Interrogation and validation of the interactome of neuronal Munc18-interacting Mint proteins with AlphaFold2.

Munc18-interacting proteins (Mints) are multidomain adaptors that regulate neuronal membrane trafficking, signaling, and neurotransmission. Mint1 and Mint2 are highly expressed in the brain with overlapping roles in the regulation of synaptic vesicle fusion required for neurotransmitter release by interacting with the essential synaptic protein Munc18-1. Here, we have used AlphaFold2 to identify and then validate the mechanisms that underpin both the specific interactions of neuronal Mint proteins with Munc18-1 as well as their wider interactome. We found that a short acidic α-helical motif within Mint1 and Mint2 is necessary and sufficient for specific binding to Munc18-1 and binds a conserved surface on Munc18-1 domain3b. In Munc18-1/2 double knockout neurosecretory cells, mutation of the Mint-binding site reduces the ability of Munc18-1 to rescue exocytosis, and although Munc18-1 can interact with Mint and Sx1a (Syntaxin1a) proteins simultaneously in vitro, we find that they have mutually reduced affinities, suggesting an allosteric coupling between the proteins. Using AlphaFold2 to then examine the entire cellular network of putative Mint interactors provides a structural model for their assembly with a variety of known and novel regulatory and cargo proteins including ADP-ribosylation factor (ARF3/ARF4) small GTPases and the AP3 clathrin adaptor complex. Validation of Mint1 interaction with a new predicted binder TJAP1 (tight junction-associated protein 1) provides experimental support that AlphaFold2 can correctly predict interactions across such large-scale datasets. Overall, our data provide insights into the diversity of interactions mediated by the Mint family and show that Mints may help facilitate a key trigger point in SNARE (soluble N-ethylmaleimide-sensitive factor attachment receptor) complex assembly and vesicle fusion.

ESCPE-1 mediates retrograde endosomal sorting of the SARS-CoV-2 host factor Neuropilin-1.

Endosomal sorting maintains cellular homeostasis by recycling transmembrane proteins and associated proteins and lipids (termed "cargoes") from the endosomal network to multiple subcellular destinations, including retrograde traffic to the trans-Golgi network (TGN). Viral and bacterial pathogens subvert retrograde trafficking machinery to facilitate infectivity. Here, we develop a proteomic screen to identify retrograde cargo proteins of the endosomal SNX-BAR sorting complex promoting exit 1 (ESCPE-1). Using this methodology, we identify Neuropilin-1 (NRP1), a recently characterized host factor for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, as a cargo directly bound and trafficked by ESCPE-1. ESCPE-1 mediates retrograde trafficking of engineered nanoparticles functionalized with the NRP1-interacting peptide of the SARS-CoV-2 spike (S) protein. CRISPR-Cas9 deletion of ESCPE-1 subunits reduces SARS-CoV-2 infection levels in cell culture. ESCPE-1 sorting of NRP1 may therefore play a role in the intracellular membrane trafficking of NRP1-interacting viruses such as SARS-CoV-2.

Structural Predictions of the SNX-RGS Proteins Suggest They Belong to a New Class of Lipid Transfer Proteins.

Recent advances in protein structure prediction using machine learning such as AlphaFold2 and RosettaFold presage a revolution in structural biology. Genome-wide predictions of protein structures are providing unprecedented insights into their architecture and intradomain interactions, and applications have already progressed towards assessing protein complex formation. Here we present detailed analyses of the sorting nexin proteins that contain regulator of G-protein signalling domains (SNX-RGS proteins), providing a key example of the ability of AlphaFold2 to reveal novel structures with previously unsuspected biological functions. These large proteins are conserved in most eukaryotes and are known to associate with lipid droplets (LDs) and sites of LD-membrane contacts, with key roles in regulating lipid metabolism. They possess five domains, including an N-terminal transmembrane domain that anchors them to the endoplasmic reticulum, an RGS domain, a lipid interacting phox homology (PX) domain and two additional domains named the PXA and PXC domains of unknown structure and function. Here we report the crystal structure of the RGS domain of sorting nexin 25 (SNX25) and show that the AlphaFold2 prediction closely matches the experimental structure. Analysing the full-length SNX-RGS proteins across multiple homologues and species we find that the distant PXA and PXC domains in fact fold into a single unique structure that notably features a large and conserved hydrophobic pocket. The nature of this pocket strongly suggests a role in lipid or fatty acid binding, and we propose that these molecules represent a new class of conserved lipid transfer proteins.

Community-Wide Experimental Evaluation of the PROSS Stability-Design Method.

Recent years have seen a dramatic improvement in protein-design methodology. Nevertheless, most methods demand expert intervention, limiting their widespread adoption. By contrast, the PROSS algorithm for improving protein stability and heterologous expression levels has been successfully applied to a range of challenging enzymes and binding proteins. Here, we benchmark the application of PROSS as a stand-alone tool for protein scientists with no or limited experience in modeling. Twelve laboratories from the Protein Production and Purification Partnership in Europe (P4EU) challenged the PROSS algorithm with 14 unrelated protein targets without support from the PROSS developers. For each target, up to six designs were evaluated for expression levels and in some cases, for thermal stability and activity. In nine targets, designs exhibited increased heterologous expression levels either in prokaryotic and/or eukaryotic expression systems under experimental conditions that were tailored for each target protein. Furthermore, we observed increased thermal stability in nine of ten tested targets. In two prime examples, the human Stem Cell Factor (hSCF) and human Cadherin-Like Domain (CLD12) from the RET receptor, the wild type proteins were not expressible as soluble proteins in E. coli, yet the PROSS designs exhibited high expression levels in E. coli and HEK293 cells, respectively, and improved thermal stability. We conclude that PROSS may improve stability and expressibility in diverse cases, and that improvement typically requires target-specific expression conditions. This study demonstrates the strengths of community-wide efforts to probe the generality of new methods and recommends areas for future research to advance practically useful algorithms for protein science.

A novel dual Ca2+ sensor system regulates Ca2+-dependent neurotransmitter release.

Ca2+-dependent neurotransmitter release requires synaptotagmins as Ca2+ sensors to trigger synaptic vesicle (SV) exocytosis via binding of their tandem C2 domains-C2A and C2B-to Ca2+. We have previously demonstrated that SNT-1, a mouse synaptotagmin-1 (Syt1) homologue, functions as the fast Ca2+ sensor in Caenorhabditis elegans. Here, we report a new Ca2+ sensor, SNT-3, which triggers delayed Ca2+-dependent neurotransmitter release. snt-1;snt-3 double mutants abolish evoked synaptic transmission, demonstrating that C. elegans NMJs use a dual Ca2+ sensor system. SNT-3 possesses canonical aspartate residues in both C2 domains, but lacks an N-terminal transmembrane (TM) domain. Biochemical evidence demonstrates that SNT-3 binds both Ca2+ and the plasma membrane. Functional analysis shows that SNT-3 is activated when SNT-1 function is impaired, triggering SV release that is loosely coupled to Ca2+ entry. Compared with SNT-1, which is tethered to SVs, SNT-3 is not associated with SV. Eliminating the SV tethering of SNT-1 by removing the TM domain or the whole N terminus rescues fast release kinetics, demonstrating that cytoplasmic SNT-1 is still functional and triggers fast neurotransmitter release, but also exhibits decreased evoked amplitude and release probability. These results suggest that the fast and slow properties of SV release are determined by the intrinsically different C2 domains in SNT-1 and SNT-3, rather than their N-termini-mediated membrane tethering. Our findings therefore reveal a novel dual Ca2+ sensor system in C. elegans and provide significant insights into Ca2+-regulated exocytosis.

Caveolae Control Contractile Tension for Epithelia to Eliminate Tumor Cells.

Epithelia are active materials where mechanical tension governs morphogenesis and homeostasis. But how that tension is regulated remains incompletely understood. We now report that caveolae control epithelial tension and show that this is necessary for oncogene-transfected cells to be eliminated by apical extrusion. Depletion of caveolin-1 (CAV1) increased steady-state tensile stresses in epithelial monolayers. As a result, loss of CAV1 in the epithelial cells surrounding oncogene-expressing cells prevented their apical extrusion. Epithelial tension in CAV1-depleted monolayers was increased by cortical contractility at adherens junctions. This reflected a signaling pathway, where elevated levels of phosphoinositide-4,5-bisphosphate (PtdIns(4,5)P2) recruited the formin, FMNL2, to promote F-actin bundling. Steady-state monolayer tension and oncogenic extrusion were restored to CAV1-depleted monolayers when tension was corrected by depleting FMNL2, blocking PtdIns(4,5)P2, or disabling the interaction between FMNL2 and PtdIns(4,5)P2. Thus, caveolae can regulate active mechanical tension for epithelial homeostasis by controlling lipid signaling to the actin cytoskeleton.

Recognising the signals for endosomal trafficking.

The endosomal compartment is a major sorting station controlling the balance between endocytic recycling and lysosomal degradation, and its homeostasis is emerging as a central factor in various neurodegenerative diseases such as Alzheimer's and Parkinson's. Membrane trafficking is generally coordinated by the recognition of specific signals in transmembrane protein cargos by different transport machineries. A number of different protein trafficking complexes are essential for sequence-specific recognition and retrieval of endosomal cargos, recycling them to other compartments and acting to counter-balance the default endosomal sorting complex required for transport-mediated degradation pathway. In this review, we provide a summary of the key endosomal transport machineries, and the molecular mechanisms by which different cargo sequences are specifically recognised.

Molecular identification of a BAR domain-containing coat complex for endosomal recycling of transmembrane proteins.

Protein trafficking requires coat complexes that couple recognition of sorting motifs in transmembrane cargoes with biogenesis of transport carriers. The mechanisms of cargo transport through the endosomal network are poorly understood. Here, we identify a sorting motif for endosomal recycling of cargoes, including the cation-independent mannose-6-phosphate receptor and semaphorin 4C, by the membrane tubulating BAR domain-containing sorting nexins SNX5 and SNX6. Crystal structures establish that this motif folds into a ß-hairpin, which binds a site in the SNX5/SNX6 phox homology domains. Over sixty cargoes share this motif and require SNX5/SNX6 for their recycling. These include cargoes involved in neuronal migration and a Drosophila snx6 mutant displays defects in axonal guidance. These studies identify a sorting motif and provide molecular insight into an evolutionary conserved coat complex, the 'Endosomal SNX-BAR sorting complex for promoting exit 1' (ESCPE-1), which couples sorting motif recognition to the BAR-domain-mediated biogenesis of cargo-enriched tubulo-vesicular transport carriers.

SDAR: a practical tool for graphical analysis of two-dimensional data.

BACKGROUND: Two-dimensional data needs to be processed and analysed in almost any experimental laboratory. Some tasks in this context may be performed with generic software such as spreadsheet programs which are available ubiquitously, others may require more specialised software that requires paid licences. Additionally, more complex software packages typically require more time by the individual user to understand and operate. Practical and convenient graphical data analysis software in Java with a user-friendly interface are rare. RESULTS: We have developed SDAR, a Java application to analyse two-dimensional data with an intuitive graphical user interface. A smart ASCII parser allows import of data into SDAR without particular format requirements. The centre piece of SDAR is the Java class GraphPanel which provides methods for generic tasks of data visualisation. Data can be manipulated and analysed with respect to the most common operations experienced in an experimental biochemical laboratory. Images of the data plots can be generated in SVG-, TIFF- or PNG-format. Data exported by SDAR is annotated with commands compatible with the Grace software. CONCLUSION: Since SDAR is implemented in Java, it is truly cross-platform compatible. The software is easy to install, and very convenient to use judging by experience in our own laboratories. It is freely available to academic users at http://www.structuralchemistry.org/pcsb/. To download SDAR, users will be asked for their name, institution and email address. A manual, as well as the source code of the GraphPanel class can also be downloaded from this site.

Alpha-1 giardin is an annexin with highly unusual calcium-regulated mechanisms.

Alpha-giardins constitute the annexin proteome (group E annexins) in the intestinal protozoan parasite Giardia and, as such, represent the evolutionary oldest eukaryotic annexins. The dominance of alpha-giardins in the cytoskeleton of Giardia with its greatly reduced actin content emphasises the importance of the alpha-giardins for the structural integrity of the parasite, which is particularly critical in the transformation stage between cyst and trophozoite. In this study, we report the crystal structures of the apo- and calcium-bound forms of α1-giardin, a protein localised to the plasma membrane of Giardia trophozoites that has recently been identified as a vaccine target. The calcium-bound crystal structure of α1-giardin revealed the presence of a type III site in the first repeat as known from other annexin structures, as well as a novel calcium binding site situated between repeats I and IV. By means of comparison, the crystal structures of three different alpha-giardins known to date indicate that these proteins engage different calcium coordination schemes, among each other, as well as compared to annexins of groups A-D. Evaluation of the calcium-dependent binding to acidic phosphoplipid membranes revealed that this process is not only mediated but also regulated by the environmental calcium concentration. Uniquely within the large family of annexins, α1-giardin disengages from the phospholipid membrane at high calcium concentrations possibly due to formation of a dimeric species. The observed behaviour is in line with changing calcium levels experienced by the parasite during excystation and may thus provide first insights into the molecular mechanisms underpinning the transformation and survival of the parasite in the host.

SBAL: a practical tool to generate and edit structure-based amino acid sequence alignments.

SUMMARY: Both alignment generation and visualization are important processes for producing biologically meaningful sequence alignments. Computational tools that combine reliable, automated and semi-automated approaches to produce secondary structure-based alignments with an appropriate visualization of the results are rare. We have developed SBAL, a tool to generate and edit secondary structure-based sequence alignments. It is easy to install and provides a user-friendly interface. Sequence alignments are displayed, with secondary structure assignments mapped to their corresponding regions in the sequence by using a simple colour scheme. The algorithm implemented for automated and semi-automated secondary structure-based alignment calculations shows a comparable performance to existing software. AVAILABILITY AND IMPLEMENTATION: SBAL has been implemented in Java to provide cross-platform compatibility. SBAL is freely available to academic users at http://www.structuralchemistry.org/pcsb/. Users will be asked for their name, institution and email address. A manual can also be downloaded from this site. The software, manual and test sets are also available as supplementary material. CONTACT: conan.wang@griffith.edu.au SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

DMAN: a Java tool for analysis of multi-well differential scanning fluorimetry experiments.

SUMMARY: Differential scanning fluorimetry (DSF) is a rapid technique that can be used in structural biology to study protein-ligand interactions. We have developed DMAN, a novel tool to analyse multi-well plate data obtained in DSF experiments. DMAN is easy to install and provides a user-friendly interface. Multi-well plate layouts can be designed by the user and experimental data can be annotated and analysed by DMAN according to the specified plate layout. Statistical tests for significance are performed automatically, and graphical tools are also provided to assist in data analysis. The modular concept of this software will allow easy development of other multi-well plate analysis applications in the future. AVAILABILITY AND IMPLEMENTATION: DMAN is implemented in Java to provide a cross-platform compatibility. It is freely available to academic users at http://www.structuralchemistry.org/pcsb/. To download DMAN, users will be asked for their name, institution and email address. A manual can also be downloaded from this site. CONTACT: conan.wang@griffith.edu.au; a.hofmann@griffith.edu.au.

Two distinct ferritin-like molecules in Pseudomonas aeruginosa: the product of the bfrA gene is a bacterial ferritin (FtnA) and not a bacterioferritin (Bfr).

Two distinct types of ferritin-like molecules often coexist in bacteria, the heme binding bacterioferritins (Bfr) and the non-heme binding bacterial ferritins (Ftn). The early isolation of a ferritin-like molecule from Pseudomonas aeruginosa suggested the possibility of a bacterioferritin assembled from two different subunits [Moore, G. R., et al. (1994) Biochem. J. 304, 493-497]. Subsequent studies demonstrated the presence of two genes encoding ferritin-like molecules in P. aeruginosa, designated bfrA and bfrB, and suggested that two distinct bacterioferritins may coexist [Ma, J.-F., et al. (1999) J. Bacteriol. 181, 3730-3742]. In this report, we present structural evidence demonstrating that the product of the bfrA gene is a ferritin-like molecule not capable of binding heme that harbors a catalytically active ferroxidase center with structural properties similar to those characteristic of bacterial and archaeal Ftns and clearly distinct from those of the ferroxidase center typical of Bfrs. Consequently, the product of the bfrA gene in P. aeruginosa is a bacterial ferritin, which we propose should be termed Pa FtnA. These results, together with the previous characterization of the product of the bfrB gene as a genuine bacterioferritin (Pa BfrB) [Weeratunga, S. J., et al. (2010) Biochemistry 49, 1160-1175], indicate the coexistence of a bacterial ferritin (Pa FtnA) and a bacterioferritin (Pa BfrB) in P. aeruginosa. In agreement with this idea, we also obtained evidence demonstrating that release of iron from Pa BfrB and Pa FtnA is likely subject to different regulation in P. aerugionsa. Whereas the efficient release of iron stored in Pa FtnA requires only the input of electrons from a ferredoxin NADP reductase (Pa Fpr), the release of iron stored in Pa BfrB requires not only electron delivery by Pa Fpr but also the presence of a "regulator", the apo form of a bacterioferritin-associated ferredoxin (apo Pa Bfd). Finally, structural analysis of iron uptake in crystallo suggests a possible pathway for the internalization of ferroxidase iron into the interior cavity of Pa FtnA.

Structural studies of bacterioferritin B from Pseudomonas aeruginosa suggest a gating mechanism for iron uptake via the ferroxidase center .

The structure of recombinant Pseudomonas aeruginosa bacterioferritin B (Pa BfrB) has been determined from crystals grown from protein devoid of core mineral iron (as-isolated) and from protein mineralized with approximately 600 iron atoms (mineralized). Structures were also obtained from crystals grown from mineralized BfrB after they had been soaked in an FeSO(4) solution (Fe soak) and in separate experiments after they had been soaked in an FeSO(4) solution followed by a soak in a crystallization solution (double soak). Although the structures consist of a typical bacterioferritin fold comprised of a nearly spherical 24-mer assembly that binds 12 heme molecules, comparison of microenvironments observed in the distinct structures provided interesting insights. The ferroxidase center in the as-isolated, mineralized, and double-soak structures is empty. The ferroxidase ligands (except His130) are poised to bind iron with minimal conformational changes. The His130 side chain, on the other hand, must rotate toward the ferroxidase center to coordinate iron. In comparison, the structure obtained from crystals soaked in an FeSO(4) solution displays a fully occupied ferroxidase center and iron bound to the internal, Fe((in)), and external, Fe((out)), surfaces of Pa BfrB. The conformation of His130 in this structure is rotated toward the ferroxidase center and coordinates an iron ion. The structures also revealed a pore on the surface of Pa BfrB that likely serves as a port of entry for Fe(2+) to the ferroxidase center. On its opposite end, the pore is capped by the side chain of His130 when it adopts its "gate-closed" conformation that enables coordination to a ferroxidase iron. A change to its "gate-open", noncoordinative conformation creates a path for the translocation of iron from the ferroxidase center to the interior cavity. These structural observations, together with findings obtained from iron incorporation measurements in solution, suggest that the ferroxidase pore is the dominant entry route for the uptake of iron by Pa BfrB. These findings, which are clearly distinct from those made with Escherichia coli Bfr [Crow, A. C., Lawson, T. L., Lewin, A., Moore, G. R., and Le Brun, N. E. (2009) J. Am. Chem. Soc. 131, 6808-6813], indicate that not all bacterioferritins operate in the same manner.

Binding of Pseudomonas aeruginosa apobacterioferritin-associated ferredoxin to bacterioferritin B promotes heme mediation of electron delivery and mobilization of core mineral iron.

The bfrB gene from Pseudomonas aeruginosa was cloned and expressed in Escherichia coli. The resultant protein (BfrB), which assembles into a 445.3 kDa complex from 24 identical subunits, binds 12 molecules of heme axially coordinated by two Met residues. BfrB, isolated with 5-10 iron atoms per protein molecule, was reconstituted with ferrous ions to prepare samples with a core mineral containing 600 +/- 40 ferric ions per BfrB molecule and approximately one phosphate molecule per iron atom. In the presence of sodium dithionite or in the presence of P. aeruginosa ferredoxin NADP reductase (FPR) and NADPH, the heme in BfrB remains oxidized, and the core iron mineral is mobilized sluggishly. In stark contrast, addition of NADPH to a solution containing BfrB, FPR, and the apo form of P. aeruginosa bacterioferritin-associated ferredoxin (apo-Bfd) results in rapid reduction of the heme in BfrB and in the efficient mobilization of the core iron mineral. Results from additional experimentation indicate that Bfd must bind to BfrB to promote heme mediation of electrons from the surface to the core to support the efficient mobilization of ferrous ions from BfrB. In this context, the thus far mysterious role of heme in bacterioferritins has been brought to the front by reconstituting BfrB with its physiological partner, apo-Bfd. These findings are discussed in the context of a model for the utilization of stored iron in which the significant upregulation of the bfd gene under low-iron conditions [Ochsner, U. A., Wilderman, P. J., Vasil, A. I., and Vasil, M. L. (2002) Mol. Microbiol. 45, 1277-1287] ensures sufficient concentrations of apo-Bfd to bind BfrB and unlock the iron stored in its core. Although these findings are in contrast to previous speculations suggesting redox mediation of electron transfer by holo-Bfd, the ability of apo-Bfd to promote iron mobilization is an economical strategy used by the cell because it obviates the need to further deplete cellular iron levels to assemble iron-sulfur clusters in Bfd before the iron stored in BfrB can be mobilized and utilized.

Biochemical and structural characterization of Pseudomonas aeruginosa Bfd and FPR: ferredoxin NADP+ reductase and not ferredoxin is the redox partner of heme oxygenase under iron-starvation conditions.

Among the 118 genes upregulated by Pseudomonas aeruginosa in response to iron starvation [Ochsner, U. A., Wilderman, P. J., Vasil, A. I., and Vasil, M. L. (2002) Mol. Microbiol. 45, 1277-1287], we focused on the products of the two genes encoding electron transfer proteins, as a means of identifying the redox partners of the heme oxygenase (pa-HO) expressed under low-iron stress conditions. Biochemical and spectroscopic investigations demonstrated that the bfd gene encodes a 73-amino acid protein (pa-Bfd) that incorporates a [2Fe-2S]2+/+ center, whereas the fpr gene encodes a 258-residue NADPH-dependent ferredoxin reductase (pa-FPR) that utilizes FAD as a cofactor. In vitro reconstitution of pa-HO catalytic activity with the newly characterized proteins led to the surprising observation that pa-FPR efficiently supports the catalytic cycle of pa-HO, without the need of a ferredoxin. In comparison, electron transfer from pa-Bfd to pa-HO is sluggish, which strongly argues against the possibility that the seven electrons needed by pa-HO to degrade biliverdin are transferred from NADPH to pa-HO in a ferredoxin (Bfd)-dependent manner. Given that pa-HO functions to release iron from exogenous heme acquired under iron-starvation conditions, the use of a flavoenzyme rather than an iron-sulfur center-containing protein to support heme degradation is an efficient use of resources in the cell. The crystal structure of pa-FPR (1.6 A resolution) showed that its fold is comparable that of the superfamily of ferredoxin reductases and most similar to the structure of Azotobacter vinelandii FPR and Escherichia coli flavodoxin reductase. The latter two enzymes interact with distinct redox partners, a ferredoxin and a flavodoxin, respectively. Hence, findings reported herein extend the range of redox partners recognized by the fold of pa-FPR to include a heme oxygenase (pa-HO).