Selectivity of Lewy body protein interactions along the aggregation pathway of α-synuclein.

The aggregation of alpha-synuclein (α-SYN) follows a cascade of oligomeric, prefibrillar and fibrillar forms, culminating in the formation of Lewy Bodies (LB), the pathological hallmarks of Parkinson's Disease. Although LB contain over 70 proteins, the potential for interactions along the aggregation pathway of α-SYN is unknown. Here we propose a map of interactions of 65 proteins against different species of α-SYN. We measured binding to monomeric α-SYN using AlphaScreen, a sensitive nano-bead luminescence assay for detection of protein interactions. To access oligomeric species, we used the pathological mutants of α-SYN (A30P, G51D and A53T) which form oligomers with distinct properties. Finally, we generated amyloid fibrils from recombinant α-SYN. Binding to oligomers and fibrils was measured by two-color coincidence detection (TCCD) on a single molecule spectroscopy setup. Overall, we demonstrate that LB components are recruited to specific steps in the aggregation of α-SYN, uncovering future targets to modulate aggregation in synucleinopathies.

MyD88 TIR domain higher-order assembly interactions revealed by microcrystal electron diffraction and serial femtosecond crystallography.

MyD88 and MAL are Toll-like receptor (TLR) adaptors that signal to induce pro-inflammatory cytokine production. We previously observed that the TIR domain of MAL (MALTIR) forms filaments in vitro and induces formation of crystalline higher-order assemblies of the MyD88 TIR domain (MyD88TIR). These crystals are too small for conventional X-ray crystallography, but are ideally suited to structure determination by microcrystal electron diffraction (MicroED) and serial femtosecond crystallography (SFX). Here, we present MicroED and SFX structures of the MyD88TIR assembly, which reveal a two-stranded higher-order assembly arrangement of TIR domains analogous to that seen previously for MALTIR. We demonstrate via mutagenesis that the MyD88TIR assembly interfaces are critical for TLR4 signaling in vivo, and we show that MAL promotes unidirectional assembly of MyD88TIR. Collectively, our studies provide structural and mechanistic insight into TLR signal transduction and allow a direct comparison of the MicroED and SFX techniques.

Rapid HIV-1 Capsid Interaction Screening Using Fluorescence Fluctuation Spectroscopy.

The HIV capsid is a multifunctional protein capsule that mediates the delivery of the viral genetic material into the nucleus of the target cell. Host cell proteins bind to a number of repeating binding sites on the capsid to regulate steps in the replication cycle. Here, we develop a fluorescence fluctuation spectroscopy method using self-assembled capsid particles as the bait to screen for fluorescence-labeled capsid-binding analytes ("prey" molecules) in solution. The assay capitalizes on the property of the HIV capsid as a multivalent interaction platform, facilitating high sensitivity detection of multiple prey molecules that have accumulated onto capsids as spikes in fluorescence intensity traces. By using a scanning stage, we reduced the measurement time to 10 s without compromising on sensitivity, providing a rapid binding assay for screening libraries of potential capsid interactors. The assay can also identify interfaces for host molecule binding by using capsids with defects in known interaction interfaces. Two-color coincidence detection using the fluorescent capsid as the bait further allows the quantification of binding levels and determination of binding affinities. Overall, the assay provides new tools for the discovery and characterization of molecules used by the HIV capsid to orchestrate infection. The measurement principle can be extended for the development of sensitive interaction assays, utilizing natural or synthetic multivalent scaffolds as analyte-binding platforms.

SARS-CoV-2 proteases PLpro and 3CLpro cleave IRF3 and critical modulators of inflammatory pathways (NLRP12 and TAB1): implications for disease presentation across species.

The genome of SARS-CoV-2 encodes two viral proteases (NSP3/papain-like protease and NSP5/3C-like protease) that are responsible for cleaving viral polyproteins during replication. Here, we discovered new functions of the NSP3 and NSP5 proteases of SARS-CoV-2, demonstrating that they could directly cleave proteins involved in the host innate immune response. We identified 3 proteins that were specifically and selectively cleaved by NSP3 or NSP5: IRF-3, and NLRP12 and TAB1, respectively. Direct cleavage of IRF3 by NSP3 could explain the blunted Type-I IFN response seen during SARS-CoV-2 infections while NSP5 mediated cleavage of NLRP12 and TAB1 point to a molecular mechanism for enhanced production of cytokines and inflammatory responThe genome of SARS-CoV-2 encodes two viral proteases (NSP3/papain-like protease and NSP5/3C-like protease) that are responsible for cleaving viral polyproteins during replication. Here, we discovered new functions of the NSP3 and NSP5 proteases of SARS-CoV-2, demonstrating that they could directly cleave proteins involved in the host innate immune response. We identified 3 proteins that were specifically and selectively cleaved by NSP3 or NSP5: IRF-3, and NLRP12 and TAB1, respectively. Direct cleavage of IRF3 by NSP3 could explain the blunted Type-I IFN response seen during SARS-CoV-2 infections while NSP5 mediated cleavage of NLRP12 and TAB1 point to a molecular mechanism for enhanced production of cytokines and inflammatory response observed in COVID-19 patients. We demonstrate that in the mouse NLRP12 protein, one of the recognition site is not cleaved in our in-vitro assay. We pushed this comparative alignment of IRF-3 and NLRP12 homologs and show that the lack or presence of cognate cleavage motifs in IRF-3 and NLRP12 could contribute to the presentation of disease in cats and tigers, for example. Our findings provide an explanatory framework for indepth studies into the pathophysiology of COVID-19.

Single-molecule detection on a portable 3D-printed microscope.

Single-molecule assays have, by definition, the ultimate sensitivity and represent the next frontier in biological analysis and diagnostics. However, many of these powerful technologies require dedicated laboratories and trained personnel and have therefore remained research tools for specialists. Here, we present a single-molecule confocal system built from a 3D-printed scaffold, resulting in a compact, plug and play device called the AttoBright. This device performs single photon counting and fluorescence correlation spectroscopy (FCS) in a simple format and is widely applicable to the detection of single fluorophores, proteins, liposomes or bacteria. The power of single-molecule detection is demonstrated by detecting single α-synuclein amyloid fibrils, that are currently evaluated as biomarkers for Parkinson's disease, with an improved sensitivity of >100,000-fold over bulk measurements.

Rational evolution of the cofactor-binding site of cytochrome P450 reductase yields variants with increased activity towards specific cytochrome P450 enzymes.

NADPH-cytochrome P450 reductase (CPR) is the natural redox partner of microsomal cytochrome P450 enzymes. CPR shows a stringent preference for NADPH over the less expensive cofactor, NADH, economically limiting its use as a biocatalyst. The complexity of cofactor-linked CPR protein dynamics and the incomplete understanding of the interaction of CPR with both cofactors and electron acceptors present challenges for the successful rational engineering of a CPR with enhanced activity with NADH. Here, we report a rational evolution approach to enhance the activity of CPR with NADH, in which mutations were introduced into the NADPH-binding flavin adenine dinucleotide (FAD) domain. Multiple CPR mutants that used NADH more effectively than the wild-type CPR in the reduction of the surrogate electron acceptor, cytochrome c were found. However, most were inactive in supporting P450 activity, arguing against the use of cytochrome c as a surrogate electron acceptor. Unexpectedly, several mutants showed significantly improved activity towards CYP2C9 (mutant 1-014) and/or CYP2A6 (mutants 1-014, 1-015, 1-053 and 1-077) using NADPH, even though the mutations were introduced at locations remote from the putative CPR-P450 interaction face. Therefore, mutations at sites in the FAD domain of CPR may be promising future engineering targets to enhance P450-mediated substrate turnover. ENZYMES: NADPH-cytochrome P450 reductase - EC 1.6.2.4; cytochrome P450 - EC 1.14.14.1.

Pathological mutations differentially affect the self-assembly and polymerisation of the innate immune system signalling adaptor molecule MyD88.

BACKGROUND: Higher-order self-assembly of proteins, or "prion-like" polymerisation, is now emerging as a simple and robust mechanism for signal amplification, in particular within the innate immune system, where the recognition of pathogens or danger-associated molecular patterns needs to trigger a strong, binary response within cells. MyD88, an important adaptor protein downstream of TLRs, is one of the most recent candidates for involvement in signalling by higher order self-assembly. In this new light, we set out to re-interpret the role of polymerisation in MyD88-related diseases and study the impact of disease-associated point mutations L93P, R196C, and L252P/L265P at the molecular level. RESULTS: We first developed new in vitro strategies to characterise the behaviour of polymerising, full-length MyD88 at physiological levels. To this end, we used single-molecule fluorescence fluctuation spectroscopy coupled to a eukaryotic cell-free protein expression system. We were then able to explore the polymerisation propensity of full-length MyD88, at low protein concentration and without purification, and compare it to the behaviours of the isolated TIR domain and death domain that have been shown to have self-assembly properties on their own. These experiments demonstrate that the presence of both domains is required to cooperatively lead to efficient polymerisation of the protein. We then characterised three pathological mutants of MyD88. CONCLUSION: We discovered that all mutations block the ability of MyD88 to polymerise fully. Interestingly, we show that, in contrast to L93P and R196C, L252P is a gain-of-function mutation, which allows the MyD88 mutant to form extremely stable oligomers, even at low nanomolar concentrations. Thus, our results shed new light on the digital "all-or-none" responses by the myddosomes and the behaviour of the oncogenic mutations of MyD88.

Homodimerization regulates an endothelial specific signature of the SOX18 transcription factor.

During embryogenesis, vascular development relies on a handful of transcription factors that instruct cell fate in a distinct sub-population of the endothelium (1). The SOXF proteins that comprise SOX7, 17 and 18, are molecular switches modulating arterio-venous and lymphatic endothelial differentiation (2,3). Here, we show that, in the SOX-F family, only SOX18 has the ability to switch between a monomeric and a dimeric form. We characterized the SOX18 dimer in binding assays in vitro, and using a split-GFP reporter assay in a zebrafish model system in vivo. We show that SOX18 dimerization is driven by a novel motif located in the vicinity of the C-terminus of the DNA binding region. Insertion of this motif in a SOX7 monomer forced its assembly into a dimer. Genome-wide analysis of SOX18 binding locations on the chromatin revealed enrichment for a SOX dimer binding motif, correlating with genes with a strong endothelial signature. Using a SOX18 small molecule inhibitor that disrupts dimerization, we revealed that dimerization is important for transcription. Overall, we show that dimerization is a specific feature of SOX18 that enables the recruitment of key endothelial transcription factors, and refines the selectivity of the binding to discrete genomic locations assigned to endothelial specific genes.

Cell-free formation and interactome analysis of caveolae.

Caveolae have been linked to the regulation of signaling pathways in eukaryotic cells through direct interactions with caveolins. Here, we describe a cell-free system based on Leishmania tarentolae (Lt) extracts for the biogenesis of caveolae and show its use for single-molecule interaction studies. Insertion of expressed caveolin-1 (CAV1) into Lt membranes was analogous to that of caveolin in native membranes. Electron tomography showed that caveolins generate domains of precise size and curvature. Cell-free caveolae were used in quantitative assays to test the interaction of membrane-inserted caveolin with signaling proteins and to determine the stoichiometry of interactions. Binding of membrane-inserted CAV1 to several proposed binding partners, including endothelial nitric-oxide synthase, was negligible, but a small number of proteins, including TRAF2, interacted with CAV1 in a phosphorylation-(CAV1Y14)-stimulated manner. In cells subjected to oxidative stress, phosphorylated CAV1 recruited TRAF2 to the early endosome forming a novel signaling platform. These findings lead to a novel model for cellular stress signaling by CAV1.

Unexpected instabilities explain batch-to-batch variability in cell-free protein expression systems.

Cell-free methods of protein synthesis offer rapid access to expressed proteins. Though the amounts produced are generally only at a small scale, these are sufficient to perform protein-protein interaction assays and tests of enzymatic activity. As such they are valuable tools for the biochemistry and bioengineering community. However the most complex, eukaryotic cell-free systems are difficult to manufacture in house and can be prohibitively expensive to obtain from commercial sources. The Leishmania tarentolae system offers a relatively cheap alternative which is capable of producing difficult to express proteins, but which is simpler to produce in large scale. However, this system suffers from batch-to-batch variability, which has been accepted as a consequence of the complexity of the extracts. Here we show an unexpected origin for the variability observed and demonstrate that small variations in a single parameter can dramatically affect expression, such that minor pipetting errors can have major effects on yields. L. tarentolae cell-free lysate activity is shown to be more stable to changes in Mg2+ concentration at a lower ratio of feed solution to lysate in the reaction than typically used, and a higher Mg2+ optimum. These changes essentially eliminate batch-to-batch variability of L. tarentolae lysate activity and permit their full potential to be realized.

Unveiling a Selective Mechanism for the Inhibition of α-Synuclein Aggregation by ß-Synuclein.

α-Synuclein (αS) is an intrinsically disordered protein that is associated with Parkinson's disease (PD) through its ability to self-assemble into oligomers and fibrils. Inhibition of this oligomerization cascade is an interesting approach to developing therapeutical strategies and ß-synuclein (ßS) has been described as a natural negative regulator of this process. However, the biological background and molecular mechanisms by which this inhibition occurs is unclear. Herein, we focused on assessing the effect of ßS on the aggregation of five αS pathological mutants linked to early-onset PD (A30P, E46K, H50Q, G51D and A53T). By coupling single molecule fluorescence spectroscopy to a cell-free protein expression system, we validated the ability of ßS to act as a chaperone of αS, effectively inhibiting its aggregation. Interestingly, we found that ßS does so in a selective manner, i.e., is a more effective inhibitor for certain αS pathological mutants-A30P and G51D-as compared to E46K, H50Q and A53T. Moreover, two-color coincidence experiments proved that this discrepancy is due to a preferential incorporation of ßS into smaller oligomers of αS. This was validated by showing that the chaperoning effect was lost when proteins were mixed after being expressed individually. This study highlights the potential of fluorescence spectroscopy to deconstruct αS aggregation cascade and its interplay with ßS.

A Split-Luciferase Reporter Recognizing GFP and mCherry Tags to Facilitate Studies of Protein-Protein Interactions.

The use of fluorescently-tagged proteins in microscopy has become routine, and anti-GFP (Green fluorescent protein) affinity matrices are increasingly used in proteomics protocols. However, some protein-protein interactions assays, such as protein complementation assays (PCA), require recloning of each protein as a fusion with the different parts of the complementation system. Here we describe a generic system where the complementation is separated from the proteins and can be directly used with fluorescently-tagged proteins. By using nanobodies and performing tests in cell-free expression systems, we accelerated the development of multiple reporters, detecting heterodimers and homodimers or oligomers tagged with GFP or mCherry. We demonstrate that the system can detect interactions at a broad range of concentrations, from low nanomolar up to micromolar.

Cytochrome P450 is present in both ferrous and ferric forms in the resting state within intact Escherichia coli and hepatocytes.

Cytochrome P450 enzymes (P450s) are exceptionally versatile monooxygenases, mediating hydroxylations of unactivated C-H bonds, epoxidations, dealkylations, and N- and S-oxidations as well as other less common reactions. In the conventional view of the catalytic cycle, based upon studies of P450s in vitro, substrate binding to the Fe(III) resting state facilitates the first 1-electron reduction of the heme. However, the resting state of P450s in vivo has not been examined. In the present study, whole cell difference spectroscopy of bacterial (CYP101A1 and CYP176A1, i.e. P450cam and P450cin) and mammalian (CYP1A2, CYP2C9, CYP2A6, CYP2C19, and CYP3A4) P450s expressed within intact Escherichia coli revealed that both Fe(III) and Fe(II) forms of the enzyme are present in the absence of substrates. The relevance of this finding was supported by similar observations of Fe(II) P450 heme in intact rat hepatocytes. Electron paramagnetic resonance (EPR) spectroscopy of the bacterial forms in intact cells showed that a proportion of the P450 in cells was in an EPR-silent form in the native state consistent with the presence of Fe(II) P450. Coexpression of suitable cognate electron donors increased the degree of endogenous reduction to over 80%. A significant proportion of intracellular P450 remained in the Fe(II) form after vigorous aeration of cells. The addition of substrates increased the proportion of Fe(II) heme, suggesting a kinetic gate to heme reduction in the absence of substrate. In summary, these observations suggest that the resting state of P450s should be regarded as a mixture of Fe(III) and Fe(II) forms in both aerobic and oxygen-limited conditions.

Structure of myelin P2 protein from equine spinal cord.

Equine P2 protein has been isolated from horse spinal cord and its structure determined to 2.1 A. Since equine myelin is a viable alternative to bovine tissue for large-scale preparations, characterization of the proteins from equine spinal cord myelin has been initiated. There is an unusually high amount of P2 protein in equine CNS myelin compared with other species. The structure was determined by molecular replacement and subsequently refined to an R value of 0.187 (Rfree=0.233). The structure contains a molecule of the detergent LDAO and HEPES buffer in the binding cavity and is otherwise analogous to other cellular retinol-binding proteins.

Analysis of the domain properties of the novel cytochrome P450 RhF.

The properties of the heme, flavin mononucleotide (FMN) and FeS domains of P450 RhF, from Rhodococcus sp. NCIMB 9784, expressed separately and in combination are analysed. The nucleotide preference, imidazole binding and reduction potentials of the heme and FMN domains are unaltered by their separation. The intact enzyme is monomeric and the flavin is confirmed to be FMN. The two one-electron reduction potentials of the FMN are -240 and -270 mV. The spectroscopic and thermodynamic properties of the FeS domain, masked in the intact enzyme, are revealed for the first time, confirming it as a 2Fe-2S ferredoxin with a reduction potential of -214 mV.

Oxo-iron clusters in a bacterial iron-trafficking protein: new roles for a conserved motif.

We report a set of three 1.8-1.9 A resolution X-ray crystal structures of Neisseria gonorrhoeae Fbp (ferric-ion binding protein): (i) open-cleft apo-Fbp containing bound phosphate, (ii) open-cleft mono-Fe Fbp capped by nitrilotriacetate, and (iii) open-cleft trinuclear oxo-iron Fbp, the first structure of an iron-cluster adduct of a transferrin. The nine independent molecules in the unit cells provide 'snapshots' of the versatile dynamic structural roles of the conserved dityrosyl iron-binding motif (Tyr195-Tyr196) which control the capture and, possibly, processing of iron. These findings have implications for understanding bacterial iron acquisition and dissimilation, and organic/mineral interfaces.

A self-sufficient cytochrome p450 with a primary structural organization that includes a flavin domain and a [2Fe-2S] redox center.

p450 RhF from Rhodococcus sp. NCIMB 9784 is the first example of a new class of cytochrome p450 in which electrons are supplied by a novel, FMN- and Fe/S-containing, reductase partner in a fused arrangement. We have previously cloned the gene encoding the enzyme and shown it to comprise an N-terminal p450 domain fused to a reductase domain that displays similarity to the phthalate family of oxygenase reductase proteins. A reductase of this type had never previously been reported to interact with a cytochrome p450. In this report we describe the purification and partial characterization of p450 RhF. We show that the enzyme is self-sufficient in catalyzing the O-dealkylation of 7-ethoxycoumarin. The p450 RhF catalyzed O-dealkylation of 7-ethoxycoumarin is inhibited by several compounds that are known inhibitors of cytochrome p450. Presteady state kinetic analysis indicates that p450 RhF shows a 500-fold preference for NAPDH over NADH in terms of Kd value (6.6 microm versus 3.7 mm, respectively). Potentiometric studies show reduction potentials of -243 mV for the two-electron reduction of the FMN and -423 mV for the heme (in the absence of substrate).

A novel protein-mineral interface.

Transferrins transport Fe3+ and other metal ions in mononuclear-binding sites. We present the first evidence that a member of the transferrin superfamily is able to recognize multi-nuclear oxo-metal clusters, small mineral fragments that are the most abundant forms of many metals in the environment. We show that the ferric ion-binding protein from Neisseria gonorrhoeae (nFbp) readily binds clusters of Fe3+, Ti4+, Zr4+ or Hf4+ in solution. The 1.7 A resolution crystal structure of Hf-nFbp reveals three distinct types of clusters in an open, positively charged cleft between two hinged protein domains. A di-tyrosyl cluster nucleation motif (Tyr195-Tyr196) is situated at the bottom of this cleft and binds either a trinuclear oxo-Hf cluster, which is capped by phosphate, or a pentanuclear cluster, which in turn can be capped with phosphate. This first high-resolution structure of a protein-mineral interface suggests a novel metal-uptake mechanism and provides a model for protein-mediated mineralization/dissimilation, which plays a critical role in geochemical processes.