Interaction Motif Research Articles

Multivalent Tau/PSD-95 interactions arrest in vitro condensates and clusters mimicking the postsynaptic density

Alzheimer’s disease begins with mild memory loss and slowly destroys memory and thinking. Cognitive impairment in Alzheimer’s disease has been associated with the localization of the microtubule-associated protein Tau at the postsynapse. However, the correlation between Tau at the postsynapse and synaptic dysfunction remains unclear. Here, we show that Tau arrests liquid-like droplets formed by the four postsynaptic density proteins PSD-95, GKAP, Shank, Homer in solution, as well as NMDA (N-methyl-D-aspartate)-receptor-associated protein clusters on synthetic membranes. Tau-mediated condensate/cluster arrest critically depends on the binding of multiple interaction motifs of Tau to a canonical GMP-binding pocket in the guanylate kinase domain of PSD-95. We further reveal that competitive binding of a high-affinity phosphorylated peptide to PSD-95 rescues the diffusional dynamics of an NMDA truncated construct, which contains the last five amino acids of the NMDA receptor subunit NR2B fused to the C-terminus of the tetrameric GCN4 coiled-coil domain, in postsynaptic density-like condensates/clusters. Taken together, our findings propose a molecular mechanism where Tau modulates the dynamic properties of the postsynaptic density.

Structural properties of the HNF-1A transactivation domain.

Hepatocyte nuclear factor 1α (HNF-1A) is a transcription factor with important gene regulatory roles in pancreatic β-cells. HNF1A gene variants are associated with a monogenic form of diabetes (HNF1A-MODY) or an increased risk for type 2 diabetes. While several pancreatic target genes of HNF-1A have been described, a lack of knowledge regarding the structure-function relationships in HNF-1A prohibits a detailed understanding of HNF-1A-mediated gene transcription, which is important for precision medicine and improved patient care. Therefore, we aimed to characterize the understudied transactivation domain (TAD) of HNF-1A in vitro. We present a bioinformatic approach to dissect the TAD sequence, analyzing protein structure, sequence composition, sequence conservation, and the existence of protein interaction motifs. Moreover, we developed the first protocol for the recombinant expression and purification of the HNF-1A TAD. Small-angle X-ray scattering and synchrotron radiation circular dichroism suggested a disordered conformation for the TAD. Furthermore, we present functional data on HNF-1A undergoing liquid-liquid phase separation, which is in line with in silico predictions and may be of biological relevance for gene transcriptional processes in pancreatic β-cells.

Two‐Dimensional Supramolecular Polymerization of a Bis‐Urea Macrocycle into a Brick‐Like Hydrogen‐Bonded Network

AbstractWe report on a dendronized bis‐urea macrocycle 1 self‐assembling via a cooperative mechanism into two‐dimensional (2D) nanosheets formed solely by alternated urea‐urea hydrogen bonding interactions. The pure macrocycle self‐assembles in bulk into one‐dimensional liquid‐crystalline columnar phases. In contrast, its self‐assembly mode drastically changes in CHCl3 or tetrachloroethane, leading to 2D hydrogen‐bonded networks. Theoretical calculations, complemented by previously reported crystalline structures, indicate that the 2D assembly is formed by a brick‐like hydrogen bonding pattern between bis‐urea macrocycles. This assembly is promoted by the swelling of the trisdodecyloxyphenyl groups upon solvation, which frustrates, due to steric effects, the formation of the thermodynamically more stable columnar macrocycle stacks. This work proposes a new design strategy to access 2D supramolecular polymers by means of a single non‐covalent interaction motif, which is of great interest for materials development.

Two-Dimensional Supramolecular Polymerization of a Bis-Urea Macrocycle into a Brick-Like Hydrogen-Bonded Network.

We report on a dendronized bis-urea macrocycle 1 self-assembling via a cooperative mechanism into two-dimensional (2D) nanosheets formed solely by alternated urea-urea hydrogen bonding interactions. The pure macrocycle self-assembles in bulk into one-dimensional liquid-crystalline columnar phases. In contrast, its self-assembly mode drastically changes in CHCl3 or tetrachloroethane, leading to 2D hydrogen-bonded networks. Theoretical calculations, complemented by previously reported crystalline structures, indicate that the 2D assembly is formed by a brick-like hydrogen bonding pattern between bis-urea macrocycles. This assembly is promoted by the swelling of the trisdodecyloxyphenyl groups upon solvation, which frustrates, due to steric effects, the formation of the thermodynamically more stable columnar macrocycle stacks. This work proposes a new design strategy to access 2D supramolecular polymers by means of a single non-covalent interaction motif, which is of great interest for materials development.

Pressure-dependent cationic associations of ionic liquids with bentonite nanoclay

Pressure-dependent IR spectroscopy is used to investigate the possibility of stabilizing ionic liquid (IL)–hydrophilic bentonite (Bent) composites with the application of high pressures. Ambient-pressure experiments suggest IL hydrophilicity/hydrophobicity impacts cation and anion interactions with Bent. For example, both the cation and anion of hydrophilic 1-butyl-3-methylimidazolium dicyanamide ([BMIM][DCA]) experience composition-dependent vibrational mode wavenumber shifts. However, anion–Bent interactions are suppressed at ambient pressure when the nanoclay is paired with hydrophobic 1-butyl-3-methylimidazolium bis(trifluoromethanesulfonyl)imide ([BMIM][NTf2]). Compression to high pressures triggers significant blue shifts for ring-bound C–H stretching bands. The rates of these changes upon pressurization indicate the two ILs experience different interaction motifs with the Bent surface. Large wavenumber shifts for C4–H and C5–H stretching modes of [BMIM][NTf2] imply preferential coordination to Bent surface sites through these groups. This presumably leaves the C2–H group available for hydrogen bonding with the NTf2 anion. The situation is different for [BMIM][DCA], where all three C–H groups on the imidazolium ring appear to interact with Bent surface sites. Depressurization reveals further differences between the two systems. Spectroscopic features are reversible for [BMIM][NTf2], whereas the C–H stretching mode blue shifts for [BMIM][DCA] are either irreversible or the relaxation is kinetically hindered upon return to ambient pressure. Taken together, the spectroscopic data strongly suggests that IL–Bent interactions are pressure sensitive, and IL assemblages along the interface may be manipulated through the application of high pressure.

Structural and biochemical insights into the interaction mechanism underlying HORMAD1 and its partner proteins

The mammalian HORMA domain-containing protein 1 (HORMAD1) regulates DNA mismatch repair and homologous recombination (HR) repair in many cancers. Here, we show that the structure of human HORMAD1 adopts a self-closed conformation and displays an intra-molecular HORMA domain-closure motif interaction mode. Structural and biochemical data suggest that the interaction modes of the peptide motifs from HORMAD2 and MCM9 with HORMAD1 are highly similar to that of HORMAD1 own closure motif. The peptide motifs from diverse binding partners of HORMAD1 share a conserved Ser-Glu-Pro sequence. Additionally, structural comparison unveiled the HORMA-peptide motif interaction mode diversity among HORMA-containing proteins. Finally, cell-based assays revealed that this HORMA-closure motif interaction pattern contributes to DNA mismatch repair and is required for HORMAD1-dependent HR repair. Together, our results provide structural and biochemical insights into the common theme and functional plasticity of the HORMA domain-containing protein family, and also reveal a universal regulation mechanism for HORMAD1.

A CK2 and SUMO-dependent, PML NB-involved regulatory mechanism controlling BLM ubiquitination and G-quadruplex resolution

The Boom syndrome helicase (BLM) unwinds a variety of DNA structures such as Guanine (G)-quadruplex. Here we reveal a role of RNF111/Arkadia and its paralog ARKL1, as well as Promyelocytic Leukemia Nuclear Bodies (PML NBs), in the regulation of ubiquitination and control of BLM protein levels. RNF111 exhibits a non-canonical SUMO targeted E3 ligase (STUBL) activity targeting BLM ubiquitination in PML NBs. ARKL1 promotes RNF111 localization to PML NBs through SUMO-interacting motif (SIM) interaction with SUMOylated RNF111, which is regulated by casein kinase 2 (CK2) phosphorylation of ARKL1 at a serine residue near the ARKL1 SIM domain. Upregulated BLM in ARKL1 or RNF111-deficient cells leads to a decrease of G-quadruplex levels in the nucleus. These results demonstrate that a CK2- and RNF111-ARKL1-dependent regulation of BLM in PML NBs plays a critical role in controlling BLM protein levels for the regulation of G-quadruplex.

The Calpain-7 protease functions together with the ESCRT-III protein IST1 within the midbody to regulate the timing and completion of abscission.

The Endosomal Sorting Complexes Required for Transport (ESCRT) machinery mediates the membrane fission step that completes cytokinetic abscission and separates dividing cells. Filaments composed of ESCRT-III subunits constrict membranes of the intercellular bridge midbody to the abscission point. These filaments also bind and recruit cofactors whose activities help execute abscission and/or delay abscission timing in response to mitotic errors via the NoCut/Abscission checkpoint. We previously showed that the ESCRT-III subunit IST1 binds the cysteine protease Calpain-7 (CAPN7) and that CAPN7 is required for both efficient abscission and NoCut checkpoint maintenance (Wenzel et al., 2022). Here, we report biochemical and crystallographic studies showing that the tandem microtubule-interacting and trafficking (MIT) domains of CAPN7 bind simultaneously to two distinct IST1 MIT interaction motifs. Structure-guided point mutations in either CAPN7 MIT domain disrupted IST1 binding in vitro and in cells, and depletion/rescue experiments showed that the CAPN7-IST1 interaction is required for (1) CAPN7 recruitment to midbodies, (2) efficient abscission, and (3) NoCut checkpoint arrest. CAPN7 proteolytic activity is also required for abscission and checkpoint maintenance. Hence, IST1 recruits CAPN7 to midbodies, where its proteolytic activity is required to regulate and complete abscission.

Zα domain proteins mediate the immune response.

The Zα domain has a compact α/β architecture containing a three-helix bundle flanked on one side by a twisted antiparallel β sheet. This domain displays a specific affinity for double-stranded nucleic acids that adopt a left-handed helical conformation. Currently, only three Zα-domain proteins have been identified in eukaryotes, specifically ADAR1, ZBP1, and PKZ. ADAR1 is a double-stranded RNA (dsRNA) binding protein that catalyzes the conversion of adenosine residues to inosine, resulting in changes in RNA structure, function, and expression. In addition to its editing function, ADAR1 has been shown to play a role in antiviral defense, gene regulation, and cellular differentiation. Dysregulation of ADAR1 expression and activity has been associated with various disease states, including cancer, autoimmune disorders, and neurological disorders. As a sensing molecule, ZBP1 exhibits the ability to recognize nucleic acids with a left-handed conformation. ZBP1 harbors a RIP homotypic interaction motif (RHIM), composed of a highly charged surface region and a leucine-rich hydrophobic core, enabling the formation of homotypic interactions between proteins with similar structure. Upon activation, ZBP1 initiates a downstream signaling cascade leading to programmed cell death, a process mediated by RIPK3 via the RHIM motif. PKZ was identified in fish, and contains two Zα domains at the N-terminus. PKZ is essential for normal growth and development and may contribute to the regulation of immune system function in fish. Interestingly, some pathogenic microorganisms also encode Zα domain proteins, such as, Vaccinia virus and Cyprinid Herpesvirus. Zα domain proteins derived from pathogenic microorganisms have been demonstrated to be pivotal contributors in impeding the host immune response and promoting virus replication and spread. This review focuses on the mammalian Zα domain proteins: ADAR1 and ZBP1, and thoroughly elucidates their functions in the immune response.

A structural comparison of salt forms of dopamine with the structures of other phenylethylamines.

The structures of four salt forms of dopamine are reported. These are dopamine [2-(3,4-dihydroxyphenyl)ethan-1-aminium] benzoate, C8H12NO2+·C7H5O2-, I, dopamine 4-nitrobenzoate, C8H12NO2+·C7H4NO4-, II, dopamine ethanedisulfonate, 2C8H12NO2+·C2H4O6S22-, III, and dopamine 4-hydroxybenzenesulfonate monohydrate, C8H12NO2+·C6H5O4S-·H2O, IV. In all four structures, the dopamine cation adopts an extended conformation. Intermolecular interaction motifs that are common in the salt forms of tyramine can be found in related dopamine structures, but hydrogen bonding in the dopamine structures appear to be more variable and less predictable than for tyramine. Packing analysis discovered three dopamine-containing groups of structures that can be described as isostructural with regards to the cation positions. Two of these groups contain both dopamine and tyramine species, and one of these is also highly variable in other ways too, containing anhydrous and hydrated forms, different anion types and ionized and neutral phenylethylamine species. As such, the group illustrates that packing behaviour can be robust and similar even where intermolecular interactions such as hydrogen bonds are very different.

Intrinsically disordered CsoS2 acts as a general molecular thread for α-carboxysome shell assembly

Carboxysomes are a paradigm of self-assembling proteinaceous organelles found in nature, offering compartmentalisation of enzymes and pathways to enhance carbon fixation. In α-carboxysomes, the disordered linker protein CsoS2 plays an essential role in carboxysome assembly and Rubisco encapsulation. Its mechanism of action, however, is not fully understood. Here we synthetically engineer α-carboxysome shells using minimal shell components and determine cryoEM structures of these to decipher the principle of shell assembly and encapsulation. The structures reveal that the intrinsically disordered CsoS2 C-terminus is well-structured and acts as a universal “molecular thread” stitching through multiple shell protein interfaces. We further uncover in CsoS2 a highly conserved repetitive key interaction motif, [IV]TG, which is critical to the shell assembly and architecture. Our study provides a general mechanism for the CsoS2-governed carboxysome shell assembly and cargo encapsulation and further advances synthetic engineering of carboxysomes for diverse biotechnological applications.

Double Cation-π Directed Two-Dimensional Metallacycle-Based Hierarchical Self-Assemblies for Dual-Mode Catalysis.

Hierarchical self-assembly of Pt(II) metallacycles for the construction of functional materials has received considerable research interest, owing to their potential to meet increasing complexity and functionality demands while being based on well-defined scaffolds. However, the fabrication of long-range-ordered Pt(II) metallacycle-based two-dimensional hierarchical self-assemblies (2D HSAs) remains a challenge, primarily because of the limitations of conventional orthogonal noncovalent interaction (NCI) motifs and the intrinsic characteristics of Pt(II) metallacycles, making the delicate self-assembly processes difficult to control. Herein, we prepare well-regulated Pt(II)-metallacycle-based 2D HSAs through a directed strategy involving double cation-π interactions derived from C3-symmetric hexagonal Pt(II) metallacycles and C2-symmetric sodium phenate monomers. Spatially confined arrays of planar Pt(II) metallacycles and the selective growth of self-assemblies at desired locations are achieved by employing strong cation-π driving forces with well-defined directionality as the second orthogonal NCI, realizing the bottom-up, three-stage construction of Pt(II)-metallacycle-based 2D HSAs. The resultant 2D HSAs are applied as dual-mode catalysis platforms, which are loaded with two different nanocatalysts, one promoting catalytic oxidation and the other promoting photocatalytic reduction.

LIM domain only 7 negatively controls nonalcoholic steatohepatitis in the setting of hyperlipidemia.

Hyperlipidemia has been extensively recognized as a high-risk factor for NASH; however, clinical susceptibility to NASH is highly heterogeneous. The key controller(s) of NASH susceptibility in patients with hyperlipidemia has not yet been elucidated. Here, we aimed to reveal the key regulators of NASH in patients with hyperlipidemia and to explore its role and underlying mechanisms. To identify the predominant suppressors of NASH in the setting of hyperlipidemia, we collected liver biopsy samples from patients with hyperlipidemia, with or without NASH, and performed RNA-sequencing analysis. Notably, decreased Lineage specific Interacting Motif domain only 7 (LMO7) expression robustly correlated with the occurrence and severity of NASH. Although overexpression of LMO7 effectively blocked hepatic lipid accumulation and inflammation, LMO7 deficiency in hepatocytes greatly exacerbated diet-induced NASH progression. Mechanistically, lysine 48 (K48)-linked ubiquitin-mediated proteasomal degradation of tripartite motif-containing 47 (TRIM47) and subsequent inactivation of the c-Jun N-terminal kinase (JNK)/p38 mitogen-activated protein kinase (MAPK) cascade are required for the protective function of LMO7 in NASH. These findings provide proof-of-concept evidence supporting LMO7 as a robust suppressor of NASH in the context of hyperlipidemia, indicating that targeting the LMO7-TRIM47 axis is a promising therapeutic strategy for NASH.

(Invited) Crystallographic Characterization of U@C2 n (2n = 82−86): Insights about Metal-Cage Interactions for Mono-Metallofullerenes

Endohedral mono-metallofullerenes are the prototypes to understand the fundamental nature and the unique interactions between the encapsulated metals and the fullerene cages. Recently, we succeeded in the synthesis, isolation, and characterization of four new U-based mono-metallofullerenes, including U@Cs (6)-C82, U@C 2(8)-C84, U@Cs (15)-C84, and U@C 1(12)-C86. Furthermore, a new finding concerning the metal positions inside the cages reveals that the encapsulated metal ions are always located on symmetry planes of the fullerene cages, as long as the fullerene cages possess mirror planes. Symmetrical patterns, such as indacene, sumanene, and phenalene, and charge transfer are found to determine the metal positions inside the fullerene cages. DFT calculations support that the metal-fullerene motif interaction determines the stability of the metal position. In fullerenes containing symmetry planes, the metal prefers to occupy a symmetrical arrangement with respect to the interacting motifs, which share one of their symmetry planes with the fullerene. This finding provides new insights into the nature of metal-cage interactions and gives new guidelines for structural determinations using crystallographic and theoretical methods.

The EDC4-XRN1 interaction controls P-body dynamics to link mRNA decapping with decay.

Deadenylation-dependent mRNA decapping and decay is the major cytoplasmic mRNA turnover pathway in eukaryotes. Many mRNA decapping and decay factors are associated with each other via protein-protein interaction motifs. For example, the decapping enzyme DCP2 and the 5'-3' exonuclease XRN1 interact with the enhancer of mRNA-decapping protein 4 (EDC4), a large scaffold that has been reported to stimulate mRNA decapping. mRNA decapping and decay factors are also found in processing bodies (P-bodies), evolutionarily conserved ribonucleoprotein granules that are often enriched with mRNAs targeted for decay, yet paradoxically are not required for mRNA decay to occur. Here, we show that disrupting the EDC4-XRN1 interaction or altering their stoichiometry inhibits mRNA decapping, with microRNA-targeted mRNAs being stabilized in a translationally repressed state. Importantly, we demonstrate that this concomitantly leads to larger P-bodies that are responsible for preventing mRNA decapping. Finally, we demonstrate that P-bodies support cell viability and prevent stress granule formation when XRN1 is limiting. Taken together, these data demonstrate that the interaction between XRN1 and EDC4 regulates P-body dynamics to properly coordinate mRNA decapping with 5'-3' decay in human cells.

Intramolecular interaction of NEP regulated by CRM1 ensures the unidirectional transport of M1 for the nuclear export of influenza viral ribonucleoprotein

IntroductionThe influenza virus genome consists of single-stranded RNAs and forms viral ribonucleoprotein (RNP) complexes. After viral genome replication in the nucleus, the viral RNP interacts with viral protein M1. The M1-viral RNP complex is exported to the cytoplasm via the CRM1-dependent pathway using NS2/NEP as an export adaptor protein. NEP is a 14 kDa protein and diffusely localizes in the nucleus and cytoplasm. Upon binding to the NLS motif of M1, NEP inhibits the nuclear accumulation of M1 and promotes the nuclear export of M1-viral RNP complex. However, the detail mechanism by which NEP binds to M1 only in the nucleus remains unclear.MethodsTo visualize the interaction of NEP with M1 in the formation of vRNP export complexes, we performed in situ proximity ligation assays. The close proximity of N-terminal and C-terminal domains of NEP was tested by split Renilla luciferase complementation assays in which the N-terminal and C-terminal fragments of Renilla luciferase were fused to the N-terminus and C-terminus of NEP, respectively.Results and discussionWe found that the intramolecular interaction of NEP inhibits the interaction of NEP with M1. The intramolecular interaction of NEP was mediated through the interaction of the N-terminal NES motif with the M1-binding domain at the C-terminus. By adding leptomycin B, a potent inhibitor of CRM1, the interaction of NEP with M1 was impaired. These results suggest that CRM1 disrupts the intramolecular interaction of NEP by recognizing the NES motif at the N-terminus of NEP, thereby promoting the interaction of NEP with M1. We also found that NEP mutant deficient in the intramolecular interaction was co-localized with M1 at the plasma membrane and did not show nuclear localization with M1. Based on these results, we propose that the intramolecular interaction of NEP regulated by CRM1 ensures the unidirectional transport of M1.

Interaction of L1CAM with LC3 Is Required for L1-Dependent Neurite Outgrowth and Neuronal Survival.

The neural cell adhesion molecule L1 (also called L1CAM or CD171) functions not only in cell migration, but also in cell survival, differentiation, myelination, neurite outgrowth, and signaling during nervous system development and in adults. The proteolytic cleavage of L1 in its extracellular domain generates soluble fragments which are shed into the extracellular space and transmembrane fragments that are internalized into the cell and transported to various organelles to regulate cellular functions. To identify novel intracellular interaction partners of L1, we searched for protein-protein interaction motifs and found two potential microtubule-associated protein 1 light-chain 3 (LC3)-interacting region (LIR) motifs within L1, one in its extracellular domain and one in its intracellular domain. By ELISA, immunoprecipitation, and proximity ligation assay using L1 mutant mice lacking the 70 kDa L1 fragment (L1-70), we showed that L1-70 interacts with LC3 via the extracellular LIR motif in the fourth fibronectin type III domain, but not by the motif in the intracellular domain. The disruption of the L1-LC3 interaction reduces L1-mediated neurite outgrowth and neuronal survival.

The cell cycle protein MAD2 facilitates endocytosis of the serotonin transporter in the neuronal soma.

Monoamine transporters retrieve serotonin (SERT), dopamine (DAT), and norepinephrine (NET) from the synaptic cleft. Transporter internalization contributes to the regulation of their surface expression. Clathrin-mediated endocytosis of plasma membrane proteins requires adaptor protein-2 (AP2), which recruits cargo to the nascent clathrin cage. However, the intracellular portions of monoamine transporters are devoid of a conventional AP2-binding site. Here, we identify a MAD2 (mitotic arrest deficient-2) interaction motif in the C-terminus of SERT, which binds the closed conformation of MAD2 and allows for the recruitment of two additional mitotic spindle assembly checkpoint (SAC) proteins, BubR1 and p31comet , and of AP2. We visualize MAD2, BubR1, and p31comet in dorsal raphe neurons, and depletion of MAD2 in primary serotonergic rat neurons decreases SERT endocytosis in the soma. Our findings do not only provide mechanistic insights into transporter internalization but also allow for rationalizing why SAC proteins are present in post-mitotic neurons.

Self-organization of primitive metabolic cycles due to non-reciprocal interactions

One of the greatest mysteries concerning the origin of life is how it has emerged so quickly after the formation of the earth. In particular, it is not understood how metabolic cycles, which power the non-equilibrium activity of cells, have come into existence in the first instances. While it is generally expected that non-equilibrium conditions would have been necessary for the formation of primitive metabolic structures, the focus has so far been on externally imposed non-equilibrium conditions, such as temperature or proton gradients. Here, we propose an alternative paradigm in which naturally occurring non-reciprocal interactions between catalysts that can partner together in a cyclic reaction lead to their recruitment into self-organized functional structures. We uncover different classes of self-organized cycles that form through exponentially rapid coarsening processes, depending on the parity of the cycle and the nature of the interaction motifs, which are all generic but have readily tuneable features.

Chalcogen-Bond-Induced Double Helix Based on Self-Assembly of a Small Planar Building Block.

As a rule, helical structures at the molecular level are formed by non-planar units. This makes the design of helices, starting from planar building blocks via self-assembly, even more fascinating. Until now, however, this has only been achieved in rare cases, where hydrogen and halogen bonds were involved. Here, we show that the carbonyl-tellurium interaction motif is suitable to assemble even small planar units into helical structures in solid phase. We found two different types of helices: both single and double helices, depending on the substitution pattern. In the double helix, the strands are connected by additional Te⋅⋅⋅Te chalcogen bonds. In the case of the single helix, a spontaneous enantiomeric resolution occurs in the crystal. This underlines the potential of the carbonyl-tellurium chalcogen bond to generate complex three-dimensional patterns.