube opening, and formation of a transmembrane channel. The data allow to detail the first steps of an otherwise undescribed infection mechanism.

HIV-1 Rev mediates the nuclear export of intron-containing viral RNA transcripts and is essential for viral replication. Rev is imported into the nucleus by the host protein importin β (Impβ), but how Rev associates with Impβ is poorly understood. Here, we report biochemical, mutational, and biophysical studies of the Impβ/Rev complex. We show that Impβ binds two Rev monomers through independent binding sites, in contrast to the 1:1 binding stoichiometry observed for most Impβ cargos. Peptide scanning data and charge-reversal mutations identify the N-terminal tip of Rev helix α2 within Rev's arginine-rich motif (ARM) as a primary Impβ-binding epitope. Cross-linking mass spectrometry and compensatory mutagenesis data combined with molecular docking simulations suggest a structural model in which one Rev monomer binds to the C-terminal half of Impβ with Rev helix α2 roughly parallel to the HEAT-repeat superhelical axis, whereas the other monomer binds to the N-terminal half. These findings shed light on the molecular basis of Rev recognition by Impβ and highlight an atypical binding behavior that distinguishes Rev from canonical cellular Impβ cargos., (© 2022 Spittler et al.)

In Angiosperms, the plastid-encoded RNA polymerase (PEP) is a multimeric enzyme, essential for the proper expression of the plastid genome during chloroplast biogenesis. It is especially required for the light initiated expression of photosynthesis genes and the subsequent build-up of the photosynthetic apparatus. The PEP complex is composed of a prokaryotic-type core of four plastid-encoded subunits and 12 nuclear-encoded PEP-associated proteins (PAPs). Among them, there are two iron superoxide dismutases, FSD2/PAP9 and FSD3/PAP4. Superoxide dismutases usually are soluble enzymes not bound into larger protein complexes. To investigate this unusual feature, we characterized PAP9 using molecular genetics, fluorescence microscopy, mass spectrometry, X-ray diffraction, and solution-state NMR. Despite the presence of a predicted nuclear localization signal within the sequence of the predicted chloroplast transit peptide, PAP9 was mainly observed within plastids. Mass spectrometry experiments with the recombinant Arabidopsis PAP9 suggested that monomers and dimers of PAP9 could be associated to the PEP complex. In crystals, PAP9 occurred as a dimeric enzyme that displayed a similar fold to that of the FeSODs or manganese SOD (MnSODs). A zinc ion, instead of the expected iron, was found to be penta-coordinated with a trigonal- bipyramidal geometry in the catalytic center of the recombinant protein. The metal coordination involves a water molecule and highly conserved residues in FeSODs. Solution-state NMR and DOSY experiments revealed an unfolded C-terminal 34 amino-acid stretch in the stand-alone protein and few internal residues interacting with the rest of the protein. We hypothesize that this C-terminal extension had appeared during evolution as a distinct feature of the FSD2/PAP9 targeting it to the PEP complex. Close vicinity to the transcriptional apparatus may allow for the protection against the strongly oxidizing aerial environment during plant conquering of terrestrial habitats., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Favier, Gans, Boeri Erba, Signor, Muthukumar, Pfannschmidt, Blanvillain and Cobessi.)

This structural and biophysical study exploited a method of perdeuterating hen egg-white lysozyme based on the expression of insoluble protein in Escherichia coli followed by in-column chemical refolding. This allowed detailed comparisons with perdeuterated lysozyme produced in the yeast Pichia pastoris , as well as with unlabelled lysozyme. Both perdeuterated variants exhibit reduced thermal stability and enzymatic activity in comparison with hydrogenated lysozyme. The thermal stability of refolded perdeuterated lysozyme is 4.9°C lower than that of the perdeuterated variant expressed and secreted in yeast and 6.8°C lower than that of the hydrogenated Gallus gallus protein. However, both perdeuterated variants exhibit a comparable activity. Atomic resolution X-ray crystallographic analyses show that the differences in thermal stability and enzymatic function are correlated with refolding and deuteration effects. The hydrogen/deuterium isotope effect causes a decrease in the stability and activity of the perdeuterated analogues; this is believed to occur through a combination of changes to hydrophobicity and protein dynamics. The lower level of thermal stability of the refolded perdeuterated lysozyme is caused by the unrestrained Asn103 peptide-plane flip during the unfolded state, leading to a significant increase in disorder of the Lys97-Gly104 region following subsequent refolding. An ancillary outcome of this study has been the development of an efficient and financially viable protocol that allows stable and active perdeuterated lysozyme to be more easily available for scientific applications., (© Ramos et al. 2021.)

S100A9, with its congener S100A8, belongs to the S100 family of calcium-binding proteins found exclusively in vertebrates. These two proteins are major constituents of neutrophils. In response to a pathological condition, they can be released extracellularly and become alarmins that induce both pro- and anti-inflammatory signals, through specific cell surface receptors. They also act as antimicrobial agents, mainly as a S100A8/A9 heterocomplex, through metal sequestration. The mechanisms whereby divalent cations modulate the extracellular functions of S100A8 and S100A9 are still unclear. Importantly, it has been proposed that these ions may affect both the ternary and quaternary structure of these proteins, thereby influencing their physiological properties. In the present study, we report the crystal structures of WT and C80A murine S100A9 (mS100A9), determined at 1.45 and 2.35 Å resolution, respectively, in the presence of calcium and zinc. These structures reveal a canonical homodimeric form for the protein. They also unravel an intramolecular disulfide bridge that stabilizes the C-terminal tail in a rigid conformation, thus shaping a second Zn-binding site per S100A9 protomer. In solution, mS100A9 apparently binds only two zinc ions per homodimer, with an affinity in the micromolar range, and aggregates in the presence of excess zinc. Using mass spectrometry, we demonstrate that mS100A9 can form both non-covalent and covalent homodimers with distinct disulfide bond patterns. Interestingly, calcium and zinc seem to affect differentially the relative proportion of these forms. We discuss how the metal-dependent interconversion between mS100A9 homodimers may explain the versatility of physiological functions attributed to the protein., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Fatty acid β-oxidation (FAO) and oxidative phosphorylation (OXPHOS) are mitochondrial redox processes that generate ATP. The biogenesis of the respiratory Complex I, a 1 MDa multiprotein complex that is responsible for initiating OXPHOS, is mediated by assembly factors including the mitochondrial complex I assembly (MCIA) complex. However, the organisation and the role of the MCIA complex are still unclear. Here we show that ECSIT functions as the bridging node of the MCIA core complex. Furthermore, cryo-electron microscopy together with biochemical and biophysical experiments reveal that the C-terminal domain of ECSIT directly binds to the vestigial dehydrogenase domain of the FAO enzyme ACAD9 and induces its deflavination, switching ACAD9 from its role in FAO to an MCIA factor. These findings provide the structural basis for the MCIA complex architecture and suggest a unique molecular mechanism for coordinating the regulation of the FAO and OXPHOS pathways to ensure an efficient energy production., (© 2020 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

The initial greening of angiosperms involves light activation of photoreceptors that trigger photomorphogenesis, followed by the development of chloroplasts. In these semi-autonomous organelles, construction of the photosynthetic apparatus depends on the coordination of nuclear and plastid gene expression. Here, we show that the expression of PAP8, an essential subunit of the plastid-encoded RNA polymerase (PEP) in Arabidopsis thaliana, is under the control of a regulatory element recognized by the photomorphogenic factor HY5. PAP8 protein is localized and active in both plastids and the nucleus, and particularly required for the formation of late photobodies. In the pap8 albino mutant, phytochrome-mediated signalling is altered, degradation of the chloroplast development repressors PIF1/PIF3 is disrupted, HY5 is not stabilized, and the expression of the photomorphogenesis regulator GLK1 is impaired. PAP8 translocates into plastids via its targeting pre-sequence, interacts with the PEP and eventually reaches the nucleus, where it can interact with another PEP subunit pTAC12/HMR/PAP5. Since PAP8 is required for the phytochrome B-mediated signalling cascade and the reshaping of the PEP activity, it may coordinate nuclear gene expression with PEP-driven chloroplastic gene expression during chloroplast biogenesis., (© 2020 The Authors.)

Temperate bacteriophages can enter one of two life cycles following infection of a sensitive host: the lysogenic or the lytic life cycle. The choice between the two alternative life cycles is dependent upon a tight regulation of promoters and their cognate regulatory proteins within the phage genome. We investigated the genetic switch of TP901-1, a bacteriophage of Lactococcus lactis , controlled by the CI repressor and the modulator of repression (MOR) antirepressor and their interactions with DNA. We determined the solution structure of MOR, and we solved the crystal structure of MOR in complex with the N-terminal domain of CI, revealing the structural basis of MOR inhibition of CI binding to the DNA operator sites. 15 N NMR Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion and rotating frame R 1ρ measurements demonstrate that MOR displays molecular recognition dynamics on two different time scales involving a repacking of aromatic residues at the interface with CI. Mutations in the CI:MOR binding interface impair complex formation in vitro, and when introduced in vivo, the bacteriophage switch is unable to choose the lytic life cycle showing that the CI:MOR complex is essential for proper functioning of the genetic switch. On the basis of sequence alignments, we show that the structural features of the MOR:CI complex are likely conserved among a larger family of bacteriophages from human pathogens implicated in transfer of antibiotic resistance., Competing Interests: The authors declare no competing interest.

Mass spectrometry (MS) is an effective approach for determining the mass of biomolecules with high accuracy, sensitivity and speed. Over the past 25 years, MS performed under non-denaturing conditions ("native MS") has been successfully exploited to investigate non-covalently associated biomolecules. Here we illustrate native MS applications aimed at studying protein-ligand interactions and structures of biomolecular assemblies, including both soluble and membrane protein complexes. Moreover, we review how the partial dissociation of holo-complexes can be used to determine the stoichiometry of subunits and their topology. We also describe "native top-down MS", an approach based on Fourier Transform MS (FT MS), whereby non-covalent interactions are preserved while covalent bonds are selectively fragmented. Overall, native MS plays an increasingly important role in integrative structural biology, helping researchers to elucidate the three dimensional architecture of intricate macromolecular complexes., Competing Interests: Declaration of Competing Interest No conflict to declare., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Native mass spectrometry is an emerging technique in biology that gives the possibility to study noncovalently bound complexes with high sensitivity and accuracy. It thus allows the characterization of macromolecular assemblies, assessing their mass and stoichiometries and mapping the interacting surfaces. In this review, we discuss the application of native mass spectrometry to dynamic molecular machines based on multiple weak interactions. In the study of these machines, it is crucial to understand which and under which conditions various complexes form at any time point. We focus on the specific example of the iron-sulfur cluster biogenesis machine because this is an archetype of a dynamic machine that requires very specific and demanding experimental conditions, such as anaerobicity and the need of retaining the fold of marginally folded proteins. We describe the advantages, challenges and current limitations of the technique by providing examples from our own experience and suggesting possible future solutions., (© 2020 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Cyt1Aa is the one of four crystalline protoxins produced by mosquitocidal bacterium Bacillus thuringiensis israelensis (Bti) that has been shown to delay the evolution of insect resistance in the field. Limiting our understanding of Bti efficacy and the path to improved toxicity and spectrum has been ignorance of how Cyt1Aa crystallizes in vivo and of its mechanism of toxicity. Here, we use serial femtosecond crystallography to determine the Cyt1Aa protoxin structure from sub-micron-sized crystals produced in Bti. Structures determined under various pH/redox conditions illuminate the role played by previously uncharacterized disulfide-bridge and domain-swapped interfaces from crystal formation in Bti to dissolution in the larval mosquito midgut. Biochemical, toxicological and biophysical methods enable the deconvolution of key steps in the Cyt1Aa bioactivation cascade. We additionally show that the size, shape, production yield, pH sensitivity and toxicity of Cyt1Aa crystals grown in Bti can be controlled by single atom substitution.

Human transthyretin (TTR) is implicated in several fatal forms of amyloidosis. Many mutations of TTR have been identified; most of these are pathogenic, but some offer protective effects. The molecular basis underlying the vastly different fibrillation behaviours of these TTR mutants is poorly understood. Here, on the basis of neutron crystallography, native mass spectrometry and modelling studies, we propose a mechanism whereby TTR can form amyloid fibrils via a parallel equilibrium of partially unfolded species that proceeds in favour of the amyloidogenic forms of TTR. It is suggested that unfolding events within the TTR monomer originate at the C-D loop of the protein, and that destabilising mutations in this region enhance the rate of TTR fibrillation. Furthermore, it is proposed that the binding of small molecule drugs to TTR stabilises non-amyloidogenic states of TTR in a manner similar to that occurring for the protective mutants of the protein.

Native mass spectrometry (MS) enables the characterization of macromolecular assemblies with high sensitivity. It can reveal the stoichiometry of subunits as well as their two-dimensional interaction network and provide information regarding the dynamic behavior of macromolecular complexes. Here, we describe the workflow to perform native MS experiments. In addition, we illustrate the quality control analysis of proteins using MS in denaturing conditions.

The chapter author provided the below additional text to be added in the acknowledgement section. This has now been updated in the revised version of the book.

Altered synaptic function has been associated with neurological and psychiatric conditions including intellectual disability, schizophrenia and autism spectrum disorder (ASD). Amongst the recently discovered synaptic proteins is p140Cap, an adaptor that localizes at dendritic spines and regulates their maturation and physiology. We recently showed that p140Cap knockout mice have cognitive deficits, impaired long-term potentiation (LTP) and long-term depression (LTD), and immature, filopodia-like dendritic spines. Only a few p140Cap interacting proteins have been identified in the brain and the molecular complexes and pathways underlying p140Cap synaptic function are largely unknown. Here, we isolated and characterized the p140Cap synaptic interactome by co-immunoprecipitation from crude mouse synaptosomes, followed by mass spectrometry-based proteomics. We identified 351 p140Cap interactors and found that they cluster to sub complexes mostly located in the postsynaptic density (PSD). p140Cap interactors converge on key synaptic processes, including transmission across chemical synapses, actin cytoskeleton remodeling and cell-cell junction organization. Gene co-expression data further support convergent functions: the p140Cap interactors are tightly co-expressed with each other and with p140Cap. Importantly, the p140Cap interactome and its co-expression network show strong enrichment in genes associated with schizophrenia, autism, bipolar disorder, intellectual disability and epilepsy, supporting synaptic dysfunction as a shared biological feature in brain diseases. Overall, our data provide novel insights into the molecular organization of the synapse and indicate that p140Cap acts as a hub for postsynaptic complexes relevant to psychiatric and neurological disorders.

Innate immune receptors NOD1 and NOD2 are activated by bacterial peptidoglycans leading to recruitment of adaptor kinase RIP2, which, upon phosphorylation and ubiquitination, becomes a scaffold for downstream effectors. The kinase domain (RIP2K) is a pharmaceutical target for inflammatory diseases caused by aberrant NOD2-RIP2 signalling. Although structures of active RIP2K in complex with inhibitors have been reported, the mechanism of RIP2K activation remains to be elucidated. Here we analyse RIP2K activation by combining crystal structures of the active and inactive states with mass spectrometric characterization of their phosphorylation profiles. The active state has Helix αC inwardly displaced and the phosphorylated Activation Segment (AS) disordered, whilst in the inactive state Helix αC is outwardly displaced and packed against the helical, non-phosphorylated AS. Biophysical measurements show that the active state is a stable dimer whilst the inactive kinase is in a monomer-dimer equilibrium, consistent with the observed structural differences at the dimer interface. We conclude that RIP2 kinase auto-phosphorylation is intimately coupled to dimerization, similar to the case of BRAF. Our results will help drug design efforts targeting RIP2 as a potential treatment for NOD2-RIP2 related inflammatory diseases.

The spontaneous formation of biological higher-order structures from smaller building blocks, called self-assembly, is a fundamental attribute of life. Although the protein self-assembly is a time-dependent process that occurs at the molecular level, its current understanding originates either from static structures of trapped intermediates or from modeling. Nuclear magnetic resonance (NMR) spectroscopy has the unique ability to monitor structural changes in real time; however, its size limitation and time-resolution constraints remain a challenge when studying the self-assembly of large biological particles. We report the application of methyl-specific isotopic labeling combined with relaxation-optimized NMR spectroscopy to overcome both size- and time-scale limitations. We report for the first time the self-assembly process of a half-megadalton protein complex that was monitored at the structural level, including the characterization of intermediate states, using a mutagenesis-free strategy. NMR was used to obtain individual kinetics data on the different transient intermediates and the formation of final native particle. In addition, complementary time-resolved electron microscopy and native mass spectrometry were used to characterize the low-resolution structures of oligomerization intermediates.

Artificial enzymes are required to catalyse non-natural reactions. Here, a hybrid catalyst was developed by embedding a novel Ru complex in the transport protein NikA. The protein scaffold activates the bound Ru complex to produce a catalyst with high regio- and stereo-selectivity. The hybrid efficiently and stably produced α-hydroxy-β-chloro chlorohydrins from alkenes (up to 180 TON with a TOF of 1050 h -1 ).

How the very first step in nucleosome assembly, deposition of histone H3-H4 as tetramers or dimers on DNA, is accomplished remains largely unclear. Here, we report that yeast chromatin assembly factor 1 (CAF1), a conserved histone chaperone complex that deposits H3-H4 during DNA replication, binds a single H3-H4 heterodimer in solution. We identify a new DNA-binding domain in the large Cac1 subunit of CAF1, which is required for high-affinity DNA binding by the CAF1 three-subunit complex, and which is distinct from the previously described C-terminal winged-helix domain. CAF1 binds preferentially to DNA molecules longer than 40 bp, and two CAF1-H3-H4 complexes concertedly associate with DNA molecules of this size, resulting in deposition of H3-H4 tetramers. While DNA binding is not essential for H3-H4 tetrasome deposition in vitro, it is required for efficient DNA synthesis-coupled nucleosome assembly. Mutant histones with impaired H3-H4 tetramerization interactions fail to release from CAF1, indicating that DNA deposition of H3-H4 tetramers by CAF1 requires a hierarchical cooperation between DNA binding, H3-H4 deposition and histone tetramerization.

Vesiculoviruses enter cells by membrane fusion, driven by a large, low-pH-induced, conformational change in the fusion glycoprotein G that involves transition from a trimeric pre-fusion toward a trimeric post-fusion state via monomeric intermediates. Here, we present the structure of the G fusion protein at intermediate pH for two vesiculoviruses, vesicular stomatitis virus (VSV) and Chandipura virus (CHAV), which is responsible for deadly encephalopathies. First, a CHAV G crystal structure shows two intermediate conformations forming a flat dimer of heterodimers. On virions, electron microscopy (EM) and tomography reveal monomeric spikes similar to one of the crystal conformations. In solution, mass spectrometry shows dimers of G. Finally, mutations at a dimer interface, involving fusion domains associated in an antiparallel manner to form an intermolecular β-sheet, affect G fusion properties. The location of the compensatory mutations restoring fusion activity strongly suggests that this interface is functionally relevant. This work reveals the range of G structural changes and suggests that G monomers can re-associate, through antiparallel interactions between fusion domains, into dimers that play a role at some early stage of the fusion process., (© 2017 The Authors.)

It is well established that the formation of transthyretin (TTR) amyloid fibrils is linked to the destabilization and dissociation of its tetrameric structure into insoluble aggregates. Isotope labeling is used for the study of TTR by NMR, neutron diffraction, and mass spectrometry (MS). Here MS, thioflavin T fluorescence, and crystallographic data demonstrate that while the X-ray structures of unlabeled and deuterium-labeled TTR are essentially identical, subunit exchange kinetics and amyloid formation are accelerated for the deuterated protein. However, a slower subunit exchange is noted in deuterated solvent, reflecting the poorer solubility of non-polar protein side chains in such an environment. These observations are important for the interpretation of kinetic studies involving deuteration. The destabilizing effects of TTR deuteration are rather similar in character to those observed for aggressive mutations of TTR such as L55P (associated with familial amyloid polyneuropathy)., (© 2016 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.)

The CI repressor from the temperate bacteriophage TP901-1 consists of two folded domains, an N-terminal helix-turn-helix DNA-binding domain (NTD) and a C-terminal oligomerization domain (CTD), which we here suggest to be further divided into CTD1 and CTD2. Full-length CI is a hexameric protein, whereas a truncated version, CI∆58, forms dimers. We identify the dimerization region of CI∆58 as CTD1 and determine its secondary structure to be helical both within the context of CI∆58 and in isolation. To our knowledge this is the first time that a helical dimerization domain has been found in a phage repressor. We also precisely determine the length of the flexible linker connecting the NTD to the CTD. Using electrophoretic mobility shift assays and native mass spectrometry, we show that CI∆58 interacts with the OL operator site as one dimer bound to both half-sites, and with much higher affinity than the isolated NTD domain thus demonstrating cooperativity between the two DNA binding domains. Finally, using small angle X-ray scattering data and state-of-the-art ensemble selection techniques, we delineate the conformational space sampled by CI∆58 in solution, and we discuss the possible role that the dynamics play in CI-repressor function.

The vacuolar protein sorting 4 AAA-ATPase (Vps4) recycles endosomal sorting complexes required for transport (ESCRT-III) polymers from cellular membranes. Here we present a 3.6-Å X-ray structure of ring-shaped Vps4 from Metallosphera sedula (MsVps4), seen as an asymmetric pseudohexamer. Conserved key interface residues are shown to be important for MsVps4 assembly, ATPase activity in vitro, ESCRT-III disassembly in vitro and HIV-1 budding. ADP binding leads to conformational changes within the protomer, which might propagate within the ring structure. All ATP-binding sites are accessible and the pseudohexamer binds six ATP with micromolar affinity in vitro. In contrast, ADP occupies one high-affinity and five low-affinity binding sites in vitro, consistent with conformational asymmetry induced on ATP hydrolysis. The structure represents a snapshot of an assembled Vps4 conformation and provides insight into the molecular motions the ring structure undergoes in a concerted action to couple ATP hydrolysis to ESCRT-III substrate disassembly.

Protein complexes play pivotal roles in cellular life. Nevertheless, their characterization remains a substantial challenge. Mass spectrometry (MS) is an emerging tool to study protein assemblies, and electrospray ionization (ESI) is often used because it preserves non-covalent interactions. Matrix-assisted laser desorption/ionization (MALDI) represents an important alternative to ESI because it is more tolerant to salts and detergents (e.g. necessary in the case of membrane complex analyses). Prior to MALDI-MS, the subunits should be crosslinked (XLed). Moreover, crosslinking (XLing) is useful when constraint distances are determined to obtain low-resolution structural information. Here we report a novel XLing approach to study protein complexes with MALDI-MS. We investigated two tetramers (i.e. alcohol dehydrogenase and aldolase) larger than 140 kDa at two pH values (7.2 and 8.0). We tested two different crosslinkers (XLers) (i.e. BS(3) and glutaraldehyde), used separately or in combination. We utilized gentle agitation and ultracentrifugation. Our data shows that the pH influenced the XLing when using a single XLer. Combining two XLers was demonstrated to be more efficient than using a reagent alone. In particular, the combination determined a higher degree of XLing and lower mass shift. This could suggest a ranking in target amino acid availability. First residues at specific distances are linked by BS(3) , then glutaraldehyde binds residues that are still available at larger distances. Ultracentrifugation and gentle agitation both provide similar degrees of XLing, but the former method determined a lower mass increment resulting from redundant XLing. To conclude, we present an efficient dual XLing approach for determining mass and stoichiometry of protein assemblies., (Copyright © 2015 John Wiley & Sons, Ltd.)

Mass spectrometry (MS) is a powerful tool for determining the mass of biomolecules with high accuracy and sensitivity. MS performed under so-called "native conditions" (native MS) can be used to determine the mass of biomolecules that associate noncovalently. Here we review the application of native MS to the study of protein-ligand interactions and its emerging role in elucidating the structure of macromolecular assemblies, including soluble and membrane protein complexes. Moreover, we discuss strategies aimed at determining the stoichiometry and topology of subunits by inducing partial dissociation of the holo-complex. We also survey recent developments in "native top-down MS", an approach based on Fourier Transform MS, whereby covalent bonds are broken without disrupting non-covalent interactions. Given recent progress, native MS is anticipated to play an increasingly important role for researchers interested in the structure of macromolecular complexes., (© 2015 The Protein Society.)

MS has emerged as an important tool to investigate noncovalent interactions between proteins and various ligands (e.g. other proteins, small molecules, or drugs). In particular, ESI under so-called "native conditions" (a.k.a. "native MS") has considerably expanded the scope of such investigations. For instance, ESI quadrupole time of flight (Q-TOF) instruments have been used to probe the precise stoichiometry of protein assemblies, the interactions between subunits and the position of subunits within the complex (i.e. defining core and peripheral subunits). This review highlights several illustrative native Q-TOF-based investigations and recent seminal contributions of top-down MS (i.e. Fourier transform (FT) MS) to the characterization of noncovalent complexes. Combined top-down and native MS, recently demonstrated in "high-mass modified" orbitrap mass spectrometers, and further improvements needed for the enhanced investigation of biologically significant noncovalent interactions by MS will be discussed., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Effectively determining masses of proteins is critical to many biological studies (e.g. for structural biology investigations). Accurate mass determination allows one to evaluate the correctness of protein primary sequences, the presence of mutations and/or post-translational modifications, the possible protein degradation, the sample homogeneity, and the degree of isotope incorporation in case of labelling (e.g. (13)C labelling). Electrospray ionisation (ESI) mass spectrometry (MS) is widely used for mass determination of denatured proteins, but its efficiency is affected by the composition of the sample buffer. In particular, the presence of salts, detergents, and contaminants severely undermines the effectiveness of protein analysis by ESI-MS. Matrix-assisted laser desorption/ionization (MALDI) MS is an attractive alternative, due to its salt tolerance and the simplicity of data acquisition and interpretation. Moreover, the mass determination of large heterogeneous proteins (bigger than 100 kDa) is easier by MALDI-MS due to the absence of overlapping high charge state distributions which are present in ESI spectra. Here we present an accessible approach for analysing proteins larger than 100 kDa by MALDI-time of flight (TOF). We illustrate the advantages of using a mixture of two matrices (i.e. 2,5-dihydroxybenzoic acid and α-cyano-4-hydroxycinnamic acid) and the utility of the thin layer method as approach for sample deposition. We also discuss the critical role of the matrix and solvent purity, of the standards used for calibration, of the laser energy, and of the acquisition time. Overall, we provide information necessary to a novice for analysing intact proteins larger than 100 kDa by MALDI-MS.

Protein phosphorylation tightly regulates specific binding of effector proteins that control many diverse biological functions of cells (e. g. signaling, migration and proliferation). p140Cap is an adaptor protein, specifically expressed in brain, testis and epithelial cells, that undergoes phosphorylation and tunes its interactions with other regulatory molecules via post-translation modification. In this work, using mass spectrometry, we found that p140Cap is in vivo phosphorylated on tyrosine (Y) within the peptide GEGLpYADPYGLLHEGR (from now on referred to as EGLYA) as well as on three serine residues. Consistently, EGLYA has the highest score of in silico prediction of p140Cap phosphorylation. To further investigate the p140Cap function, we performed site specific mutagenesis on tyrosines inserted in EGLYA and EPLYA, a second sequence with the same highest score of phosphorylation. The mutant protein, in which both EPLYA/EGLYA tyrosines were converted to phenylalanine, was no longer tyrosine phosphorylated, despite the presence of other tyrosine residues in p140Cap sequence. Moreover, this mutant lost its ability to bind the C-terminal Src kinase (Csk), previously shown to interact with p140Cap by Far Western analysis. In addition, we found that in vitro and in HEK-293 cells, the Abelson kinase is the major kinase involved in p140Cap tyrosine phosphorylation on the EPLYA and EGLYA sequences. Overall, these data represent an original attempt to in vivo characterise phosphorylated residues of p140Cap. Elucidating the function of p140Cap will provide novel insights into its biological activity not only in normal cells, but also in tumors.

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) has been demonstrated to be a valuable tool to investigate noncovalent interactions of biomolecules. The direct detection of noncovalent assemblies is often more troublesome than with electrospray ionization. Using dedicated sample preparation techniques and carefully optimized instrumental parameters, a number of biomolecule assemblies were successfully analyzed. For complexes dissociating under MALDI conditions, covalent stabilization with chemical cross-linking is a suitable alternative. Indirect methods allow the detection of noncovalent assemblies by monitoring the fading of binding partners or altered H/D exchange patterns.

Repair of DNA double-strand breaks (DSBs) is essential for cell survival and maintaining genome integrity. DSBs are repaired in a stepwise manner by homologous recombination. Here, we focused on the early steps of DSB repair, including DSB recognition, which is still only poorly understood. In prokaryotes, this process has been proposed to involve the RecN protein, a member of the structural maintenance of chromosome (SMC) protein family, which include key eukaryotic and prokaryotic proteins such as cohesin, condensin, and Rad50. An extensive high- and low-resolution structural analysis of Deinococcus radiodurans RecN using a combination of protein crystallography and small-angle X-ray scattering enabled us to assemble a quasi-atomic model of the entire RecN protein, representing the complete structure of a SMC-like protein. These results, together with a thorough biochemical and mutational study of RecN, allow us to propose a model for the role of RecN in DSB repair., (Copyright © 2012 Elsevier Ltd. All rights reserved.)