BAR domain scaffolds in dynamin-mediated membrane fission.

Biological membranes undergo constant remodeling by membrane fission and fusion to change their shape and to exchange material between subcellular compartments. During clathrin-mediated endocytosis, the dynamic assembly and disassembly of protein scaffolds comprising members of the bin-amphiphysin-rvs (BAR) domain protein superfamily constrain the membrane into distinct shapes as the pathway progresses toward fission by the GTPase dynamin. In this Review, we discuss how BAR domain protein assembly and disassembly are controlled in space and time and which structural and biochemical features allow the tight regulation of their shape and function to enable dynamin-mediated membrane fission.

Structural insights into the activation mechanism of antimicrobial GBP1.

The dynamin-related human guanylate-binding protein 1 (GBP1) mediates host defenses against microbial pathogens. Upon GTP binding and hydrolysis, auto-inhibited GBP1 monomers dimerize and assemble into soluble and membrane-bound oligomers, which are crucial for innate immune responses. How higher-order GBP1 oligomers are built from dimers, and how assembly is coordinated with nucleotide-dependent conformational changes, has remained elusive. Here, we present cryo-electron microscopy-based structural data of soluble and membrane-bound GBP1 oligomers, which show that GBP1 assembles in an outstretched dimeric conformation. We identify a surface-exposed helix in the large GTPase domain that contributes to the oligomerization interface, and we probe its nucleotide- and dimerization-dependent movements that facilitate the formation of an antimicrobial protein coat on a gram-negative bacterial pathogen. Our results reveal a sophisticated activation mechanism for GBP1, in which nucleotide-dependent structural changes coordinate dimerization, oligomerization, and membrane binding to allow encapsulation of pathogens within an antimicrobial protein coat.

FIP200 Claw Domain Binding to p62 Promotes Autophagosome Formation at Ubiquitin Condensates.

The autophagy cargo receptor p62 facilitates the condensation of misfolded, ubiquitin-positive proteins and their degradation by autophagy, but the molecular mechanism of p62 signaling to the core autophagy machinery is unclear. Here, we show that disordered residues 326-380 of p62 directly interact with the C-terminal region (CTR) of FIP200. Crystal structure determination shows that the FIP200 CTR contains a dimeric globular domain that we designated the "Claw" for its shape. The interaction of p62 with FIP200 is mediated by a positively charged pocket in the Claw, enhanced by p62 phosphorylation, mutually exclusive with the binding of p62 to LC3B, and it promotes degradation of ubiquitinated cargo by autophagy. Furthermore, the recruitment of the FIP200 CTR slows the phase separation of ubiquitinated proteins by p62 in a reconstituted system. Our data provide the molecular basis for a crosstalk between cargo condensation and autophagosome formation.

Structural analysis of PLD3 reveals insights into the mechanism of lysosomal 5' exonuclease-mediated nucleic acid degradation.

The phospholipase D (PLD) family is comprised of enzymes bearing phospholipase activity towards lipids or endo- and exonuclease activity towards nucleic acids. PLD3 is synthesized as a type II transmembrane protein and proteolytically cleaved in lysosomes, yielding a soluble active form. The deficiency of PLD3 leads to the slowed degradation of nucleic acids in lysosomes and chronic activation of nucleic acid-specific intracellular toll-like receptors. While the mechanism of PLD phospholipase activity has been extensively characterized, not much is known about how PLDs bind and hydrolyze nucleic acids. Here, we determined the high-resolution crystal structure of the luminal N-glycosylated domain of human PLD3 in its apo- and single-stranded DNA-bound forms. PLD3 has a typical phospholipase fold and forms homodimers with two independent catalytic centers via a newly identified dimerization interface. The structure of PLD3 in complex with an ssDNA-derived thymidine product in the catalytic center provides insights into the substrate binding mode of nucleic acids in the PLD family. Our structural data suggest a mechanism for substrate binding and nuclease activity in the PLD family and provide the structural basis to design immunomodulatory drugs targeting PLD3.

Development of selective inhibitors of phosphatidylinositol 3-kinase C2α.

Phosphatidylinositol 3-kinase type 2α (PI3KC2α) and related class II PI3K isoforms are of increasing biomedical interest because of their crucial roles in endocytic membrane dynamics, cell division and signaling, angiogenesis, and platelet morphology and function. Herein we report the development and characterization of PhosphatidylInositol Three-kinase Class twO INhibitors (PITCOINs), potent and highly selective small-molecule inhibitors of PI3KC2α catalytic activity. PITCOIN compounds exhibit strong selectivity toward PI3KC2α due to their unique mode of interaction with the ATP-binding site of the enzyme. We demonstrate that acute inhibition of PI3KC2α-mediated synthesis of phosphatidylinositol 3-phosphates by PITCOINs impairs endocytic membrane dynamics and membrane remodeling during platelet-dependent thrombus formation. PITCOINs are potent and selective cell-permeable inhibitors of PI3KC2α function with potential biomedical applications ranging from thrombosis to diabetes and cancer.

Structure and assembly of the mitochondrial membrane remodelling GTPase Mgm1.

Balanced fusion and fission are key for the proper function and physiology of mitochondria1,2. Remodelling of the mitochondrial inner membrane is mediated by the dynamin-like protein mitochondrial genome maintenance 1 (Mgm1) in fungi or the related protein optic atrophy 1 (OPA1) in animals3-5. Mgm1 is required for the preservation of mitochondrial DNA in yeast6, whereas mutations in the OPA1 gene in humans are a common cause of autosomal dominant optic atrophy-a genetic disorder that affects the optic nerve7,8. Mgm1 and OPA1 are present in mitochondria as a membrane-integral long form and a short form that is soluble in the intermembrane space. Yeast strains that express temperature-sensitive mutants of Mgm19,10 or mammalian cells that lack OPA1 display fragmented mitochondria11,12, which suggests that Mgm1 and OPA1 have an important role in inner-membrane fusion. Consistently, only the mitochondrial outer membrane-not the inner membrane-fuses in the absence of functional Mgm113. Mgm1 and OPA1 have also been shown to maintain proper cristae architecture10,14; for example, OPA1 prevents the release of pro-apoptotic factors by tightening crista junctions15. Finally, the short form of OPA1 localizes to mitochondrial constriction sites, where it presumably promotes mitochondrial fission16. How Mgm1 and OPA1 perform their diverse functions in membrane fusion, scission and cristae organization is at present unknown. Here we present crystal and electron cryo-tomography structures of Mgm1 from Chaetomium thermophilum. Mgm1 consists of a GTPase (G) domain, a bundle signalling element domain, a stalk, and a paddle domain that contains a membrane-binding site. Biochemical and cell-based experiments demonstrate that the Mgm1 stalk mediates the assembly of bent tetramers into helical filaments. Electron cryo-tomography studies of Mgm1-decorated lipid tubes and fluorescence microscopy experiments on reconstituted membrane tubes indicate how the tetramers assemble on positively or negatively curved membranes. Our findings convey how Mgm1 and OPA1 filaments dynamically remodel the mitochondrial inner membrane.

Quantification and demonstration of the collective constriction-by-ratchet mechanism in the dynamin molecular motor.

Dynamin oligomerizes into helical filaments on tubular membrane templates and, through constriction, cleaves them in a GTPase-driven way. Structural observations of GTP-dependent cross-bridges between neighboring filament turns have led to the suggestion that dynamin operates as a molecular ratchet motor. However, the proof of such mechanism remains absent. Particularly, it is not known whether a powerful enough stroke is produced and how the motor modules would cooperate in the constriction process. Here, we characterized the dynamin motor modules by single-molecule Förster resonance energy transfer (smFRET) and found strong nucleotide-dependent conformational preferences. Integrating smFRET with molecular dynamics simulations allowed us to estimate the forces generated in a power stroke. Subsequently, the quantitative force data and the measured kinetics of the GTPase cycle were incorporated into a model including both a dynamin filament, with explicit motor cross-bridges, and a realistic deformable membrane template. In our simulations, collective constriction of the membrane by dynamin motor modules, based on the ratchet mechanism, is directly reproduced and analyzed. Functional parallels between the dynamin system and actomyosin in the muscle are seen. Through concerted action of the motors, tight membrane constriction to the hemifission radius can be reached. Our experimental and computational study provides an example of how collective motor action in megadalton molecular assemblies can be approached and explicitly resolved.

Competitively disrupting the neutrophil-specific receptor-autoantigen CD177:proteinase 3 membrane complex reduces anti-PR3 antibody-induced neutrophil activation.

CD177 is a neutrophil-specific receptor presenting the proteinase 3 (PR3) autoantigen on the neutrophil surface. CD177 expression is restricted to a neutrophil subset, resulting in CD177pos/mPR3high and CD177neg/mPR3low populations. The CD177pos/mPR3high subset has implications for antineutrophil cytoplasmic autoantibody (ANCA)-associated autoimmune vasculitis, wherein patients harbor PR3-specific ANCAs that activate neutrophils for degranulation. Here, we generated high-affinity anti-CD177 monoclonal antibodies, some of which interfered with PR3 binding to CD177 (PR3 "blockers") as determined by surface plasmon resonance spectroscopy and used them to test the effect of competing PR3 from the surface of CD177pos neutrophils. Because intact anti-CD177 antibodies also caused neutrophil activation, we prepared nonactivating Fab fragments of a PR3 blocker and nonblocker that bound specifically to CD177pos neutrophils. We observed that Fab blocker clone 40, but not nonblocker clone 80, dose-dependently reduced anti-PR3 antibody binding to CD177pos neutrophils. Importantly, preincubation with clone 40 significantly reduced respiratory burst in primed neutrophils challenged with either monoclonal antibodies to PR3 or PR3-ANCA immunoglobulin G from ANCA-associated autoimmune vasculitis patients. After separating the two CD177/mPR3 neutrophil subsets from individual donors by magnetic sorting, we found that PR3-ANCAs provoked significantly more superoxide production in CD177pos/mPR3high than in CD177neg/mPR3low neutrophils, and that anti-CD177 Fab clone 40 reduced the superoxide production of CD177pos cells to the level of the CD177neg cells. Our data demonstrate the importance of the CD177:PR3 membrane complex in maintaining a high ANCA epitope density and thereby underscore the contribution of CD177 to the severity of PR3-ANCA diseases.

EHD2-mediated restriction of caveolar dynamics regulates cellular fatty acid uptake.

Eps15-homology domain containing protein 2 (EHD2) is a dynamin-related ATPase located at the neck of caveolae, but its physiological function has remained unclear. Here, we found that global genetic ablation of EHD2 in mice leads to increased lipid droplet size in fat tissue. This organismic phenotype was paralleled at the cellular level by increased fatty acid uptake via a caveolae- and CD36-dependent pathway that also involves dynamin. Concomitantly, elevated numbers of detached caveolae were found in brown and white adipose tissue lacking EHD2, and increased caveolar mobility in mouse embryonic fibroblasts. EHD2 expression itself was down-regulated in the visceral fat of two obese mouse models and obese patients. Our data suggest that EHD2 controls a cell-autonomous, caveolae-dependent fatty acid uptake pathway and imply that low EHD2 expression levels are linked to obesity.

A SEPT1-based scaffold is required for Golgi integrity and function.

Compartmentalization of membrane transport and signaling processes is of pivotal importance to eukaryotic cell function. While plasma membrane compartmentalization and dynamics are well known to depend on the scaffolding function of septin GTPases, the roles of septins at intracellular membranes have remained largely elusive. Here, we show that the structural and functional integrity of the Golgi depends on its association with a septin 1 (SEPT1)-based scaffold, which promotes local microtubule nucleation and positioning of the Golgi. SEPT1 function depends on the Golgi matrix protein GM130 (also known as GOLGA2) and on centrosomal proteins, including CEP170 and components of γ-tubulin ring complex (γ-Turc), to facilitate the perinuclear concentration of Golgi membranes. Accordingly, SEPT1 depletion triggers a massive fragmentation of the Golgi ribbon, thereby compromising anterograde membrane traffic at the level of the Golgi.

Autocrine LTA signaling drives NF-κB and JAK-STAT activity and myeloid gene expression in Hodgkin lymphoma.

Persistent NF-κB activation is a hallmark of the malignant Hodgkin/Reed-Sternberg (HRS) cells in classical Hodgkin lymphoma (cHL). Genomic lesions, Epstein-Barr virus infection, soluble factors, and tumor-microenvironment interactions contribute to this activation. Here, in an unbiased approach to identify the cHL cell-secreted key factors for NF-κB activation, we have dissected the secretome of cultured cHL cells by chromatography and subsequent mass spectrometry. We identified lymphotoxin-α (LTA) as the causative factor for autocrine and paracrine activation of canonical and noncanonical NF-κB in cHL cell lines. In addition to inducing NF-κB, LTA promotes JAK2/STAT6 signaling. LTA and its receptor TNFRSF14 are transcriptionally activated by noncanonical NF-κB, creating a continuous feedback loop. Furthermore, LTA shapes the expression of cytokines, receptors, immune checkpoint ligands and adhesion molecules, including CSF2, CD40, PD-L1/PD-L2, and VCAM1. Comparison with single-cell gene-activity profiles of human hematopoietic cells showed that LTA induces genes restricted to the lymphoid lineage, as well as those largely restricted to the myeloid lineage. Thus, LTA sustains autocrine NF-κB activation, impacts activation of several signaling pathways, and drives expression of genes essential for microenvironmental interactions and lineage ambiguity. These data provide a robust rationale for targeting LTA as a treatment strategy for cHL patients.

Crystal structure of the dynamin tetramer.

The mechanochemical protein dynamin is the prototype of the dynamin superfamily of large GTPases, which shape and remodel membranes in diverse cellular processes. Dynamin forms predominantly tetramers in the cytosol, which oligomerize at the neck of clathrin-coated vesicles to mediate constriction and subsequent scission of the membrane. Previous studies have described the architecture of dynamin dimers, but the molecular determinants for dynamin assembly and its regulation have remained unclear. Here we present the crystal structure of the human dynamin tetramer in the nucleotide-free state. Combining structural data with mutational studies, oligomerization measurements and Markov state models of molecular dynamics simulations, we suggest a mechanism by which oligomerization of dynamin is linked to the release of intramolecular autoinhibitory interactions. We elucidate how mutations that interfere with tetramer formation and autoinhibition can lead to the congenital muscle disorders Charcot-Marie-Tooth neuropathy and centronuclear myopathy, respectively. Notably, the bent shape of the tetramer explains how dynamin assembles into a right-handed helical oligomer of defined diameter, which has direct implications for its function in membrane constriction.

Membrane fission by dynamin: what we know and what we need to know.

The large GTPase dynamin is the first protein shown to catalyze membrane fission. Dynamin and its related proteins are essential to many cell functions, from endocytosis to organelle division and fusion, and it plays a critical role in many physiological functions such as synaptic transmission and muscle contraction. Research of the past three decades has focused on understanding how dynamin works. In this review, we present the basis for an emerging consensus on how dynamin functions. Three properties of dynamin are strongly supported by experimental data: first, dynamin oligomerizes into a helical polymer; second, dynamin oligomer constricts in the presence of GTP; and third, dynamin catalyzes membrane fission upon GTP hydrolysis. We present the two current models for fission, essentially diverging in how GTP energy is spent. We further discuss how future research might solve the remaining open questions presently under discussion.

Structure of myxovirus resistance protein a reveals intra- and intermolecular domain interactions required for the antiviral function.

Human myxovirus resistance protein 1 (MxA) is an interferon-induced dynamin-like GTPase that acts as a cell-autonomous host restriction factor against many viral pathogens including influenza viruses. To study the molecular principles of its antiviral activity, we determined the crystal structure of nucleotide-free MxA, which showed an extended three-domain architecture. The central bundle signaling element (BSE) connected the amino-terminal GTPase domain with the stalk via two hinge regions. MxA oligomerized in the crystal via the stalk and the BSE, which in turn interacted with the stalk of the neighboring monomer. We demonstrated that the intra- and intermolecular domain interplay between the BSE and stalk was essential for oligomerization and the antiviral function of MxA. Based on these results, we propose a structural model for the mechano-chemical coupling in ring-like MxA oligomers as the principle mechanism for this unique antiviral effector protein.

Structural insights into the activation mechanism of dynamin-like EHD ATPases.

Eps15 (epidermal growth factor receptor pathway substrate 15)-homology domain containing proteins (EHDs) comprise a family of dynamin-related mechano-chemical ATPases involved in cellular membrane trafficking. Previous studies have revealed the structure of the EHD2 dimer, but the molecular mechanisms of membrane recruitment and assembly have remained obscure. Here, we determined the crystal structure of an amino-terminally truncated EHD4 dimer. Compared with the EHD2 structure, the helical domains are 50° rotated relative to the GTPase domain. Using electron paramagnetic spin resonance (EPR), we show that this rotation aligns the two membrane-binding regions in the helical domain toward the lipid bilayer, allowing membrane interaction. A loop rearrangement in GTPase domain creates a new interface for oligomer formation. Our results suggest that the EHD4 structure represents the active EHD conformation, whereas the EHD2 structure is autoinhibited, and reveal a complex series of domain rearrangements accompanying activation. A comparison with other peripheral membrane proteins elucidates common and specific features of this activation mechanism.

Polymer-like Model to Study the Dynamics of Dynamin Filaments on Deformable Membrane Tubes.

Peripheral membrane proteins with intrinsic curvature can act both as sensors of membrane curvature and shape modulators of the underlying membranes. A well-studied example of such proteins is the mechanochemical GTPase dynamin, which assembles into helical filaments around membrane tubes and catalyzes their scission in a GTPase-dependent manner. It is known that the dynamin coat alone, without GTP, can constrict membrane tubes to radii of ∼10 nm, indicating that the intrinsic shape and elasticity of dynamin filaments should play an important role in membrane remodeling. However, molecular and dynamic understanding of the process is lacking. Here, we develop a dynamical polymer-chain model for a helical elastic filament bound on a deformable membrane tube of conserved mass, accounting for thermal fluctuations in the filament and lipid flows in the membrane. The model is based on the locally cylindrical helix approximation for dynamin. We obtain the elastic parameters of the dynamin filament by molecular dynamics simulations of its tetrameric building block and also from coarse-grained structure-based simulations of a 17-dimer filament. The results show that the stiffness of dynamin is comparable to that of the membrane. We determine equilibrium shapes of the filament and the membrane and find that mostly the pitch of the filament, not its radius, is sensitive to variations in membrane tension and stiffness. The close correspondence between experimental estimates of the inner tube radius and those predicted by the model suggests that dynamin's "stalk" region is responsible for its GTP-independent membrane-shaping ability. The model paves the way for future mesoscopic modeling of dynamin with explicit motor function.

Effects of allelic variations in the human myxovirus resistance protein A on its antiviral activity.

Only a minority of patients infected with seasonal influenza A viruses exhibit a severe or fatal outcome of infection, but the reasons for this inter-individual variability in influenza susceptibility are unclear. To gain further insights into the molecular mechanisms underlying this variability, we investigated naturally occurring allelic variations of the myxovirus resistance 1 (MX1) gene coding for the influenza restriction factor MxA. The interferon-induced dynamin-like GTPase consists of an N-terminal GTPase domain, a bundle signaling element, and a C-terminal stalk responsible for oligomerization and viral target recognition. We used online databases to search for variations in the MX1 gene. Deploying in vitro approaches, we found that non-synonymous variations in the GTPase domain cause the loss of antiviral and enzymatic activities. Furthermore, we showed that these amino acid substitutions disrupt the interface for GTPase domain dimerization required for the stimulation of GTP hydrolysis. Variations in the stalk were neutral or slightly enhanced or abolished MxA antiviral function. Remarkably, two other stalk variants altered MxA's antiviral specificity. Variations causing the loss of antiviral activity were found only in heterozygous carriers. Interestingly, the inactive stalk variants blocked the antiviral activity of WT MxA in a dominant-negative way, suggesting that heterozygotes are phenotypically MxA-negative. In contrast, the GTPase-deficient variants showed no dominant-negative effect, indicating that heterozygous carriers should remain unaffected. Our results demonstrate that naturally occurring mutations in the human MX1 gene can influence MxA function, which may explain individual variations in influenza virus susceptibility in the human population.

Quantitative GTPase Affinity Purification Identifies Rho Family Protein Interaction Partners.

Although Rho GTPases are essential molecular switches involved in many cellular processes, an unbiased experimental comparison of their interaction partners was not yet performed. Here, we develop quantitative GTPase affinity purification (qGAP) to systematically identify interaction partners of six Rho GTPases (Cdc42, Rac1, RhoA, RhoB, RhoC, and RhoD), depending on their nucleotide loading state. The method works with cell line or tissue-derived protein lysates in combination with SILAC-based or label-free quantification, respectively. We demonstrate that qGAP identifies known and novel binding partners that can be validated in an independent assay. Our interaction network for six Rho GTPases contains many novel binding partners, reveals highly promiscuous interaction of several effectors, and mirrors evolutionary relationships among Rho GTPases.

Structural insights into oligomerization and mitochondrial remodelling of dynamin 1-like protein.

Dynamin 1-like protein (DNM1L) mediates fission of mitochondria and peroxisomes, and dysfunction of DNM1L has been implicated in several neurological disorders. To study the molecular basis of mitochondrial remodelling, we determined the crystal structure of DNM1L that is comprised of a G domain, a bundle signalling element and a stalk. DNM1L assembled via a central stalk interface, and mutations in this interface disrupted dimerization and interfered with membrane binding and mitochondrial targeting. Two sequence stretches at the tip of the stalk were shown to be required for ordered assembly of DNM1L on membranes and its function in mitochondrial fission. In the crystals, DNM1L dimers further assembled via a second, previously undescribed, stalk interface to form a linear filament. Mutations in this interface interfered with liposome tubulation and mitochondrial remodelling. Based on these results and electron microscopy reconstructions, we propose an oligomerization mode for DNM1L which differs from that of dynamin and might be adapted to the remodelling of mitochondria.

Phosphorylation of PACSIN2 by protein kinase C triggers the removal of caveolae from the plasma membrane.

PACSIN2, a membrane-sculpting BAR domain protein, localizes to caveolae. Here, we found that protein kinase C (PKC) phosphorylates PACSIN2 at serine 313, thereby decreasing its membrane binding and tubulation capacities. Concomitantly, phosphorylation decreased the time span for which caveolae could be tracked at the plasma membrane (the 'tracking duration'). Analyses of the phospho-mimetic S313E mutant suggested that PACSIN2 phosphorylation was sufficient to reduce caveolar-tracking durations. Both hypotonic treatment and isotonic drug-induced PKC activation increased PACSIN2 phosphorylation at serine 313 and shortened caveolar-tracking durations. Caveolar-tracking durations were also reduced upon the expression of other membrane-binding-deficient PACSIN2 mutants or upon RNA interference (RNAi)-mediated PACSIN2 depletion, pointing to a role for PACSIN2 levels in modulating the lifetime of caveolae. Interestingly, the decrease in membrane-bound PACSIN2 was inversely correlated with the recruitment and activity of dynamin 2, a GTPase that mediates membrane scission. Furthermore, expression of EHD2, which stabilizes caveolae and binds to PACSIN2, restored the tracking durations of cells with reduced PACSIN2 levels. These findings suggest that the PACSIN2 phosphorylation decreases its membrane-binding activity, thereby decreasing its stabilizing effect on caveolae and triggering dynamin-mediated removal of caveolae.