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Cytokine receptor-like factor 2 (CRLF2) and interleukin-7 receptor α (IL-7Rα) form a receptor for thymic stromal lymphopoietin (TSLP). A somatic mutation consisting of the substitution of five amino acids (SLLLL) in the transmembrane domain of CRLF2 with three amino acids, including glutamic acid, isoleucine, and methionine (insEIM), which has been identified in acute lymphocytic leukemia, causes the TSLP-independent dimerization with IL-7Rα and activation. However, the dimerization mechanism remains unclear. In this study, we examined the involvement of the amino acids in the transmembrane domains of EIM CRLF2 and IL-7Rα in TSLP-independent activation. HEK293 cells were transfected with vectors encoding CRLF2 and IL-7Rα, or their mutants, in which the amino acid of the transmembrane domain was replaced with alanine. STAT5 phosphorylation was detected using western blotting, and receptor dimerization was analyzed using the NanoBiT assay. The substitution of glutamic acid within the insEIM mutation for alanine failed to cause the STAT5 phosphorylation in the absence of TSLP. Moreover, the alanine substation of the specific leucine residues in the transmembrane domains of both CRLF2 and IL-7Rα abrogated the TSLP-independent signal transduction and dimerization. The mutation of IL-7Rα W264 partially reduced the phosphorylation of STAT5 without affecting receptor dimerization. These results suggest that the amino acids in the transmembrane domains of EIM CRLF2 and IL-7Rα play at least three possible functions: interaction through hydrogen bonds, hydrophobic interaction, and signal transduction. Our findings contribute to a better understanding of the function of the transmembrane domains of cytokine receptors in their dimerization and signal transduction., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

The functions of proteins generally depend on their assembly into complexes. During evolution, some complexes have transitioned from homomers encoded by a single gene to heteromers encoded by duplicate genes. This transition could occur without adaptive evolution through intermolecular compensatory mutations. Here, we experimentally duplicated and evolved a homodimeric enzyme to determine whether and how this could happen. We identified hundreds of deleterious mutations that inactivate individual homodimers but produce functional enzymes when coexpressed as duplicated proteins that heterodimerize. The structure of one such heteromer reveals how both losses of function are buffered through the introduction of asymmetry in the complex that allows them to subfunctionalize. Constructive neutral evolution can thus occur by gene duplication followed by only one deleterious mutation per duplicate.

Progesterone receptor membrane component 1 (PGRMC1) binds heme via a surface-exposed site and displays some structural resemblance to cytochrome b5 despite their different functions. In the case of PGRMC1, it is the protein interaction with drug-metabolizing cytochrome P450s and the epidermal growth factor receptor that has garnered the most attention. These interactions are thought to result in a compromised ability to metabolize common chemotherapy agents and to enhance cancer cell proliferation. X-ray crystallography and immunoprecipitation data have suggested that heme-mediated PGRMC1 dimers are important for facilitating these interactions. However, more recent studies have called into question the requirement of heme binding for PGRMC1 dimerization. Our study employs spectroscopic and computational methods to probe and define heme binding and its impact on PGRMC1 dimerization. Fluorescence, electron paramagnetic resonance and circular dichroism spectroscopies confirm heme binding to apo-PGRMC1 and were used to demonstrate the stabilizing effect of heme on the wild-type protein. We also utilized variants (C129S and Y113F) to precisely define the contributions of disulfide bonds and direct heme coordination to PGRMC1 dimerization. Understanding the key factors involved in these processes has important implications for downstream protein-protein interactions that may influence the metabolism of chemotherapeutic agents. This work opens avenues for deeper exploration into the physiological significance of the truncated-PGRMC1 model and developing design principles for potential therapeutics to target PGRMC1 dimerization and downstream interactions.

Synonymous mutations in messenger RNAs (mRNAs) can reduce protein-protein binding substantially without changing the protein's amino acid sequence. Here, we use coarse-grain simulations of protein synthesis, post-translational dynamics, and dimerization to understand how synonymous mutations can influence the dimerization of two E. coli homodimers, oligoribonuclease and ribonuclease T. We synthesize each protein from its wildtype, fastest- and slowest-translating synonymous mRNAs in silico and calculate the ensemble-averaged interaction energy between the resulting dimers. We find synonymous mutations alter oligoribonuclease's dimer properties. Relative to wildtype, the dimer interaction energy becomes 4% and 10% stronger, respectively, when translated from its fastest- and slowest-translating mRNAs. Ribonuclease T dimerization, however, is insensitive to synonymous mutations. The structural and kinetic origin of these changes are misfolded states containing non-covalent lasso-entanglements, many of which structurally perturb the dimer interface, and whose probability of occurrence depends on translation speed. These entangled states are kinetic traps that persist for long time scales. Entanglements cause altered dimerization energies for oligoribonuclease, as there is a large association (odds ratio: 52) between the co-occurrence of non-native self-entanglements and weak-binding dimer conformations. Simulated at all-atom resolution, these entangled structures persist for long timescales, indicating the conclusions are independent of model resolution. Finally, we show that regions of the protein we predict to have changes in entanglement are also structurally perturbed during refolding, as detected by limited-proteolysis mass spectrometry. Thus, non-native changes in entanglement at dimer interfaces is a mechanism through which oligomer structure and stability can be altered., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Hepcidin, a peptide hormone that negatively regulates iron metabolism, is expressed by bone morphogenetic protein (BMP) signaling. Erythroferrone (ERFE) is an extracellular protein that binds and inhibits BMP ligands, thus positively regulating iron import by indirectly suppressing hepcidin. This allows for rapid erythrocyte regeneration after blood loss. ERFE belongs to the C1Q/TNF-related protein family and is suggested to adopt multiple oligomeric forms: a trimer, a hexamer, and a high molecular weight species. The molecular basis for how ERFE binds BMP ligands and how the different oligomeric states impact BMP inhibition are poorly understood. In this study, we demonstrated that ERFE activity is dependent on the presence of stable dimeric or trimeric ERFE and that larger species are dispensable for BMP inhibition. Additionally, we used an in silico approach to identify a helix, termed the ligand-binding domain, that was predicted to bind BMPs and occlude the type I receptor pocket. We provide evidence that the ligand-binding domain is crucial for activity through luciferase assays and surface plasmon resonance analysis. Our findings provide new insight into how ERFE oligomerization impacts BMP inhibition, while identifying critical molecular features of ERFE essential for binding BMP ligands., Competing Interests: Conflict of interest T. B. T. is a consultant/advisor for Keros Therapeutics and Oviva Therapeutics. All other authors declare they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Huntington's disease is caused by a polyglutamine (polyQ) expansion in the huntingtin protein. Huntingtin exon 1 (Httex1), as well as other naturally occurring N-terminal huntingtin fragments with expanded polyQ are prone to aggregation, forming potentially cytotoxic oligomers and fibrils. Antibodies and other N-terminal huntingtin binders are widely explored as biomarkers and possible aggregation-inhibiting therapeutics. A monoclonal antibody, MW1, is known to preferentially bind to huntingtin fragments with expanded polyQ lengths, but the molecular basis of the polyQ length specificity remains poorly understood. Using solution NMR, electron paramagnetic resonance, and other biophysical methods, we investigated the structural features of the Httex1-MW1 interaction. Rather than recognizing residual α-helical structure, which is promoted by expanded Q-lengths, MW1 caused the formation of a new, non-native, conformation in which the entire polyQ is largely extended. This non-native polyQ structure allowed the formation of large mixed Httex1-MW1 multimers (600-2900 kD), when Httex1 with pathogenic Q-length (Q46) was used. We propose that these multivalent, entropically favored interactions, are available only to proteins with longer Q-lengths and represent a major factor governing the Q-length preference of MW1. The present study reveals that it is possible to target proteins with longer Q-lengths without having to stabilize a natively favored conformation. Such mechanisms could be exploited in the design of other Q-length specific binders., Competing Interests: Conflict of interest The authors declare no conflict of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

The cystic fibrosis transmembrane conductance regulator (CFTR) is an anion channel that regulates salt and fluid homeostasis across epithelial membranes 1 . Alterations in CFTR cause cystic fibrosis, a fatal disease without a cure 2,3 . Electrophysiological properties of CFTR have been analysed for decades 4-6 . The structure of CFTR, determined in two globally distinct conformations, underscores its evolutionary relationship with other ATP-binding cassette transporters. However, direct correlations between the essential functions of CFTR and extant structures are lacking at present. Here we combine ensemble functional measurements, single-molecule fluorescence resonance energy transfer, electrophysiology and kinetic simulations to show that the two nucleotide-binding domains (NBDs) of human CFTR dimerize before channel opening. CFTR exhibits an allosteric gating mechanism in which conformational changes within the NBD-dimerized channel, governed by ATP hydrolysis, regulate chloride conductance. The potentiators ivacaftor and GLPG1837 enhance channel activity by increasing pore opening while NBDs are dimerized. Disease-causing substitutions proximal (G551D) or distal (L927P) to the ATPase site both reduce the efficiency of NBD dimerization. These findings collectively enable the framing of a gating mechanism that informs on the search for more efficacious clinical therapies., (© 2023. The Author(s).)

The CTLH (C-terminal to lissencephaly-1 homology motif) complex is a multisubunit RING E3 ligase with poorly defined substrate specificity and flexible subunit composition. Two key subunits, muskelin and Wdr26, specify two alternative CTLH complexes that differ in quaternary structure, thereby allowing the E3 ligase to presumably target different substrates. With the aid of different biophysical and biochemical techniques, we characterized CTLH complex assembly pathways, focusing not only on Wdr26 and muskelin but also on RanBP9, Twa1, and Armc8β subunits, which are critical to establish the scaffold of this E3 ligase. We demonstrate that the ability of muskelin to tetramerize and the assembly of Wdr26 into dimers define mutually exclusive oligomerization modules that compete with nanomolar affinity for RanBP9 binding. The remaining scaffolding subunits, Armc8β and Twa1, strongly interact with each other and with RanBP9, again with nanomolar affinity. Our data demonstrate that RanBP9 organizes subunit assembly and prevents higher order oligomerization of dimeric Wdr26 and the Armc8β-Twa1 heterodimer through its tight binding. Combined, our studies define alternative assembly pathways of the CTLH complex and elucidate the role of RanBP9 in governing differential oligomeric assemblies, thereby advancing our mechanistic understanding of CTLH complex architectures., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Replication licensing, a prerequisite of DNA replication, helps to ensure once-per-cell-cycle genome duplication. Some DNA replication-initiation proteins are sequentially loaded onto replication origins to form pre-replicative complexes (pre-RCs). ORC and Noc3p bind replication origins throughout the cell cycle, providing a platform for pre-RC assembly. We previously reported that cell cycle-dependent ORC dimerization is essential for the chromatin loading of the symmetric MCM double-hexamers. Here, we used Saccharomyces cerevisiae separation-of-function NOC3 mutants to confirm the separable roles of Noc3p in DNA replication and ribosome biogenesis. We also show that an essential and cell cycle-dependent Noc3p dimerization cycle regulates the ORC dimerization cycle. Noc3p dimerizes at the M-to-G 1 transition and de-dimerizes in S-phase. The Noc3p dimerization cycle coupled with the ORC dimerization cycle enables replication licensing, protects nascent sister replication origins after replication initiation, and prevents re-replication. This study has revealed a new mechanism of replication licensing and elucidated the molecular mechanism of Noc3p as a mediator of ORC dimerization in pre-RC formation., (© 2023 Amin et al.)

A wide variety of oligomeric structures are formed during the aggregation of proteins associated with neurodegenerative diseases. Such soluble oligomers are believed to be key toxic species in the related disorders; therefore, identification of the structural determinants of toxicity is of upmost importance. Here, we analysed toxic oligomers of α-synuclein and its pathological variants in order to identify structural features that could be related to toxicity and found a novel structural polymorphism within G51D oligomers. These G51D oligomers can adopt a variety of β-sheet-rich structures with differing degrees of α-helical content, and the helical structural content of these oligomers correlates with the level of induced cellular dysfunction in SH-SY5Y cells. This structure-function relationship observed in α-synuclein oligomers thus presents the α-helical structure as another potential structural determinant that may be linked with cellular toxicity in amyloid-related proteins.

Ubiquitination and ADP-ribosylation are post-translational modifications that play major roles in pathways including the DNA damage response and viral infection. The enzymes responsible for these modifications are therefore potential targets for therapeutic intervention. DTX3L is an E3 Ubiquitin ligase that forms a heterodimer with PARP9. In addition to its ubiquitin ligase activity, DTX3L-PARP9 also acts as an ADP-ribosyl transferase for Gly76 on the C-terminus of ubiquitin. NAD+-dependent ADP-ribosylation of ubiquitin by DTX3L-PARP9 prevents ubiquitin from conjugating to protein substrates. To gain insight into how DTX3L-PARP9 generates these post-translational modifications, we produced recombinant forms of DTX3L and PARP9 and studied their physical interactions. We show the DTX3L D3 domain (230-510) mediates the interaction with PARP9 with nanomolar affinity and an apparent 1 : 1 stoichiometry. We also show that DTX3L and PARP9 assemble into a higher molecular weight oligomer, and that this is mediated by the DTX3L N-terminal region (1-200). Lastly, we show that ADP-ribosylation of ubiquitin at Gly76 is reversible in vitro by several Macrodomain-type hydrolases. Our study provides a framework to understand how DTX3L-PARP9 mediates ADP-ribosylation and ubiquitination through both intra- and inter-subunit interactions., (© 2022 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Human death receptors control apoptotic events during cell differentiation, cell homeostasis and the elimination of damaged or infected cells. Receptor activation involves ligand-induced structural reorganizations of preformed receptor trimers. Here we show that the death receptor transmembrane domains only have a weak intrinsic tendency to homo-oligomerize within a membrane, and thus these domains potentially do not significantly contribute to receptor trimerization. However, mutation of Pro183 in the human CD95/Fas receptor transmembrane helix results in a dramatically increased interaction propensity, as shown by genetic assays. The increased interaction of the transmembrane domain is coupled with a decreased ligand-sensitivity of cells expressing the Fas receptor, and thus in a decreased number of apoptotic events. Mutation of Pro183 likely results in a substantial rearrangement of the self-associated Fas receptor transmembrane trimer, which likely abolishes further signaling of the apoptotic signal but may activate other signaling pathways. Our study shows that formation of a stable Fas receptor transmembrane helix oligomer does not per se result in receptor activation., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Heterodimerization of RNA binding proteins Nrd1 and Nab3 is essential to communicate the RNA recognition in the nascent transcript with the Nrd1 recognition of the Ser 5 -phosphorylated Rbp1 C-terminal domain in RNA polymerase II. The structure of a Nrd1-Nab3 chimera reveals the basis of heterodimerization, filling a missing gap in knowledge of this system. The free form of the Nrd1 interaction domain of Nab3 (NRID) forms a multi-state three-helix bundle that is clamped in a single conformation upon complex formation with the Nab3 interaction domain of Nrd1 (NAID). The latter domain forms two long helices that wrap around NRID, resulting in an extensive protein-protein interface that would explain the highly favorable free energy of heterodimerization. Mutagenesis of some conserved hydrophobic residues involved in the heterodimerization leads to temperature-sensitive phenotypes, revealing the importance of this interaction in yeast cell fitness. The Nrd1-Nab3 structure resembles the previously reported Rna14/Rna15 heterodimer structure, which is part of the poly(A)-dependent termination pathway, suggesting that both machineries use similar structural solutions despite they share little sequence homology and are potentially evolutionary divergent., (© 2022 Chaves-Arquero et al.)

The APOBEC3 (A3) family of single-stranded DNA cytidine deaminases are host restriction factors that inhibit lentiviruses, such as HIV-1, in the absence of the Vif protein that causes their degradation. Deamination of cytidine in HIV-1 (-)DNA forms uracil that causes inactivating mutations when uracil is used as a template for (+)DNA synthesis. For APOBEC3C (A3C), the chimpanzee and gorilla orthologues are more active than human A3C, and we determined that Old World Monkey A3C from rhesus macaque (rh) is not active against HIV-1. Biochemical, virological, and coevolutionary analyses combined with molecular dynamics simulations showed that the key amino acids needed to promote rhA3C antiviral activity, 44, 45, and 144, also promoted dimerization and changes to the dynamics of loop 1, near the enzyme active site. Although forced evolution of rhA3C resulted in a similar dimer interface with hominid A3C, the key amino acid contacts were different. Overall, our results determine the basis for why rhA3C is less active than human A3C and establish the amino acid network for dimerization and increased activity. Based on identification of the key amino acids determining Old World Monkey antiviral activity we predict that other Old World Monkey A3Cs did not impart anti-lentiviral activity, despite fixation of a key residue needed for hominid A3C activity. Overall, the coevolutionary analysis of the A3C dimerization interface presented also provides a basis from which to analyze dimerization interfaces of other A3 family members., Competing Interests: Declaration of interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Most of the known Drosophila architectural proteins interact with an important cofactor, CP190, that contains three domains (BTB, M, and D) that are involved in protein-protein interactions. The highly conserved N-terminal CP190 BTB domain forms a stable homodimer that interacts with unstructured regions in the three best-characterized architectural proteins: dCTCF, Su(Hw), and Pita. Here, we identified two new CP190 partners, CG4730 and CG31365, that interact with the BTB domain. The CP190 BTB resembles the previously characterized human BCL6 BTB domain, which uses its hydrophobic groove to specifically associate with unstructured regions of several transcriptional repressors. Using GST pull-down and yeast two-hybrid assays, we demonstrated that mutations in the hydrophobic groove strongly affect the affinity of CP190 BTB for the architectural proteins. In the yeast two-hybrid assay, we found that architectural proteins use various mechanisms to improve the efficiency of interaction with CP190. Pita and Su(Hw) have two unstructured regions that appear to simultaneously interact with hydrophobic grooves in the BTB dimer. In dCTCF and CG31365, two adjacent regions interact simultaneously with the hydrophobic groove of the BTB and the M domain of CP190. Finally, CG4730 interacts with the BTB, M, and D domains of CP190 simultaneously. These results suggest that architectural proteins use different mechanisms to increase the efficiency of interaction with CP190.

The BAM is a macromolecular machine responsible for the folding and the insertion of integral proteins into the outer membrane of diderm Gram-negative bacteria. In Escherichia coli , it consists of a transmembrane β-barrel subunit, BamA, and four outer membrane lipoproteins (BamB-E). Using BAM-specific antibodies, in E. coli cells, the complex is shown to localize in the lateral wall in foci. The machinery was shown to be enriched at midcell with specific cell cycle timing. The inhibition of septation by aztreonam did not alter the BAM midcell localization substantially. Furthermore, the absence of late cell division proteins at midcell did not impact BAM timing or localization. These results imply that the BAM enrichment at the site of constriction does not require an active cell division machinery. Expression of the Tre1 toxin, which impairs the FtsZ filamentation and therefore midcell localization, resulted in the complete loss of BAM midcell enrichment. A similar effect was observed for YidC, which is involved in the membrane insertion of cell division proteins in the inner membrane. The presence of the Z-ring is needed for preseptal peptidoglycan (PG) synthesis. As BAM was shown to be embedded in the PG layer, it is possible that BAM is inserted preferentially simultaneously with de novo PG synthesis to facilitate the insertion of OMPs in the newly synthesized outer membrane.

Human calprotectin (CP, S100A8/S100A9 oligomer, MRP8/MRP14 oligomer) is an abundant innate immune protein that contributes to the host metal-withholding response. Its ability to sequester transition metal nutrients from microbial pathogens depends on a complex interplay of Ca(II) binding and self-association, which converts the αβ heterodimeric apo protein into a Ca(II)-bound (αβ) 2 heterotetramer that displays enhanced transition metal affinities, antimicrobial activity, and protease stability. A paucity of structural data on the αβ heterodimer has hampered molecular understanding of how Ca(II) binding enables CP to exert its metal-sequestering innate immune function. We report solution NMR data that reveal how Ca(II) binding affects the structure and dynamics of the CP αβ heterodimer. These studies provide a structural model in which the apo αβ heterodimer undergoes conformational exchange and switches between two states, a tetramerization-incompetent or "inactive" state and a tetramerization-competent or "active" state. Ca(II) binding to the EF-hands of the αβ heterodimer causes the active state to predominate, resulting in self-association and formation of the (αβ) 2 heterotetramer. Moreover, Ca(II) binding causes local and allosteric ordering of the His 3 Asp and His 6 metal-binding sites. Ca(II) binding to the noncanonical EF-hand of S100A9 positions (A9)D30 and organizes the His 3 Asp site. Remarkably, Ca(II) binding causes allosteric effects in the C-terminal region of helix α IV of S100A9, which stabilize the α-helicity at positions H91 and H95 and thereby organize the functionally versatile His 6 site. Collectively, this study illuminates the molecular basis for how CP responds to high extracellular Ca(II) concentrations, which enables its metal-sequestering host-defense function.

The objective of this study was to determine the expression and functional role of a long noncoding RNA (lncRNA) MIAT (myocardial infarction-associated transcript) in leiomyoma pathogenesis. Leiomyoma compared with myometrium (n = 66) expressed significantly more MIAT that was independent of race/ethnicity and menstrual cycle phase but dependent on MED12 (mediator complex subunit 12) mutation status. Leiomyomas bearing the MED12 mutation expressed higher levels of MIAT and lower levels of microRNA 29 family (miR-29a, -b, and -c) compared with MED12 wild-type leiomyomas. Using luciferase reporter activity and RNA immunoprecipitation analysis, MIAT was shown to sponge the miR-29 family. In a 3-dimensional spheroid culture system, transient transfection of MIAT siRNA in leiomyoma smooth muscle cell (LSMC) spheroids resulted in upregulation of miR-29 family and downregulation of miR-29 targets, collagen type I (COL1A1), collagen type III (COL3A1), and TGF-β3 (transforming growth factor β-3). Treatment of LSMC spheroids with TGF-β3 induced COL1A1, COL3A1, and MIAT levels, but repressed miR-29 family expression. Knockdown of MIAT in LSMC spheroids blocked the effects of TGF-β3 on the induction of COL1A1 and COL3A1 expression. Collectively, these results underscore the physiological significance of MIAT in extracellular matrix accumulation in leiomyoma., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Endocrine Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The design of allosteric modulators to control protein function is a key objective in drug discovery programs. Altering functionally essential allosteric residue networks provides unique protein family subtype specificity, minimizes unwanted off-target effects, and helps avert resistance acquisition typically plaguing drugs that target orthosteric sites. In this work, we used protein engineering and dimer interface mutations to positively and negatively modulate the immunosuppressive activity of the proapoptotic human galectin-7 (GAL-7). Using the PoPMuSiC and BeAtMuSiC algorithms, mutational sites and residue identity were computationally probed and predicted to either alter or stabilize the GAL-7 dimer interface. By designing a covalent disulfide bridge between protomers to control homodimer strength and stability, we demonstrate the importance of dimer interface perturbations on the allosteric network bridging the two opposite glycan-binding sites on GAL-7, resulting in control of induced apoptosis in Jurkat T cells. Molecular investigation of G16X GAL-7 variants using X-ray crystallography, biophysical, and computational characterization illuminates residues involved in dimer stability and allosteric communication, along with discrete long-range dynamic behaviors involving loops 1, 3, and 5. We show that perturbing the protein-protein interface between GAL-7 protomers can modulate its biological function, even when the overall structure and ligand-binding affinity remains unaltered. This study highlights new avenues for the design of galectin-specific modulators influencing both glycan-dependent and glycan-independent interactions., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Protein arginine methylation is a posttranslational modification catalyzed by the protein arginine methyltransferase (PRMT) enzyme family. Dysregulated protein arginine methylation is linked to cancer and a variety of other human diseases. PRMT1 is the predominant PRMT isoform in mammalian cells and acts in pathways regulating transcription, DNA repair, apoptosis, and cell proliferation. PRMT1 dimer formation, which is required for methyltransferase activity, is mediated by interactions between a structure called the dimerization arm on one monomer and a surface of the Rossman Fold of the other monomer. Given the link between PRMT1 dysregulation and disease and the link between PRMT1 dimerization and activity, we searched the Catalogue of Somatic Mutations in Cancer (COSMIC) database to identify potential inactivating mutations occurring in the PRMT1 dimerization arm. We identified three mutations that correspond to W215L, Y220N, and M224V substitutions in human PRMT1V2 (isoform 1) (W197L, Y202N, M206V in rat PRMT1V1). Using a combination of site-directed mutagenesis, analytical ultracentrifugation, native PAGE, and activity assays, we found that these conservative substitutions surprisingly disrupt oligomer formation and substantially impair both S-adenosyl-L-methionine (AdoMet) binding and methyltransferase activity. Molecular dynamics simulations suggest that these substitutions introduce novel interactions within the dimerization arm that lock it in a conformation not conducive to dimer formation. These findings provide a clear, if putative, rationale for the contribution of these mutations to impaired arginine methylation in cells and corresponding health consequences., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

The tumour necrosis factor receptor superfamily (TNFRSF) members contain cysteine-rich domains (CRD) in their extracellular regions, and the membrane-distal CRD1 forms homologous interactions in the absence of ligand. The CRD1 is therefore termed a pre-ligand assembly domain (PLAD). The role of PLAD-PLAD interactions in the induction of signalling as a consequence of TNF-TNFR binding led to the development of soluble PLAD domains as antagonists of TNFR activation. In the present study, we generated recombinant wild-type (WT) PLAD of TNFR1 and mutant forms with single alanine substitutions of amino acid residues thought to be critical for the formation of homologous dimers and/or trimers of PLAD (K19A, T31A, D49A and D52A). These mutated PLADs were compared with WT PLAD as antagonists of TNF-induced apoptosis or the activation of inflammatory signalling pathways. Unlike WT PLAD, the mutated PLADs showed little or no homologous interactions, confirming the importance of particular amino acids as contact residues in the PLAD binding region. However, as with WT PLAD, the mutated PLADs functioned as antagonists of TNF-induced TNFR1 activity leading to induction of cell death or NF-κB signalling. Indeed, some of the mutant PLADs, and K19A PLAD in particular, showed enhanced antagonistic activity compared with WT PLAD. This is consistent with the reduced formation of homologous multimers by these PLAD mutants effectively increasing the concentration of PLAD available to bind and antagonize WT TNFR1 when compared to WT PLAD acting as an antagonist. This may have implications for the development of antagonistic PLADs as therapeutic agents., (© 2021 John Wiley & Sons Ltd.)

G-protein coupled receptors (GPCRs) are known to form homo- and hetero- oligomers which are considered critical to modulate their function. However, studying the existence and functional implication of these complexes is not straightforward as controversial results are obtained depending on the method of analysis employed. Here, we use a quantitative single molecule super-resolution imaging technique named qPAINT to quantify complex formation within an example GPCR. qPAINT, based upon DNA-PAINT, takes advantage of the binding kinetics between fluorescently labelled DNA imager strands to complementary DNA docking strands coupled to protein targeting antibodies to quantify the protein copy number in nanoscale dimensions. We demonstrate qPAINT analysis via a novel pipeline to study the oligomerization of the purinergic receptor Y2 (P2Y 2 ), a rhodopsin-like GPCR, highly expressed in the pancreatic cancer cell line AsPC-1, under control, agonistic and antagonistic conditions. Results reveal that whilst the density of P2Y 2 receptors remained unchanged, antagonistic conditions displayed reduced percentage of oligomers, and smaller numbers of receptors in complexes. Yet, the oligomeric state of the receptors was not affected by agonist treatment, in line with previous reports. Understanding P2Y 2 oligomerization under agonistic and antagonistic conditions will contribute to unravelling P2Y 2 mechanistic action and therapeutic targeting.

Glycation of α-synuclein (αSyn), as occurs with aging, has been linked to the progression of Parkinson's disease (PD) through the promotion of advanced glycation end-products and the formation of toxic oligomers that cannot be properly cleared from neurons. DJ-1, an antioxidative protein that plays a critical role in PD pathology, has been proposed to repair glycation in proteins, yet a mechanism has not been elucidated. In this study, we integrate solution nuclear magnetic resonance (NMR) spectroscopy and liquid atomic force microscopy (AFM) techniques to characterize glycated N-terminally acetylated-αSyn (glyc-ac-αSyn) and its interaction with DJ-1. Glycation of ac-αSyn by methylglyoxal increases oligomer formation, as visualized by AFM in solution, resulting in decreased dynamics of the monomer amide backbone around the Lys residues, as measured using NMR. Upon addition of DJ-1, this NMR signature of glyc-ac-αSyn monomers reverts to a native ac-αSyn-like character. This phenomenon is reversible upon removal of DJ-1 from the solution. Using relaxation-based NMR, we have identified the binding site on DJ-1 for glycated and native ac-αSyn as the catalytic pocket and established that the oxidation state of the catalytic cysteine is imperative for binding. Based on our results, we propose a novel mechanism by which DJ-1 scavenges glyc-ac-αSyn oligomers without chemical deglycation, suppresses glyc-ac-αSyn monomer-oligomer interactions, and releases free glyc-ac-αSyn monomers in solution. The interference of DJ-1 with ac-αSyn oligomers may promote free ac-αSyn monomer in solution and suppress the propagation of toxic oligomer and fibril species. These results expand the understanding of the role of DJ-1 in PD pathology by acting as a scavenger for aggregated αSyn.

Background: Insulin's discovery 100 years ago and its ongoing use since that time to treat diabetes belies the molecular complexity of its structure and that of its receptor. Advances in single-particle cryo-electron microscopy have over the past three years revolutionized our understanding of the atomic detail of insulin-receptor interactions., Scope of Review: This review describes the three-dimensional structure of insulin and its receptor and details on how they interact. This review also highlights the current gaps in our structural understanding of the system., Major Conclusions: A near-complete picture has been obtained of the hormone receptor interactions, providing new insights into the kinetics of the interactions and necessitating a revision of the extant two-site cross-linking model of hormone receptor engagement. How insulin initially engages the receptor and the receptor's traversed trajectory as it undergoes conformational changes associated with activation remain areas for future investigation., (Copyright © 2021 The Author. Published by Elsevier GmbH.. All rights reserved.)

The CD8αβ heterodimer plays a crucial role in the stabilization between major histocompatibility complex class I molecules (MHC-I) and the T cell receptor (TCR). The interaction between CD8 and MHC-I can be regulated by posttranslational modifications, which are proposed to play an important role in the development of CD8 T cells. One modification that has been proposed to control CD8 coreceptor function is ribosylation. Utilizing NAD + , the ecto-enzyme adenosine diphosphate (ADP) ribosyl transferase 2.2 (ART2.2) catalyzes the addition of ADP-ribosyl groups onto arginine residues of CD8α or β chains and alters the interaction between the MHC and TCR complexes. To date, only interactions between modified CD8 and classical MHC-I (MHC-Ia), have been investigated and the interaction with non-classical MHC (MHC-Ib) has not been explored. Here, we show that ADP-ribosylation of CD8 facilitates the binding of the liver-restricted nonclassical MHC, H2-Q10, independent of the associated TCR or presented peptide, and propose that this highly regulated binding imposes an additional inhibitory leash on the activation of CD8-expressing cells in the presence of NAD + . These findings highlight additional important roles for nonclassical MHC-I in the regulation of immune responses., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Bacillus subtilis can form structurally complex biofilms on solid or liquid surfaces, which requires expression of genes for matrix production. The transcription of these genes is activated by regulatory protein RemA, which binds to poorly conserved, repetitive DNA regions but lacks obvious DNA-binding motifs or domains. Here, we present the structure of the RemA homologue from Geobacillus thermodenitrificans, showing a unique octameric ring with the potential to form a 16-meric superstructure. These results, together with further biochemical and in vivo characterization of B. subtilis RemA, suggests that the protein can wrap DNA around its ring-like structure through a LytTR-related domain., (© 2021. The Author(s).)

Despite the continuous discovery of host and guest proteins in membraneless organelles, complex host-guest interactions hinder the understanding of the molecular grammar governing liquid-liquid phase separation. In this study, we characterized the localization and dynamic properties of guest proteins in liquid droplets using single-molecule fluorescence microscopy. Eighteen guest proteins of different sizes, structures, and oligomeric states were examined in host p53 liquid droplets. Recruitment did not significantly depend on the structural properties of the guest proteins, but was moderately correlated with their length, total charge, and number of R and Y residues. In contrast, the diffusion of disordered guest proteins was comparable to that of host p53, whereas that of folded proteins varied widely. Molecular dynamics simulations suggest that folded proteins diffuse within the voids of the liquid droplet while interacting weakly with neighboring host proteins, whereas disordered proteins adapt their structures to form tight interactions with the host proteins. Our study provides insights into the key molecular principles of the localization and dynamics of guest proteins in liquid droplets., (© 2021. The Author(s).)

CLEC12A is a myeloid inhibitory receptor that negatively regulates inflammation in mouse models of autoimmune and autoinflammatory arthritis. Reduced CLEC12A expression enhances myeloid cell activation and inflammation in CLEC12A knock-out mice with collagen antibody-induced or gout-like arthritis. Similarly to other C-type lectin receptors, CLEC12A harbours a stalk domain between its ligand binding and transmembrane domains. While it is presumed that the cysteines in the stalk domain have multimerisation properties, their role in CLEC12A expression and/or signaling remain unknown. We thus used site-directed mutagenesis to determine whether the stalk domain cysteines play a role in CLEC12A expression, internalisation, oligomerisation, and/or signaling. Mutation of C118 blocks CLEC12A transport through the secretory pathway diminishing its cell-surface expression. In contrast, mutating C130 does not affect CLEC12A cell-surface expression but increases its oligomerisation, inducing ligand-independent phosphorylation of the receptor. Moreover, we provide evidence that CLEC12A dimerisation is regulated in a redox-dependent manner. We also show that antibody-induced CLEC12A cross-linking induces flotillin oligomerisation in insoluble membrane domains in which CLEC12A signals. Taken together, these data indicate that the stalk cysteines in CLEC12A differentially modulate this inhibitory receptor's expression, oligomerisation and signaling, suggestive of the regulation of CLEC12A in a redox-dependent manner during inflammation.

The DNA mismatch repair (MMR) factor Mlh1-Pms1 contains long intrinsically disordered regions (IDRs) whose exact functions remain elusive. We performed cross-linking mass spectrometry to identify interactions within Mlh1-Pms1 and used this information to insert FRB and FKBP dimerization domains into their IDRs. Baker's yeast strains bearing these constructs were grown with rapamycin to induce dimerization. A strain containing FRB and FKBP domains in the Mlh1 IDR displayed a complete defect in MMR when grown with rapamycin. but removing rapamycin restored MMR functions. Strains in which FRB was inserted into the IDR of one MLH subunit and FKBP into the other subunit were also MMR defective. The MLH complex containing FRB and FKBP domains in the Mlh1 IDR displayed a rapamycin-dependent defect in Mlh1-Pms1 endonuclease activity. In contrast, linking the Mlh1 and Pms1 IDRs through FRB-FKBP dimerization inappropriately activated Mlh1-Pms1 endonuclease activity. We conclude that dynamic and coordinated rearrangements of the MLH IDRs both positively and negatively regulate how the MLH complex acts in MMR. The application of the FRB-FKBP dimerization system to interrogate in vivo functions of a critical repair complex will be useful for probing IDRs in diverse enzymes and to probe transient loss of MMR on demand., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Polypyrimidine tract-binding protein (PTB) is an RNA binding protein existing both as dimer and monomer and shuttling between nucleus and cytoplasm. However, the regulation of PTB dimerization and the relationship between their functions and subcellular localization are unknown. Here we find that PTB presents as dimer and monomer in nucleus and cytoplasm respectively, and a disulfide bond involving Cysteine 23 is critical for the dimerization of PTB. Additionally, protein disulfide isomerase (PDI) is identified to be the enzyme that catalyzes the de-dimerization of PTB, which is dependent on the CGHC active site of the a' domain of PDI. Furthermore, upon DNA damage induced by topoisomerase inhibitors, PTB is demonstrated to be de-dimerized with cytoplasmic accumulation. Finally, cytoplasmic PTB is found to associate with the ribosome and enhances the translation of p53. Collectively, these findings uncover a previously unrecognized mechanism of PTB dimerization, and shed light on the de-dimerization of PTB functionally linking to cytoplasmic localization and translational regulation., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

In the current study, zebrafish TNF-α1 (zTNF-α1) was crystallized, and the structure was analyzed. The zTNF-α1 trimer is composed of three monomers whose height and width are 50 Å and 60 Å, respectively. Compared with human TNF-α, zTNF-α1 shows only ~30% amino acid identity, the EF loop of each monomer lacks three amino acids, the CD loop is increased by four amino acids, and the AA'' loop is increased by one amino acid. In addition, an A″-β-chain is added to the zTNF-α1 monomer, forming two β-sheet layers with 6:5 β-chains. The top of the trimer is missing three amino acids and the inner coil because the EF loop seals the central hole at the top, forming a unique structure. In conclusion, the results elucidated the structure of the zTNF-α1 trimer, providing immunological knowledge for studying TNF-α function in the zebrafish animal model and structural information for exploring TNF-α family evolution., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Fibril formation and aggregation of α-synuclein are important for the pathogenesis of neurodegenerative disorders including Parkinson's disease. In familial Parkinson's disease, the G51D mutation of α-synuclein causes severe symptoms and rapid progression. α-Synuclein, an intrinsically disordered protein, was shown to adopt an α-helical tetrameric state that resists fibrillation and aggregation. Here, we isolated the stable dimeric state of recombinant wild-type (WT) α-synuclein and G51D α-synuclein protein. Using circular dichroism spectroscopy, we determined that the α-synuclein dimer and monomer structures were unfolded. The WT α-synuclein dimer was more resistant to fibril formation than the monomer. However, the fibril formation rate of the G51D α-synuclein dimer was similar to that of the G51D α-synuclein monomer. The fibril morphology and properties of the G51D α-synuclein monomer were different from those of the WT α-synuclein monomer and dimer and G51D α-synuclein dimer. Additionally, G51D α-synuclein monomer fibrils were more cytotoxic than other fibrils. Our findings indicate that the structural differences between G51D α-synuclein monomer fibrils and other fibrils are critically responsible for its severe neurotoxicity in familial Parkinson's disease., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The Wnt/β-catenin pathway is a highly conserved, frequently mutated developmental and cancer pathway. Its output is defined mainly by β-catenin's phosphorylation- and ubiquitylation-dependent proteasomal degradation, initiated by the multi-protein β-catenin destruction complex. The precise mechanisms underlying destruction complex function have remained unknown, largely because of the lack of suitable in vitro systems. Here we describe the in vitro reconstitution of an active human β-catenin destruction complex from purified components, recapitulating complex assembly, β-catenin modification, and degradation. We reveal that AXIN1 polymerization and APC promote β-catenin capture, phosphorylation, and ubiquitylation. APC facilitates β-catenin's flux through the complex by limiting ubiquitylation processivity and directly interacts with the SCF β-TrCP E3 ligase complex in a β-TrCP-dependent manner. Oncogenic APC truncation variants, although part of the complex, are functionally impaired. Nonetheless, even the most severely truncated APC variant promotes β-catenin recruitment. These findings exemplify the power of biochemical reconstitution to interrogate the molecular mechanisms of Wnt/β-catenin signaling., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Protein aggregation in the outermost layers of the cornea, which can lead to cloudy vision and in severe cases blindness, is linked to mutations in the extracellular matrix protein transforming growth factor-β-induced protein (TGFBIp). Among the most frequent pathogenic mutations are R124H and R555W, both associated with granular corneal dystrophy (GCD) characterized by the early-onset formation of amorphous aggregates. The molecular mechanisms of protein aggregation in GCD are largely unknown. In this study, we determined the crystal structures of R124H, R555W, and the lattice corneal dystrophy-associated A546T. Although there were no changes in the monomeric TGFBIp structure of any mutant that would explain their propensity to aggregate, R124H and R555W demonstrated a new dimer interface in the crystal packing, which is not present in wildtype TGFBIp or A546T. This interface, as seen in both the R124H and R555W structures, involves residue 124 of the first TGFBIp molecule and 555 in the second. The interface is not permitted by the Arg124 and Arg555 residues of wildtype TGFBIp and may play a central role in the aggregation exhibited by R124H and R555W in vivo. Using cross-linking mass spectrometry and in-line size exclusion chromatography-small-angle X-ray scattering, we characterized a dimer formed by wildtype and mutant TGFBIps in solution. Dimerization in solution also involves interactions between the N- and C-terminal domains of two TGFBIp molecules but was not identical to the crystal packing dimerization. TGFBIp-targeted interventions that disrupt the R124H/R555W crystal packing dimer interface might offer new therapeutic opportunities to treat patients with GCD., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Cellular growth and proliferation are primarily dictated by the mechanistic target of rapamycin complex 1 (mTORC1), which balances nutrient availability against the cell's anabolic needs. Central to the activity of mTORC1 is the RagA-RagC GTPase heterodimer, which under favorable conditions recruits the complex to the lysosomal surface to promote its activity. The RagA-RagC heterodimer has a unique architecture in that both subunits are active GTPases. To promote mTORC1 activity, the RagA subunit is loaded with GTP and the RagC subunit is loaded with GDP, while the opposite nucleotide-loading configuration inhibits this signaling pathway. Despite its unique molecular architecture, how the Rag GTPase heterodimer maintains the oppositely loaded nucleotide state remains elusive. Here, we applied structure-function analysis approach to the crystal structures of the Rag GTPase heterodimer and identified a key hydrogen bond that stabilizes the GDP-loaded state of the Rag GTPases. This hydrogen bond is mediated by the backbone carbonyl of Asn30 in the nucleotide-binding domain of RagA or Lys84 of RagC and the hydroxyl group on the side chain of Thr210 in the C-terminal roadblock domain of RagA or Ser266 of RagC, respectively. Eliminating this interdomain hydrogen bond abolishes the ability of the Rag GTPase to maintain its functional state, resulting in a distorted response to amino acid signals. Our results reveal that this long-distance interdomain interaction within the Rag GTPase is required for the maintenance and regulation of the mTORC1 nutrient-sensing pathway., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Glycoprotein VI (GPVI) is the major signaling receptor for collagen on platelets. We have raised 54 nanobodies (Nb), grouped into 33 structural classes based on their complementary determining region 3 loops, against recombinant GPVI-Fc (dimeric GPVI) and have characterized their ability to bind recombinant GPVI, resting and activated platelets, and to inhibit platelet activation by collagen. Nbs from 6 different binding classes showed the strongest binding to recombinant GPVI-Fc, suggesting that there was not a single dominant class. The most potent 3, Nb2, 21, and 35, inhibited collagen-induced platelet aggregation with nanomolar half maximal inhibitory concentration (IC50) values and inhibited platelet aggregation under flow. The binding KD of the most potent Nb, Nb2, against recombinant monomeric and dimeric GPVI was 0.6 and 0.7 nM, respectively. The crystal structure of monomeric GPVI in complex with Nb2 revealed a binding epitope adjacent to the collagen-related peptide (CRP) binding groove within the D1 domain. In addition, a novel conformation of GPVI involving a domain swap between the D2 domains was observed. The domain swap is facilitated by the outward extension of the C-C' loop, which forms the domain swap hinge. The functional significance of this conformation was tested by truncating the hinge region so that the domain swap cannot occur. Nb2 was still able to displace collagen and CRP binding to the mutant, but signaling was abolished in a cell-based NFAT reporter assay. This demonstrates that the C-C' loop region is important for GPVI signaling but not ligand binding and suggests the domain-swapped structure may represent an active GPVI conformation., (© 2021 by The American Society of Hematology.)

The characterization of molecular mechanisms underlying the taste-sensing system of chickens will add to our understanding of their feeding behaviors in poultry farming. In the mammalian taste system, the heterodimer of taste receptor type 1 members 1/3 (T1R1/T1R3) functions as an umami (amino acid) taste receptor. Here, we analyzed the expression patterns of T1R1 and T1R3 in the taste cells of chickens, labeled by the molecular markers for chicken taste buds (vimentin and α-gustducin). We observed that α-gustducin was expressed in some of the chicken T1R3-positive taste bud cells but rarely expressed in the T1R1-positive and T2R7-positive taste bud cells. These results raise the possibility that there is another second messenger signaling system in chicken taste sensory cells. We also observed that T1R3 and α-gustducin were expressed mostly in the vimentin-positive taste bud cells, whereas T1R1 and bitter taste receptor (i.e., taste receptor type 2 member 7, T2R7) were expressed largely in the vimentin-negative taste bud cells in chickens. In addition, we observed that T1R1 and T1R3 were co-expressed in about 5% of chickens' taste bud cells, which express T1R1 or T1R3. These results suggest that the heterodimer of T1R1 and T1R3 is rarely formed in chickens' taste bud cells, and they provide comparative insights into the expressional regulation of taste receptors in the taste bud cells of vertebrates.

The interplay between α-synuclein and dopamine derivatives is associated with oxidative stress-dependent neurodegeneration in Parkinson's disease (PD). The formation in the dopaminergic neurons of intraneuronal inclusions containing aggregates of α-synuclein is a typical hallmark of PD. Even though the biochemical events underlying the aberrant aggregation of α-synuclein are not completely understood, strong evidence correlates this process with the levels of dopamine metabolites. In vitro, 3,4-dihydroxyphenylacetaldehyde (DOPAL) and the other two metabolites, 3,4-dihydroxyphenylacetic acid (DOPAC) and 3,4-dihydroxyphenylethanol (DOPET), share the property to inhibit the growth of mature amyloid fibrils of α-synuclein. Although this effect occurs with the formation of differently toxic products, the molecular basis of this inhibition is still unclear. Here, we provide information on the effect of DOPAC on the aggregation properties of α-synuclein and its ability to interact with membranes. DOPAC inhibits α-synuclein aggregation, stabilizing monomer and inducing the formation of dimers and trimers. DOPAC-induced oligomers did not undergo conformational transition in the presence of membranes, and penetrated the cell, where they triggered autophagic processes. Cellular assays showed that DOPAC reduced cytotoxicity and ROS production induced by α-synuclein aggregates. Our findings show that the early radicals resulting from DOPAC autoxidation produced covalent modifications of the protein, which were not by themselves a primary cause of either fibrillation or membrane binding inhibition. These findings are discussed in the light of the potential mechanism of DOPAC protection against the toxicity of α-synuclein aggregates to better understand protein and catecholamine biology and to eventually suggest a scaffold that can help in the design of candidate molecules able to interfere in α-synuclein aggregation.

Amyloid-β (Aβ) 1-40 and 1-42 peptides are key mediators of synaptic and cognitive dysfunction in Alzheimer's disease (AD). Whereas in AD, Aβ is found to act as a pro-epileptogenic factor even before plaque formation, amyloid pathology has been detected among patients with epilepsy with increased risk of developing AD. Among Aβ aggregated species, soluble oligomers are suggested to be responsible for most of Aβ's toxic effects. Aβ oligomers exert extracellular and intracellular toxicity through different mechanisms, including interaction with membrane receptors and the formation of ion-permeable channels in cellular membranes. These damages, linked to an unbalance between excitatory and inhibitory neurotransmission, often result in neuronal hyperexcitability and neural circuit dysfunction, which in turn increase Aβ deposition and facilitate neurodegeneration, resulting in an Aβ-driven vicious loop. In this review, we summarize the most representative literature on the effects that oligomeric Aβ induces on synaptic dysfunction and network disorganization.

von Willebrand disease (VWD), the most prevalent congenital bleeding disorder, arises from a deficiency in von Willebrand factor (VWF), which has crucial roles in hemostasis. The present study investigated functional consequences and underlying pathomolecular mechanisms of several VWF propeptide (VWFpp) missense variants detected in our cohort of VWD patients for the first time. Transient expression experiments in HEK293T cells demonstrated that four out of the six investigated missense variants (p.Gly55Glu, p.Val86Glu, p.Trp191Arg, and p.Cys608Trp) severely impaired secretion. Their cotransfections with the wild-type partly corrected VWF secretion, displaying loss of large/intermediate multimers. Immunostaining of the transfected HEK293 cells illustrated the endoplasmic reticulum (ER) retention of the VWF variants. Docking of the COP I and COP II cargo recruitment proteins, ADP-ribosylation factor 1 and Sec24, onto the N-terminal VWF model (D1D2D'D3) revealed that these variants occur at VWFpp putative interfaces, which can hinder VWF loading at the ER exit quality control. Furthermore, quantitative and automated morphometric exploration of the three-dimensional immunofluorescence images showed changes in the number/size of the VWF storage organelles, Weibel-Palade body (WPB)-like vesicles. The result of this study highlighted the significance of the VWFpp variants on anterograde ER-Golgi trafficking of VWF as well as the biogenesis of WPB-like vesicles., (© 2021 The Authors. Human Mutation published by Wiley Periodicals LLC.)

Many proteins have a multimeric structure and are composed of two or more identical subunits. While this can be advantageous for the host organism, it can be a challenge when targeting specific residues in biochemical analyses. In vitro splitting and re-dimerization to circumvent this problem is a tedious process that requires stable proteins. We present an in vivo approach to transform homodimeric proteins into apparent heterodimers, which then can be purified using two-step affinity-tag purification. This opens the door to both practical applications such as smFRET to probe the conformational dynamics of homooligomeric proteins and fundamental research into the mechanism of protein multimerization, which is largely unexplored for membrane proteins. We show that expression conditions are key for the formation of heterodimers and that the order of the differential purification and reconstitution of the protein into nanodiscs is important for a functional ABC-transporter complex.

Secreted class 3 semaphorins (Sema3s) form tripartite complexes with the plexin receptor and neuropilin coreceptor, which are both transmembrane proteins that together mediate semaphorin signal for neuronal axon guidance and other processes. Despite extensive investigations, the overall architecture of and the molecular interactions in the Sema3/plexin/neuropilin complex are incompletely understood. Here we present the cryo-EM structure of a near intact extracellular region complex of Sema3A, PlexinA4 and Neuropilin 1 (Nrp1) at 3.7 Å resolution. The structure shows a large symmetric 2:2:2 assembly in which each subunit makes multiple interactions with others. The two PlexinA4 molecules in the complex do not interact directly, but their membrane proximal regions are close to each other and poised to promote the formation of the intracellular active dimer for signaling. The structure reveals a previously unknown interface between the a2b1b2 module in Nrp1 and the Sema domain of Sema3A. This interaction places the a2b1b2 module at the top of the complex, far away from the plasma membrane where the transmembrane regions of Nrp1 and PlexinA4 embed. As a result, the region following the a2b1b2 module in Nrp1 must span a large distance to allow the connection to the transmembrane region, suggesting an essential role for the long non-conserved linkers and the MAM domain in neuropilin in the semaphorin/plexin/neuropilin complex.

NF2/Merlin is an upstream regulator of hippo pathway, and it has two states: an auto-inhibited "closed" state and an active "open" form. Previous studies showed that Drosophila Merlin adopts a more closed conformation. However, the molecular mechanism of conformational regulation remains poorly understood. Here, we first confirmed the strong interaction between FERM and the C-terminal domain (CTD) of Merlin, and then determined the crystal structure of the FERM/CTD complex, which reveals the structural basis of Merlin adopting a more closed conformation compared to its human cognate NF2. Interestingly, we found that the conserved lipid-binding site of Merlin might be masked by a linker. Confocal analyses confirmed that all putative lipid-binding site are very important for the membranal location of Merlin. More, we found that the phosphomimic Thr616Asp mutation weakens the interaction between FERM and CTD of Merlin. Collectively, the crystal structure of the FERM/CTD complex not only provides a mechanistic explanation of functionally dormant conformation of Merlin may also serve as a foundation for revealing the mechanism of conformational regulation of Merlin., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Ebola virus (EBOV) glycoprotein (GP) can be recognized by neutralizing antibodies (NAbs) and is the main target for vaccine design. Here, we first investigate the contribution of the stalk and heptad repeat 1-C (HR1 C ) regions to GP metastability. Specific stalk and HR1 C modifications in a mucin-deleted form (GPΔmuc) increase trimer yield, whereas alterations of HR1 C exert a more complex effect on thermostability. Crystal structures are determined to validate two rationally designed GPΔmuc trimers in their unliganded state. We then display a modified GPΔmuc trimer on reengineered protein nanoparticles that encapsulate a layer of locking domains (LD) and a cluster of helper T-cell epitopes. In mice and rabbits, GP trimers and nanoparticles elicit cross-ebolavirus NAbs, as well as non-NAbs that enhance pseudovirus infection. Repertoire sequencing reveals quantitative profiles of vaccine-induced B-cell responses. This study demonstrates a promising vaccine strategy for filoviruses, such as EBOV, based on GP stabilization and nanoparticle display.

Ribosomal RNA synthesis is the rate-limiting step in ribosome biogenesis. In eukaryotes, RNA polymerase I (Pol I) is responsible for transcribing the ribosomal DNA genes that reside in the nucleolus. Aberrations in Pol I activity have been linked to the development of multiple cancers and other genetic diseases. Therefore, it is key that we understand the mechanisms of Pol I transcription. Recent studies have demonstrated that there are many differences between Pol I transcription in yeast and mammals. Our goal is to highlight the similarities and differences between the polymerase-associated factors (PAFs) in yeast and mammalian cells. We focus on the PAF heterodimer A49/34 in yeast and PAF53/49 in mammals. Recent studies have demonstrated that while the structures between the yeast and mammalian orthologs are very similar, they may function differently during Pol I transcription, and their patterns of regulation are different.

The tumor suppressor p53 is mutated in approximately half of all human cancers. p53 can induce apoptosis through mitochondrial membrane permeabilization by interacting with and antagonizing the anti-apoptotic proteins BCL-xL and BCL-2. However, the mechanisms by which p53 induces mitochondrial apoptosis remain elusive. Here, we report a 2.5 Å crystal structure of human p53/BCL-xL complex. In this structure, two p53 molecules interact as a homodimer, and bind one BCL-xL molecule to form a ternary complex with a 2:1 stoichiometry. Mutations at the p53 dimer interface or p53/BCL-xL interface disrupt p53/BCL-xL interaction and p53-mediated apoptosis. Overall, our current findings of the bona fide structure of p53/BCL-xL complex reveal the molecular basis of the interaction between p53 and BCL-xL, and provide insight into p53-mediated mitochondrial apoptosis.

Sarcomeres, the basic contractile units of striated muscle cells, contain arrays of thin (actin) and thick (myosin) filaments that slide past each other during contraction. The Ig-like domain-containing protein myotilin provides structural integrity to Z-discs-the boundaries between adjacent sarcomeres. Myotilin binds to Z-disc components, including F-actin and α-actinin-2, but the molecular mechanism of binding and implications of these interactions on Z-disc integrity are still elusive. To illuminate them, we used a combination of small-angle X-ray scattering, cross-linking mass spectrometry, and biochemical and molecular biophysics approaches. We discovered that myotilin displays conformational ensembles in solution. We generated a structural model of the F-actin:myotilin complex that revealed how myotilin interacts with and stabilizes F-actin via its Ig-like domains and flanking regions. Mutant myotilin designed with impaired F-actin binding showed increased dynamics in cells. Structural analyses and competition assays uncovered that myotilin displaces tropomyosin from F-actin. Our findings suggest a novel role of myotilin as a co-organizer of Z-disc assembly and advance our mechanistic understanding of myotilin's structural role in Z-discs., Competing Interests: The authors have declared that no competing interests exist.

Icosahedral viral capsids assemble with high fidelity from a large number of identical buildings blocks. The mechanisms that enable individual capsid proteins to form stable oligomeric units (capsomers) while affording structural adaptability required for further assembly into capsids are mostly unknown. Understanding these mechanisms requires knowledge of the capsomers' dynamics, especially for viruses where no additional helper proteins are needed during capsid assembly like for the Mavirus virophage that despite its complexity (triangulation number T = 27) can assemble from its major capsid protein (MCP) alone. This protein forms the basic building block of the capsid namely a trimer (MCP 3 ) of double-jelly roll protomers with highly intertwined N-terminal arms of each protomer wrapping around the other two at the base of the capsomer, secured by a clasp that is formed by part of the C-terminus. Probing the dynamics of the capsomer with HDX mass spectrometry we observed differences in conformational flexibility between functional elements of the MCP trimer. While the N-terminal arm and clasp regions show above average deuterium incorporation, the two jelly-roll units in each protomer also differ in their structural plasticity, which might be needed for efficient assembly. Assessing the role of the N-terminal arm in maintaining capsomer stability showed that its detachment is required for capsomer dissociation, constituting a barrier towards capsomer monomerisation. Surprisingly, capsomer dissociation was irreversible since it was followed by a global structural rearrangement of the protomers as indicated by computational studies showing a rearrangement of the N-terminus blocking part of the capsomer forming interface., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

EGFR is established as a driver of lung cancer, yet the regulatory machinery underlying its oncogenic activity is not fully understood. PTEN-induced kinase 1 (PINK1) kinase is a key player in mitochondrial quality control, although its role in lung cancer and EGFR regulation is unclear. In this study, we show that PINK1 physically directly interacts with EGFR via the PINK1 C-terminal domain (PINK1-CTD) and the EGFR tyrosine kinase domain. This interaction constituted an endogenous steric hindrance to receptor dimerization and inhibited EGFR-mediated lung carcinogenesis. Depletion of PINK1 from lung cancer cells promoted EGFR dimerization, receptor activation, EGFR downstream signaling, and tumor growth. In contrast, overexpression of PINK1 or PINK1-CTD suppressed EGFR dimerization, activation, downstream signaling, and tumor growth. These findings identify key elements in the EGFR regulatory cascade and illustrate a new direction for the development of anti-EGFR therapeutics, suggesting translational potential of the PINK1-CTD in lung cancer. SIGNIFICANCE: This study identifies PINK1 as a critical tumor suppressor that impedes EGFR dimerization and highlights PINK1-CTD as a potential therapeutic agent in EGFR-driven lung cancer., (©2021 American Association for Cancer Research.)

G protein-coupled receptors (GPCRs) represent a major group of drug targets with tremendous pharmacological value. Signals arising from GPCRs are primarily transduced via two functional components of their corresponding G proteins, the Gα subunit and the Gβγ dimer that dissociate from each other upon activation of the heterotrimer (Gαβγ). The Gβγ dimer has become an increasingly popular subject in GPCR signaling, owing to its numerous effectors and notable roles in signal integration. Because Gβγ dimers participate in a wide range of intracellular processes that regulate cellular physiology, they are often implicated in the pathology of various diseases. Yet, one caveat to the current 'Dissociation Model' on GPCR signaling is that unequivocal Gβγ signals are biasedly detected with G i/o -coupled receptors, while Gβγ signals from G s - or G q -coupled receptors seem to play an auxiliary role. In this review, we revisit the evidence for or against the 'Dissociation Model' and discuss in detail several hypotheses that may explain such disparity and provide alternative interpretations to accommodate the 'biased Gβγ signals' observed in different biological systems. The issue of whether unique combinations of Gβγ dimer can confer signaling specificity is also discussed in the context of physiological relevance., (© 2020 Federation of European Biochemical Societies.)