UNASSIGNED: Chromosomal translocations involving neurotrophic receptor tyrosine kinases (NTRKs) have been identified in 20 % of soft tissue sarcomas. This work focuses on the EML4-NTRK3 translocation identified in cases of Infantile Fibrosarcoma, which contains the coiled-coil multimerization domain of Echinoderm Microtubule-like protein 4 (EML4) fused with the tyrosine kinase domain of Neurotrophic Receptor Tyrosine Kinase 3 (NTRK3). The aim of the study was to test the importance of tyrosine kinase activity and multimerization for the oncogenic activity of EML4-NTRK3.
UNASSIGNED: These studies examined EML4-NTRK3 proteins containing a kinase-dead or WT kinase domain, together with mutations in specific salt bridge residues within the coiled-coil domain. Biological activity was assayed using focus assays in NIH3T3 cells. The MAPK/ERK, JAK/STAT3 and PI3K/AKT pathways were analyzed for downstream activation of signaling pathways. Localization of EML4-NTRK3 proteins was examined by immunofluorescence microscopy, and the ability of the EML4 coiled-coil domain to drive protein multimerization was examined by biochemical assays.
UNASSIGNED: Activation of EML4-NTRK3 relies on both the tyrosine kinase activity of NTRK3 and salt-bridge stabilization within the coiled-coil domain of EML4. The tyrosine kinase activity of NTRK3 is essential for the biological activation of EML4-NTRK3. Furthermore, EML4-NTRK3 activates downstream signaling pathways MAPK/ERK, JAK/STAT3 and PKC/PLCγ. The disruption of three specific salt bridge interactions within the EML4 coiled-coil domain of EML4-NTRK3 blocks downstream activation, biological activity, and the ability to hetero-multimerize with EML4. We also demonstrate that EML4-NTRK3 is localized in the cytoplasm and fails to associate with microtubules.
UNASSIGNED: These data suggest potential therapeutic strategies for Infantile Fibrosarcoma cases bearing EML4-NTRK3 fusion through inhibition of salt bridge interactions and disruption of multimerization.

Cohesin is key to eukaryotic genome organization and acts throughout the cell cycle in an ATP-dependent manner. The mechanisms underlying cohesin ATPase activity are poorly understood. Here, we characterize distinct steps of the human cohesin ATPase cycle and show that the SMC1A and SMC3 ATPase domains undergo specific but concerted structural rearrangements along this cycle. Specifically, whereas the proximal coiled coil of the SMC1A ATPase domain remains conformationally stable, that of the SMC3 displays an intrinsic flexibility. The ATP-dependent formation of the heterodimeric SMC1A/SMC3 ATPase module (engaged state) favors this flexibility, which is counteracted by NIPBL and DNA binding (clamped state). Opening of the SMC3/RAD21 interface (open-engaged state) stiffens the SMC3 proximal coiled coil, thus constricting together with that of SMC1A the ATPase module DNA-binding chamber. The plasticity of the ATP-dependent interface between the SMC1A and SMC3 ATPase domains enables these structural rearrangements while keeping the ATP gate shut. VIDEO ABSTRACT.

Soluble N-ethylmaleimide-sensitive factor Attachment protein REceptor (SNARE) proteins catalyze the fusion process of vesicles with target membranes in eukaryotic cells. To do this, they assemble in a zipper-like fashion into stable complexes between the membranes. Structural studies have shown that the complexes consist of four different helices, which we subdivide into Qa-, Qb-, Qc-, and R-helix on the basis of their sequence signatures. Using a combination of biochemistry, modeling and molecular dynamics, we investigated how the four different types are arranged in a complex. We found that there is a matching pattern in the core of the complex that dictates the position of the four fundamental SNARE types in the bundle, resulting in a QabcR complex. In the cell, several different cognate QabcR-SNARE complexes catalyze the different transport steps between the compartments of the endomembrane system. Each of these cognate QabcR complexes is compiled from a repertoire of about 20 SNARE subtypes. Our studies show that exchange within the four types is largely tolerated structurally, although some non-cognate exchanges lead to structural imbalances. This suggests that SNARE complexes have evolved for a catalytic mechanism, a mechanism that leaves little scope for selectivity beyond the QabcR rule.

The intermitochondrial cement (IMC) is a prominent germ granule that locates among clustered mitochondria in mammalian germ cells. Serving as a key platform for Piwi-interacting RNA (piRNA) biogenesis; however, how the IMC assembles among mitochondria remains elusive. Here, we identify that Tudor domain-containing 1 (TDRD1) triggers IMC assembly via phase separation. TDRD1 phase separation is driven by the cooperation of its tetramerized coiled-coil domain and dimethylarginine-binding Tudor domains but is independent of its intrinsically disordered region. TDRD1 is recruited to mitochondria by MILI and sequentially enhances mitochondrial clustering and triggers IMC assembly via phase separation to promote piRNA processing. TDRD1 phase separation deficiency in mice disrupts IMC assembly and piRNA biogenesis, leading to transposon de-repression and spermatogenic arrest. Moreover, TDRD1 phase separation is conserved in vertebrates but not in invertebrates. Collectively, our findings demonstrate a role of phase separation in germ granule formation and establish a link between membrane-bound organelles and membrane-less organelles.

Septins are cytoskeletal proteins and their interaction with membranes is crucial for their role in various cellular processes. Septins have polybasic regions (PB1 and PB2) which are important for lipid interaction. Earlier, we and others have highlighted the role of the septin C-terminal domain (CTD) to membrane interaction. However, detailed information on residues/group of residues important for such feature is lacking. In this study, we investigate the lipid-binding profile of Schistosoma mansoni Septin10 (SmSEPT10) using PIP strip and Langmuir monolayer adsorption assays. Our findings highlight the CTD as the primary domain responsible for lipid interaction in SmSEPT10, showing binding to phosphatidylinositol phosphates. SmSEPT10 CTD contains a conserved polybasic region (PB3) present in both animals and fungi septins, and a Lys (K367) within its putative amphipathic helix (AH) that we demonstrate as important for lipid binding. PB3 deletion or mutation of this Lys (K367A) strongly impairs lipid interaction. Remarkably, we observe that the AH within a construct lacking the final 43 amino acid residues is insufficient for lipid binding. Furthermore, we investigate the homocomplex formed by SmSEPT10 CTD in solution by cross-linking experiments, CD spectroscopy, SEC-MALS and SEC-SAXS. Taken together, our studies define the lipid-binding region in SmSEPT10 and offer insights into the molecular basis of septin-membrane binding. This information is particularly relevant for less-studied non-human septins, such as SmSEPT10.

Post-translational modifications (PTMs) such as phosphorylation and dephosphorylation can rapidly alter protein surface chemistry and structural conformation, which can switch protein-protein interactions (PPIs) within signaling networks. Recently, de novo-designed phosphorylation-responsive protein switches have been created that harness kinase- and phosphatase-mediated phosphorylation to modulate PPIs. PTM-driven protein switches are promising tools for investigating PTM dynamics in living cells, developing biocompatible nanodevices, and engineering signaling pathways to program cell behavior. However, little is known about the physical and kinetic constraints of PTM-driven protein switches, which limits their practical application. In this study, we present a framework to evaluate two-component PTM-driven protein switches based on four performance metrics: effective concentration, dynamic range, response time, and reversibility. Our computational models reveal an intricate relationship between the binding kinetics, phosphorylation kinetics, and switch concentration that governs the sensitivity and reversibility of PTM-driven protein switches. Building upon the insights of the interaction modeling, we built and evaluated novel phosphorylation-driven protein switches consisting of phosphorylation-sensitive coiled coils as sensor domains fused to fluorescent proteins as actuator domains. By modulating the phosphorylation state of the switches with a specific protein kinase and phosphatase, we demonstrate fast, reversible transitions between \"on\" and \"off\" states. The response of the switches linearly correlated to the kinase concentration, demonstrating its potential as a biosensor for kinase measurements in real time. As intended, the switches responded to specific kinase activity with an increase in the fluorescence signal and our model could be used to distinguish between two mechanisms of switch activation: dimerization or a structural rearrangement. The protein switch kinetics model developed here should enable PTM-driven switches to be designed with ideal performance for specific applications.

Self-assembly of designed molecules has enabled the construction of a variety of functional nanostructures. Specifically, adaptable self-assembly has demonstrated several advantageous features for smart materials. Here, we demonstrate that an α-helical coiled coil conjugated with a dendrimer can adapt to spatial restriction due to the strong steric repulsion between dendrimer chains. The adaptable transformation of a tetrameric coiled coil to a trimeric coiled coil can be confirmed using analytical ultracentrifugation upon conjugation of the dendrimer to the coiled coil-forming building block. Interestingly, circular dichroism spectroscopy analysis of the dendrimer conjugate revealed an unconventional trend: the multimerization of the coiled coil is inversely dependent on concentration. This result implies that the spatial crowding between the bulky dendritic chains is significantly stronger than that between linear chains, thereby affecting the overall assembly process. We further illustrated the application potential by decorating the surface of gold nanorods (AuNRs) with the adaptable coiled coil. The dendrimer-coiled coil peptide conjugate can be utilized to fabricate organic-inorganic nanohybrids with enhanced colloidal and thermal stabilities. This study demonstrates that the coiled coil can engage in the adaptable mode of self-assembly with the potential to form dynamic peptide-based materials.

Efforts to develop an HIV-1 vaccine include those focusing on conserved structural elements as the target of broadly neutralizing monoclonal antibodies. MAb D5 binds to a highly conserved hydrophobic pocket on the gp41 N-heptad repeat (NHR) coiled coil and neutralizes through prevention of viral fusion and entry. Assessment of 17-mer and 36-mer NHR peptides presenting the D5 epitope in rodent immunogenicity studies showed that the longer peptide elicited higher titers of neutralizing antibodies, suggesting that neutralizing epitopes outside of the D5 pocket may exist. Although the magnitude and breadth of neutralization elicited by NHR-targeting antigens are lower than that observed for antibodies directed to other epitopes on the envelope glycoprotein complex, it has been shown that NHR-directed antibodies are potentiated in TZM-bl cells containing the FcγRI receptor. Herein, we report the design and evaluation of covalently stabilized trimeric 51-mer peptides encompassing the complete gp41 NHR. We demonstrate that these peptide trimers function as effective antiviral entry inhibitors and retain the ability to present the D5 epitope. We further demonstrate in rodent and nonhuman primate immunization studies that our 51-mer constructs elicit a broader repertoire of neutralizing antibody and improved cross-clade neutralization of primary HIV-1 isolates relative to 17-mer and 36-mer NHR peptides in A3R5 and FcγR1-enhanced TZM-bl assays. These results demonstrate that sensitive neutralization assays can be used for structural enhancement of moderately potent neutralizing epitopes. Finally, we present expanded trimeric peptide designs which include unique low-molecular-weight scaffolds that provide versatility in our immunogen presentation strategy.

The therapeutic potential of bispecific antibodies is becoming widely recognised, with over a hundred formats already described. For many applications, enhanced tissue penetration is sought, so bispecifics with low molecular weight may offer a route to enhanced potency. Here we report the design of bi- and tri-specific antibody-based constructs with molecular weights as low as 14.5 and 22 kDa respectively.
Autonomous bovine ultra-long CDR H3 (knob domain peptide) modules have been engineered with artificial coiled-coil stalks derived from Sin Nombre orthohantavirus nucleocapsid protein and human Beclin-1, and joined in series to produce bi- and tri-specific antibody-based constructs with exceptionally low molecular weights.
Knob domain peptides with coiled-coil stalks retain high, independent antigen binding affinity, exhibit exceptional levels of thermal stability, and can be readily joined head-to-tail yielding the smallest described multi-specific antibody format. The resulting constructs are able to bind simultaneously to all their targets with no interference.
Compared to existing bispecific formats, the reduced molecular weight of the knob domain fusions may enable enhanced tissue penetration and facilitate binding to cryptic epitopes that are inaccessible to conventional antibodies. Furthermore, they can be easily produced at high yield as recombinant products and are free from the heavy-light chain mispairing issue. Taken together, our approach offers an efficient route to modular construction of minimalistic bi- and multi-specifics, thereby further broadening the therapeutic scope for knob domain peptides.

Nucleotide-binding domain and leucine-rich repeat (NLR) proteins are a prominent class of intracellular immune receptors in plants. However, our understanding of plant NLR structure and function is limited to the evolutionarily young flowering plant clade. Here, we describe an extended spectrum of NLR diversity across divergent plant lineages and demonstrate the structural and functional similarities of N-terminal domains that trigger immune responses. We show that the broadly distributed coiled-coil (CC) and toll/interleukin-1 receptor (TIR) domain families of nonflowering plants retain immune-related functions through translineage activation of cell death in the angiosperm Nicotiana benthamiana. We further examined a CC subfamily specific to nonflowering lineages and uncovered an essential N-terminal MAEPL motif that is functionally comparable with motifs in resistosome-forming CC-NLRs. Consistent with a conserved role in immunity, the ectopic activation of CCMAEPL in the nonflowering liverwort Marchantia polymorpha led to profound growth inhibition, defense gene activation, and signatures of cell death. Moreover, comparative transcriptomic analyses of CCMAEPL activity delineated a common CC-mediated immune program shared across evolutionarily divergent nonflowering and flowering plants. Collectively, our findings highlight the ancestral nature of NLR-mediated immunity during plant evolution that dates its origin to at least ∼500 million years ago.