Subunit Interactions Research Articles

De novo variants in LRRC8C resulting in constitutive channel activation cause a human multisystem disorder

AbstractVolume-regulated anion channels (VRACs) are multimeric proteins composed of different paralogs of the LRRC8 family. They are activated in response to hypotonic swelling, but little is known about their specific functions. We studied two human individuals with the same congenital syndrome affecting blood vessels, brain, eyes, and bones. The LRRC8C gene harbored de novo variants in both patients, located in a region of the gene encoding the boundary between the pore and a cytoplasmic domain, which is depleted of sequence variations in control subjects. When studied by cryo-EM, both LRRC8C mutant proteins assembled as their wild-type counterparts, but showed increased flexibility, suggesting a destabilization of subunit interactions. When co-expressed with the obligatory LRRC8A subunit, the mutants exhibited enhanced activation, resulting in channel activity even at isotonic conditions in which wild-type channels are closed. We conclude that structural perturbations of LRRC8C impair channel gating and constitute the mechanistic basis of the dominant gain-of-function effect of these pathogenic variants. The pleiotropic phenotype of this novel clinical entity associated with monoallelic LRRC8C variants indicates the fundamental roles of VRACs in different tissues and organs.

Anxiolytic Effect of Sesamol, Possibly Through the GABAkine Interaction Pathway.

Plant-based components have helped generate novel lead molecules and scaffolds for anxiety research in psychopharmacology. The present study examined the anxiolytic properties of sesamol (SES), a phenolic lignan derived from Sesamum indicum, employing both in vivo and computational methods to understand its mechanisms of action. In this experiment, adult Swiss albino mice received various doses of SES (25 and 50 mg/kg, p.o.) orally. Afterward, a series of behavioral assessments, including open field, swing, hole cross, and light-dark testing, were conducted. The impact of the GABAergic agonist diazepam (DZP-1 mg/kg, i.p.) along with the antagonist flumazenil (FLU-0.1 mg/kg, i.p.) has been studied as provided concurrently with the SES-50 group. Computational studies were performed to comprehend the interaction between SES and GABAA receptor subunits (α2 and α3). The results of our investigation revealed that SES dose-dependently and significantly (p < 0.05) reduced the number of square crosses, hole crosses, swings, grooming, and rearing along with a reduction of light residence time in animals. When combined with DZP, SES-50 significantly reduced all these parameters, while altering with FLU-0.1. The molecular docking analysis showed that the SES has a relatively good binding score (-5.03 ± 0.15 and -5.25 ± 0.23 kcal/mol) with GABAA receptor α2 and α3 subunits, respectively. The SES triggers anxiolytic effects via GABAA receptor α2 and α3 subunit interactions. Furthermore, precise and comprehensive preclinical research must be considered to validate potential SES targets for anxiolytic impact, clinical trial efficacy, and safety.

Unique pharmacology of mGlu homo- and heterodimers.

Metabotropic glutamate receptors (mGlus) are obligate dimer G protein coupled receptors that can all homodimerize and heterodimerize in select combinations. Responses of mGlu heterodimers to selective ligands, including orthosteric agonists and allosteric modulators, are largely unknown. The pharmacological properties of each group II and III mGlu homodimer (except mGlu6) and several heterodimers were examined when stochastically assembled in HEK293T cells, or specifically measured using an improved G protein mediated BRET assay employing complimented fragments of NanoLuciferase. Stochastically assembled receptors adopted unique signaling characteristics. Some favored the potency, efficacy or signaling kinetics of a dominant subunit, while others exhibited blended profiles reflective of a combination of homo- and heterodimers at various ratios of expressed receptor. Finally, group II and III mGlu dimers were examined for responses to selective agonists and allosteric modulators. Effects of glutamate and selective group II and III orthosteric agonists were found to result in unique concentration response profiles when examining each combination of group II and II mGlu. Effects of select allosteric modulators were examined for each mGlu2 containing dimer as well as several group III dimer pairs. Likewise, allosteric modulator effects were often unique across dimers containing the targeted subunit of the ligand being tested. Results demonstrate that mGlu dimers respond uniquely to selective ligands, and show that the mGlu family is not governed by generalizable rules dictating consequences of dimeric subunit interactions leading to signaling consequences.

Mechanism of NACHO-mediated assembly of pentameric ligand-gated ion channels.

Pentameric ligand-gated ion channels (pLGICs) are cell surface receptors of crucial importance for animal physiology 1-4 . This diverse protein family mediates the ionotropic signals triggered by major neurotransmitters and includes γ -aminobutyric acid receptors (GABA A Rs) and acetylcholine receptors (nAChRs). Receptor function is fine-tuned by a myriad of endogenous and pharmacological modulators 3 . A functional pLGIC is built from five homologous, sometimes identical, subunits, each containing a β-scaffold extracellular domain (ECD), a four-helix transmembrane domain (TMD) and intracellular loops of variable length. Although considerable progress has been made in understanding pLGICs in structural and functional terms, the molecular mechanisms that enable their assembly at the endoplasmic reticulum (ER) 5 in a vast range of potential subunit configurations 6 remain unknown. Here, we identified candidate pLGICs assembly factors selectively associated with an unassembled GABA A R subunit. Focusing on one of the candidates, we determined the cryo-EM structure of an assembly intermediate containing two α1 subunits of GABA A R each bound to an ER-resident membrane protein NACHO. The structure showed how NACHO shields the principal (+) transmembrane interface of α1 subunits containing an immature extracellular conformation. Crosslinking and structure-prediction revealed an adjacent surface on NACHO for β2 subunit interactions to guide stepwise oligimerisation. Mutations of either subunit-interacting surface on NACHO also impaired the formation of homopentameric α7 nAChRs, pointing to a generic framework for pLGIC assembly. Our work provides the foundation for understanding the regulatory principles underlying pLGIC structural diversity.

Explicit description of viral capsid subunit shapes by unfolding dihedrons

Viral capsid assembly and the design of capsid-based nanocontainers critically depend on understanding the shapes and interfaces of constituent protein subunits. However, a comprehensive framework for characterizing these features is still lacking. Here, we introduce a novel approach based on spherical tiling theory that explicitly describes the 2D shapes and interfaces of subunits in icosahedral capsids. Our method unfolds spherical dihedrons defined by icosahedral symmetry axes, enabling systematic characterization of all possible subunit geometries. Applying this framework to real T = 1 capsid structures reveals distinct interface groups within this single classification, with variations in interaction patterns around 3-fold and 5-fold symmetry axes. We validate our classification through molecular docking simulations, demonstrating its consistency with physical subunit interactions. This analysis suggests different assembly pathways for capsid nucleation. Our general framework is applicable to other triangular numbers, paving the way for broader studies in structural virology and nanomaterial design.

PPP1R2 stimulates protein phosphatase-1 through stabilisation of dynamic subunit interactions

Protein Ser/Thr phosphatase PP1 is always associated with one or two regulatory subunits or RIPPOs. One of the earliest evolved RIPPOs is PPP1R2, also known as Inhibitor-2. Since its discovery nearly 5 decades ago, PPP1R2 has been variously described as an inhibitor, activator or (metal) chaperone of PP1, but it is still unknown how PPP1R2 affects the function of PP1 in intact cells. Here, using specific research tools, we demonstrate that PPP1R2 stabilises a subgroup of PP1 holoenzymes, exemplified by PP1:RepoMan, thereby promoting the dephosphorylation of their substrates. Mechanistically, the recruitment of PPP1R2 disrupts an inhibitory, fuzzy interaction between the C-terminal tail and catalytic domain of PP1, and generates an additional C-terminal RepoMan-interaction site. The resulting holoenzyme is further stabilized by a direct PPP1R2:RepoMan interaction, which renders it refractory to competitive disruption by RIPPOs that do not interact with PPP1R2. Our data demonstrate that PPP1R2 modulates the function of PP1 by altering the balance between holoenzymes through stabilisation of specific subunit interactions.

β3-adrenergic agonist counters oxidative stress and Na+-K+ pump inhibitory S-glutathionylation of placental cells: Implications for preeclampsia.

Oxidative stress from placental ischemia/reperfusion and hypoxia/reoxygenation (H/R) in preeclampsia is accompanied by Na+-K+ pump inhibition and S-glutathionylation of its β1 subunit (GSS-β1), a modification that inhibits the pump. β3-adrenergic receptor (β3-AR) agonists can reverse GSS-β1. We examined effects of the agonist CL316,243 on GSS-β1 and sources of H/R-induced oxidative stress in immortalized first trimester human trophoblast (HTR-8/SVneo) and freshly isolated placental explants from normal term pregnancies. H/R increased GSS-β1 and, reflecting compromised α1/β1 subunit interaction, it reduced α1/β1 pump subunit co-immunoprecipitation. H/R increased p47phox/p22phox NADPH oxidase subunit co-immunoprecipitation reflecting membrane translocation of cytosolic p47phox that is needed to activate NADPH oxidase. Fluorescence of O2•--sensitive dihydroethidium increased in parallel. H/R increased S-glutathionylation of endothelial nitric oxide synthase (GSS-eNOS) that uncouples NO synthesis towards synthesis of O2•- and reduced trophoblast migration. Oxidative stress induced by tumor necrosis factor α (TNF-α) increased soluble fms-like tyrosine kinase receptor 1 (sFlt-1) trophoblast release, a marker of preeclampsia, and reduced trophoblast integration into endothelial cellular networks. CL316,243 eliminated H/R-induced GSS-β1 and decreases of α1/β1 subunit coimmunoprecipitation, eliminated NADPH oxidase activation and increases in GSS-eNOS, restored trophoblast migration, eliminated increased sFlt-1 release and restored trophoblast integration in endothelial cell networks. H/R induced GSS-β1, α1/β1 subunit co-immunoprecipitation and NADPH oxidase activation of placental explants reflected effects of H/R for trophoblasts and CL316,243 eliminated these changes. We conclude a β3-AR agonist counters key pathophysiological features of preeclampsia in vitro. β3 agonists already in human use for another purpose are potential candidates for re-purposing to treat preeclampsia.

Unique biogenesis and kinetics of hornwort Rubiscos revealed by synthetic biology systems

Hornworts are the only land plants that employ a pyrenoid to optimize Rubisco’s CO2 fixation. Yet, hornwort Rubisco remains poorly characterized. Here we assemble the hornwort Anthoceros agrestis Rubisco (AaRubisco) using the Arabidopsis thaliana SynBio expression system and observed the formation of stalled intermediates, prompting us to develop a new SynBio system with A. agrestis cognate chaperones. We successfully assembled AaRubisco and Rubisco from three other hornwort species. Unlike A. thaliana Rubisco, AaRubisco assembly is not dependent on RbcX or Raf2. Kinetic characterization reveals that hornwort Rubiscos exhibit a range of catalytic rates (3-10 s-1), but with similar affinity (∼30 μM) and specificity (∼70) for CO2. In other words, hornwort Rubiscos do not comply with the long held canonical catalytic trade-off observed in other land plants, providing experimental support that Rubisco kinetics may be phylogenetically constrained. Unexpectedly, we observed a 50% increase in AaRubisco catalytic rates when RbcX was removed from our SynBio system, without any reduction in specificity. Structural biology, biochemistry and proteomic analysis suggest that subtle differences in Rubisco large subunit interactions, when RbcX is absent during biogenesis, increases the accessibility of active sites and catalytic turnover rate. This study uncovered a previously unknown Rubisco kinetic parameter space and provides a SynBio chassis to expand the survey of other Rubisco kinetics. Our discovery could thus reshape the approaches for engineering Rubisco with superior kinetics.

Broad-Spectrum Antiviral Agents against SARS-CoV-2 Variants Inhibit the Conserved Viral Protein Nsp1-RNA Interaction.

The ongoing global threats posed by COVID-19 pandemic, catalyzed by SARS-CoV-2, underscores the pressing need for effective antiviral strategies. The viral non-structural protein 1 (Nsp1) significantly influences pathogenicity by impeding host protein expression and enhancing viral RNA translation through its interaction with the stem-loop 1 (SL1) in the 5' untranslated region (UTR). We have developed a novel dual-luciferase reporter assay, designed to investigate the critical Nsp1-SL1 interaction, and identified P23E02 as a potential inhibitor. Our investigation, combining molecular docking studies and alanine mutagenesis, has unveiled that P23E02 disrupts Nsp1-40S ribosomal subunit interaction, liberating translational inhibition and empowering host antiviral responses. P23E02 exhibits antiviral efficacy against various sarbecoviruses, making it a promising candidate for combatting COVID-19 and related diseases. This study underscores the therapeutic potential of targeting the Nsp1/SL1 axis and lays the foundation for innovative antiviral interventions, ultimately fortifying global preparedness against future viral threats.

Broad‐Spectrum Antiviral Agents against SARS‐CoV‐2 Variants Inhibit the Conserved Viral Protein Nsp1–RNA Interaction

AbstractThe ongoing global threats posed by COVID‐19 pandemic, catalyzed by SARS‐CoV‐2, underscores the pressing need for effective antiviral strategies. The viral non‐structural protein 1 (Nsp1) significantly influences pathogenicity by impeding host protein expression and enhancing viral RNA translation through its interaction with the stem‐loop 1 (SL1) in the 5′ untranslated region (UTR). We have developed a novel dual‐luciferase reporter assay, designed to investigate the critical Nsp1–SL1 interaction, and identified P23E02 as a potential inhibitor. Our investigation, combining molecular docking studies and alanine mutagenesis, has unveiled that P23E02 disrupts Nsp1–40S ribosomal subunit interaction, liberating translational inhibition and empowering host antiviral responses. P23E02 exhibits antiviral efficacy against various sarbecoviruses, making it a promising candidate for combatting COVID‐19 and related diseases. This study underscores the therapeutic potential of targeting the Nsp1/SL1 axis and lays the foundation for innovative antiviral interventions, ultimately fortifying global preparedness against future viral threats.

Fluorescence lifetime sorting reveals tunable enzyme interactions within cytoplasmic condensates.

Ribonucleoprotein (RNP) condensates partition RNA and protein into multiple liquid phases. The multiphasic feature of condensate-enriched components creates experimental challenges for distinguishing membraneless condensate functions from the surrounding dilute phase. We combined fluorescence lifetime imaging microscopy (FLIM) with phasor plot filtering and segmentation to resolve condensates from the dilute phase. Condensate-specific lifetimes were used to track protein-protein interactions by measuring FLIM-Förster resonance energy transfer (FRET). We used condensate FLIM-FRET to evaluate whether mRNA decapping complex subunits can form decapping-competent interactions within P-bodies. Condensate FLIM-FRET revealed the presence of core subunit interactions within P-bodies under basal conditions and the disruption of interactions between the decapping enzyme (Dcp2) and a critical cofactor (Dcp1A) during oxidative stress. Our results show a context-dependent plasticity of the P-body interaction network, which can be rewired within minutes in response to stimuli. Together, our FLIM-based approaches provide investigators with an automated and rigorous method to uncover and track essential protein-protein interaction dynamics within RNP condensates in live cells.

Amylin receptor subunit interactions are modulated by agonists and determine signaling.

Three amylin receptors (AMYRs) mediate the metabolic actions of the peptide hormone amylin and are drug targets for diabetes and obesity. AMY1R, AMY2R, and AMY3R are heterodimers consisting of the G protein-coupled calcitonin receptor (CTR) paired with a RAMP1, -2, or -3 accessory subunit, respectively, which increases amylin potency. Little is known about AMYR subunit interactions and their role in signaling. Here, we show that the AMYRs have distinct basal subunit equilibriums that are modulated by peptide agonists and determine the cAMP signaling phenotype. Using a novel biochemical assay that resolves the AMYR heterodimers and free subunits, we found that the AMY1/2R subunit equilibriums favored free CTR and RAMP1/2, and rat amylin and αCGRP agonists promoted subunit association. A stronger CTR-RAMP3 transmembrane domain interface yielded a more stable AMY3R, and human and salmon calcitonin agonists promoted AMY3R dissociation. Similar changes in subunit association-dissociation were observed in live cell membranes, and G protein coupling and cAMP signaling assays showed how these altered signaling. Our findings reveal regulation of heteromeric GPCR signaling through subunit interaction dynamics.

Structural biology of shelterin and telomeric chromatin: the pieces and an unfinished puzzle.

The six-subunit shelterin complex binds to mammalian telomeres and protects them from triggering multiple DNA damage response pathways. The loss of this protective function by shelterin can have detrimental effects on cells. In this review, we first discuss structural studies of shelterin, detailing the contributions of each subunit and inter-subunit interactions in protecting chromosome ends. We then examine the influence of telomeric chromatin dynamics on the function of shelterin at telomeres. These studies provide valuable insights and underscore the challenges that future research must tackle to attain high-resolution structures of shelterin.

Enhancing the stress resistance of nitrile hydratase from Rhodococcus ruber via SpyTag/SpyCatcher-mediated α- and β- subunits ligation.

Nitrile Hydratase (NHase) is one of the most important industrial enzyme widely used in the petroleum exploitation field. The enzyme, composed of two unrelated α- and β-subunits, catalyzes the conversion of acrylonitrile to acrylamide, releasing a significant amount of heat and generating the organic solvent product, acrylamide. Both the heat and acrylamide solvent have an impact on the structural stability of NHase and its catalytic activity. Therefore, enhancing the stress resistance of NHase to toxic substances is meaningful for the petroleum industry. To improve the thermo-stability and acrylamide tolerance of NHase, the two subunits were fused in vivo using SpyTag and SpyCatcher, which were attached to the termini of each subunit in various combinations. Analysis of the engineered strains showed that the C-terminus of β-NHase is a better fusion site than the N-terminus, while the C-terminus of α-NHase is the most suitable site for fusion with a larger protein. Fusion of SpyTag and SpyCatcher to the C-terminus of β-NHase and α-NHase, respectively, led to improved acrylamide tolerance and a slight enhancement in the thermo-stability of one of the engineered strains, NBSt. These results indicate that in vivo ligation of different subunits using SpyTag/SpyCatcher is a valuable strategy for enhancing subunit interaction and improving stress tolerance.

Understanding the biosynthesis of human IgM SAM-6 through a combinatorial expression of mutant subunits that affect product assembly and secretion.

Polymeric IgMs are secreted from plasma cells abundantly despite their structural complexity and intricate multimerization steps. To gain insights into IgM's assembly mechanics that underwrite such high-level secretion, we characterized the biosynthetic process of a natural human IgM, SAM-6, using a heterologous HEK293(6E) cell platform that allowed the production of IgMs both in hexameric and pentameric forms in a controlled fashion. By creating a series of mutant subunits that differentially disrupt secretion, folding, and specific inter-chain disulfide bond formation, we assessed their effects on various aspects of IgM biosynthesis in 57 different subunit chain combinations, both in hexameric and pentameric formats. The mutations caused a spectrum of changes in steady-state subcellular subunit distribution, ER-associated inclusion body formation, intracellular subunit detergent solubility, covalent assembly, secreted IgM product quality, and secretion output. Some mutations produced differential effects on product quality depending on whether the mutation was introduced to hexameric IgM or pentameric IgM. Through this systematic combinatorial approach, we consolidate diverse overlapping knowledge on IgM biosynthesis for both hexamers and pentamers, while unexpectedly revealing that the loss of certain inter-chain disulfide bonds, including the one between μHC and λLC, is tolerated in polymeric IgM assembly and secretion. The findings highlight the differential roles of underlying non-covalent protein-protein interactions in hexamers and pentamers when orchestrating the initial subunit interactions and maintaining the polymeric IgM product integrity during ER quality control steps, secretory pathway trafficking, and secretion.

G protein β subunits regulate Cav3.3 T-type channel activity and current kinetics via interaction with the Cav3.3 C-terminus

Ca2+ influx through Cav3.3 T-type channel plays crucial roles in neuronal excitability and is subject to regulation by various signaling molecules. However, our understanding of the partners of Cav3.3 and the related regulatory pathways remains largely limited. To address this quest, we employed the rat Cav3.3 C-terminus as bait in yeast-two-hybrid screenings of a cDNA library, identifying rat Gβ2 as an interaction partner. Subsequent assays revealed that the interaction of Gβ2 subunit was specific to the Cav3.3 C-terminus. Through systematic dissection of the C-terminus, we pinpointed a 22 amino acid sequence (amino acids 1789–1810) as the Gβ2 interaction site. Coexpression studies of rat Cav3.3 with various Gβγ compositions were conducted in HEK-293 cells. Patch clamp recordings revealed that coexpression of Gβ2γ2 reduced Cav3.3 current density and accelerated inactivation kinetics. Interestingly, the effects were not unique to Gβ2γ2, but were mimicked by Gβ2 alone as well as other Gβγ dimers, with similar potencies. Deletion of the Gβ2 interaction site abolished the effects of Gβ2γ2. Importantly, these Gβ2 effects were reproduced in human Cav3.3. Overall, our findings provide evidence that Gβ(γ) complexes inhibit Cav3.3 channel activity and accelerate the inactivation kinetics through the Gβ interaction with the Cav3.3 C-terminus.

VALYL-TRNA SYNTHETASE INTERACTS WITH Β-SUBUNIT OF THE EUKARYOTIC TRANSLATION ELONGATION FACTOR COMPLEX eEF1B

The aim of this study was to investigate the interaction of the N-terminal domain of the valyl-tRNA synthetase with α, β, and γ subunits of the eEF1B translation elongation factor complex. Methods: for this purpose, all 4 proteins were synthesized in bacterial cells and purified to homogeneity by a combination of chromatographic methods. The interaction of the eEF1B complex subunits with the N-terminal domain of the valyl-tRNA synthetase was verified by gel filtration and in vitro pull-down assays. Protein fractions collected at these stages were analyzed by SDS-PAGE. Results: according to the gel filtration results, eEF1Bα and eEF1Bγ subunits do not form a stable complex with the valine-tRNA synthetase domain. The potential for complexation of the eEF1Bβ subunit was evaluated by pull-down assay, which showed that this protein does interact with the valyl-tRNA synthetase. Conclusions: we concluded that the eEF1Bα and eEF1Bγ subunits do not interact with the valyl-tRNA synthetase compared to the eEF1Bβ protein. The N-terminal domain of the valyl-tRNA synthetase is necessary and sufficient for this interaction.

Structural Evolution of Bacterial Polyphosphate Degradation Enzyme for Phosphorus Cycling.

Living organisms ranging from bacteria to animals have developed their own ways to accumulate and store phosphate during evolution, in particular as the polyphosphate (polyP) granules in bacteria. Degradation of polyP into phosphate is involved in phosphorus cycling, and exopolyphosphatase (PPX) is the key enzyme for polyP degradation in bacteria. Thus, understanding the structure basis of PPX is crucial to reveal the polyP degradation mechanism. Here, it is found that PPX structure varies in the length of ɑ-helical interdomain linker (ɑ-linker) across various bacteria, which is negatively correlated with their enzymatic activity and thermostability - those with shorter ɑ-linkers demonstrate higher polyP degradation ability. Moreover, the artificial DrPPX mutants with shorter ɑ-linker tend to have more compact pockets for polyP binding and stronger subunit interactions, as well as higher enzymatic efficiency (kcat/Km) than that of DrPPX wild type. In Deinococcus-Thermus, the PPXs from thermophilic species possess a shorter ɑ-linker and retain their catalytic ability at high temperatures (70°C), which may facilitate the thermophilic species to utilize polyP in high-temperature environments. These findings provide insights into the interdomain linker length-dependent evolution of PPXs, which shed light on enzymatic adaption for phosphorus cycling during natural evolution and rational design of enzyme.

Peptides Targeting the IF1-ATP Synthase Complex Modulate the Permeability Transition Pore in Cancer HeLa Cells.

The mitochondrial protein IF1 is upregulated in many tumors and acts as a pro-oncogenic protein through its interaction with the ATP synthase and the inhibition of apoptosis. We have recently characterized the molecular nature of the IF1-Oligomycin Sensitivity Conferring Protein (OSCP) subunit interaction; however, it remains to be determined whether this interaction could be targeted for novel anti-cancer therapeutic intervention. We generated mitochondria-targeting peptides to displace IF1 from the OSCP interaction. The use of one selective peptide led to displacement of the inhibitor IF1 from ATP synthase, as shown by immunoprecipitation. NMR spectroscopy analysis, aimed at clarifying whether these peptides were able to directly bind to the OSCP protein, identified a second peptide which showed affinity for the N-terminal region of this subunit overlapping the IF1 binding region. In situ treatment with the membrane-permeable derivatives of these peptides in HeLa cells, that are silenced for the IF1 inhibitor protein, showed significant inhibition in mitochondrial permeability transition and no effects on mitochondrial respiration. These peptides mimic the effects of the IF1 inhibitor protein in cancer HeLa cells and confirm that the IF1-OSCP interaction inhibits apoptosis. A third peptide was identified which counteracts the anti-apoptotic role of IF1, showing that OSCP is a promising target for anti-cancer therapies.

Retromer-mediated recruitment of the WASH complex involves discrete interactions between VPS35, VPS29, and FAM21.

Endosomal trafficking ensures the proper distribution of lipids and proteins to various cellular compartments, facilitating intracellular communication, nutrient transport, waste disposal, and the maintenance of cell structure. Retromer, a peripheral membrane protein complex, plays an important role in this process by recruiting the associated actin-polymerizing WASH complex to establish distinct sorting domains. The WASH complex is recruited through the interaction of the VPS35 subunit of retromer with the WASH complex subunit FAM21. Here, we report the identification of two separate fragments of FAM21 that interact with VPS35, along with a third fragment that binds to the VPS29 subunit of retromer. The crystal structure of VPS29 bound to a peptide derived from FAM21 shows a distinctive sharp bend that inserts into a conserved hydrophobic pocket with a binding mode similar to that adopted by other VPS29 effectors. Interestingly, despite the network of interactions between FAM21 and retromer occurring near the Parkinson's disease-linked mutation (D620N) in VPS35, this mutation does not significantly impair the direct association with FAM21 in vitro.