The WAVE regulatory complex links diverse receptors to the actin cytoskeleton.

The WAVE regulatory complex (WRC) controls actin cytoskeletal dynamics throughout the cell by stimulating the actin-nucleating activity of the Arp2/3 complex at distinct membrane sites. However, the factors that recruit the WRC to specific locations remain poorly understood. Here, we have identified a large family of potential WRC ligands, consisting of ∼120 diverse membrane proteins, including protocadherins, ROBOs, netrin receptors, neuroligins, GPCRs, and channels. Structural, biochemical, and cellular studies reveal that a sequence motif that defines these ligands binds to a highly conserved interaction surface of the WRC formed by the Sra and Abi subunits. Mutating this binding surface in flies resulted in defects in actin cytoskeletal organization and egg morphology during oogenesis, leading to female sterility. Our findings directly link diverse membrane proteins to the WRC and actin cytoskeleton and have broad physiological and pathological ramifications in metazoans.

The bacterial effector VopL organizes actin into filament-like structures.

VopL is an effector protein from Vibrio parahaemolyticus that nucleates actin filaments. VopL consists of a VopL C-terminal domain (VCD) and an array of three WASP homology 2 (WH2) motifs. Here, we report the crystal structure of the VCD dimer bound to actin. The VCD organizes three actin monomers in a spatial arrangement close to that found in the canonical actin filament. In this arrangement, WH2 motifs can be modeled into the binding site of each actin without steric clashes. The data suggest a mechanism of nucleation wherein VopL creates filament-like structures, organized by the VCD with monomers delivered by the WH2 array, that can template addition of new subunits. Similarities with Arp2/3 complex and formin proteins suggest that organization of monomers into filament-like structures is a general and central feature of actin nucleation.

Structure of human chromatin-remodelling PBAF complex bound to a nucleosome.

DNA wraps around the histone octamer to form nucleosomes1, the repeating unit of chromatin, which create barriers for accessing genetic information. Snf2-like chromatin remodellers couple the energy of ATP binding and hydrolysis to reposition and recompose the nucleosome, and have vital roles in various chromatin-based transactions2,3. Here we report the cryo-electron microscopy structure of the 12-subunit human chromatin-remodelling polybromo-associated BRG1-associated factor (PBAF) complex bound to the nucleosome. The motor subunit SMARCA4 engages the nucleosome in the active conformation, which reveals clustering of multiple disease-associated mutations at the interfaces that are essential for chromatin-remodelling activity. SMARCA4 recognizes the H2A-H2B acidic pocket of the nucleosome through three arginine anchors of the Snf2 ATP coupling (SnAc) domain. PBAF shows notable functional modularity, and most of the auxiliary subunits are interwoven into three lobe-like submodules for nucleosome recognition. The PBAF-specific auxiliary subunit ARID2 acts as the structural core for assembly of the DNA-binding lobe, whereas PBRM1, PHF10 and BRD7 are collectively incorporated into the lobe for histone tail binding. Together, our findings provide mechanistic insights into nucleosome recognition by PBAF and a structural basis for understanding SMARCA4-related human diseases.

Structure of the NuA4 acetyltransferase complex bound to the nucleosome.

Deoxyribonucleic acid in eukaryotes wraps around the histone octamer to form nucleosomes1, the fundamental unit of chromatin. The N termini of histone H4 interact with nearby nucleosomes and play an important role in the formation of high-order chromatin structure and heterochromatin silencing2-4. NuA4 in yeast and its homologue Tip60 complex in mammalian cells are the key enzymes that catalyse H4 acetylation, which in turn regulates chromatin packaging and function in transcription activation and DNA repair5-10. Here we report the cryo-electron microscopy structure of NuA4 from Saccharomyces cerevisiae bound to the nucleosome. NuA4 comprises two major modules: the catalytic histone acetyltransferase (HAT) module and the transcription activator-binding (TRA) module. The nucleosome is mainly bound by the HAT module and is positioned close to a polybasic surface of the TRA module, which is important for the optimal activity of NuA4. The nucleosomal linker DNA carrying the upstream activation sequence is oriented towards the conserved, transcription activator-binding surface of the Tra1 subunit, which suggests a potential mechanism of NuA4 to act as a transcription co-activator. The HAT module recognizes the disk face of the nucleosome through the H2A-H2B acidic patch and nucleosomal DNA, projecting the catalytic pocket of Esa1 to the N-terminal tail of H4 and supporting its function in selective acetylation of H4. Together, our findings illustrate how NuA4 is assembled and provide mechanistic insights into nucleosome recognition and transcription co-activation by a HAT.

Mechanism of DNA translocation underlying chromatin remodelling by Snf2.

Chromatin remodellers include diverse enzymes with distinct biological functions, but nucleosome-sliding activity appears to be a common theme1,2. Among the remodelling enzymes, Snf2 serves as the prototype to study the action of this protein family. Snf2 and related enzymes share two conserved RecA-like lobes3, which by themselves are able to couple ATP hydrolysis to chromatin remodelling. The mechanism by which these enzymes couple ATP hydrolysis to translocate the nucleosome along the DNA remains unclear2,4-8. Here we report the structures of Saccharomyces cerevisiae Snf2 bound to the nucleosome in the presence of ADP and ADP-BeFx. Snf2 in the ADP-bound state adopts an open conformation similar to that in the apo state, and induces a one-base-pair DNA bulge at superhelix location 2 (SHL2), with the tracking strand showing greater distortion than the guide strand. The DNA distortion propagates to the proximal end, leading to staggered translocation of the two strands. The binding of ADP-BeFx triggers a closed conformation of the enzyme, resetting the nucleosome to a relaxed state. Snf2 shows altered interactions with the DNA in different nucleotide states, providing the structural basis for DNA translocation. Together, our findings suggest a fundamental mechanism for the DNA translocation that underlies chromatin remodelling.

A Unifying Mechanism of DNA Translocation Underlying Chromatin Remodeling.

Chromatin remodelers alter the position and composition of nucleosomes, and play key roles in the regulation of chromatin structure and various chromatin-based transactions. Recent cryo-electron microscopy (cryo-EM) and single-molecule fluorescence resonance energy transfer (smFRET) studies have shed mechanistic light on the fundamental question of how the remodeling enzymes couple with ATP hydrolysis to slide nucleosomes. Structures of the chromatin remodeler Snf2 bound to the nucleosome reveal the conformational cycle of the enzyme and the induced DNA distortion. Investigations on ISWI, Chd1, and INO80 support a unifying fundamental mechanism of DNA translocation. Finally, studies of the SWR1 complex suggest that the enzyme distorts the DNA abnormally to achieve histone exchange without net DNA translocation.

The NuA4 Core Complex Acetylates Nucleosomal Histone H4 through a Double Recognition Mechanism.

NuA4 catalyzes the acetylation of nucleosomes at histone H4, which is a well-established epigenetic event, controlling many genomic processes in Saccharomyces cerevisiae. Here we report the crystal structures of the NuA4 core complex and a cryoelectron microscopy structure with the nucleosome. The structures show that the histone-binding pocket of the enzyme is rearranged, suggesting its activation. The enzyme binds the histone tail mainly through the target lysine residue, with a preference for a small residue at the -1 position. The complex engages the nucleosome at the dish face and orients its catalytic pocket close to the H4 tail to achieve selective acetylation. The combined data reveal a space-sequence double recognition mechanism of the histone tails by a modifying enzyme in the context of the nucleosome.

Mechanistic insights into central spindle assembly mediated by the centralspindlin complex.

The central spindle spatially and temporally regulates the formation of division plane during cytokinesis in animal cells. The heterotetrameric centralspindlin complex bundles microtubules to assemble the central spindle, the mechanism of which is poorly understood. Here, we determined the crystal structures of the molecular backbone of ZEN-4/CYK-4 centralspindlin from Caenorhabditis elegans, which revealed the detailed mechanism of complex formation. The molecular backbone of centralspindlin has the intrinsic propensity to undergo liquid-liquid phase separation. The condensation of centralspindlin requires two patches of basic residues at ZEN-4 and multiple acidic residues at the intrinsically disordered region of CYK-4, explaining the synergy of the two subunits for the function. These complementary charged residues were critical for the microtubule bundling activity of centralspindlin in vitro and for the assembly of the central spindle in vivo. Together, our findings provide insights into the mechanism of central spindle assembly mediated by centralspindlin through charge-driven macromolecular condensation.

Mechanism of chromatin remodelling revealed by the Snf2-nucleosome structure.

Chromatin remodellers are helicase-like, ATP-dependent enzymes that alter chromatin structure and nucleosome positions to allow regulatory proteins access to DNA. Here we report the cryo-electron microscopy structure of chromatin remodeller Switch/sucrose non-fermentable (SWI2/SNF2) from Saccharomyces cerevisiae bound to the nucleosome. The structure shows that the two core domains of Snf2 are realigned upon nucleosome binding, suggesting activation of the enzyme. The core domains contact each other through two induced Brace helices, which are crucial for coupling ATP hydrolysis to chromatin remodelling. Snf2 binds to the phosphate backbones of one DNA gyre of the nucleosome mainly through its helicase motifs within the major domain cleft, suggesting a conserved mechanism of substrate engagement across different remodellers. Snf2 contacts the second DNA gyre via a positively charged surface, providing a mechanism to anchor the remodeller at a fixed position of the nucleosome. Snf2 locally deforms nucleosomal DNA at the site of binding, priming the substrate for the remodelling reaction. Together, these findings provide mechanistic insights into chromatin remodelling.

Structure and regulation of human epithelial cell transforming 2 protein.

Epithelial cell transforming 2 (Ect2) protein activates Rho GTPases and controls cytokinesis and many other cellular processes. Dysregulation of Ect2 is associated with various cancers. Here, we report the crystal structure of human Ect2 and complementary mechanistic analyses. The data show the C-terminal PH domain of Ect2 folds back and blocks the canonical RhoA-binding site at the catalytic center of the DH domain, providing a mechanism of Ect2 autoinhibition. Ect2 is activated by binding of GTP-bound RhoA to the PH domain, which suggests an allosteric mechanism of Ect2 activation and a positive-feedback loop reinforcing RhoA signaling. This bimodal RhoA binding of Ect2 is unusual and was confirmed with Förster resonance energy transfer (FRET) and hydrogen-deuterium exchange mass spectrometry (HDX-MS) analyses. Several recurrent cancer-associated mutations map to the catalytic and regulatory interfaces, and dysregulate Ect2 in vitro and in vivo. Together, our findings provide mechanistic insights into Ect2 regulation in normal cells and under disease conditions.

Structure and regulation of the chromatin remodeller ISWI.

ISWI is a member of the SWI2/SNF2 family of chromatin remodellers, which also includes Snf2, Chd1, and Ino80. ISWI is the catalytic subunit of several chromatin remodelling complexes, which mobilize nucleosomes along genomic DNA, promoting replication progression, transcription repression, heterochromatin formation, and many other nuclear processes. The ATPase motor of ISWI is an autonomous remodelling machine, whereas its carboxy (C)-terminal HAND-SAND-SLIDE (HSS) domain functions in binding extranucleosomal linker DNA. The activity of the catalytic core of ISWI is inhibited by the regulatory AutoN and NegC domains, which are in turn antagonized by the H4 tail and extranucleosomal DNA, respectively, to ensure the appropriate chromatin landscape in cells. How AutoN and NegC inhibit ISWI and regulate its nucleosome-centring activity remains elusive. Here we report the crystal structures of ISWI from the thermophilic yeast Myceliophthora thermophila and its complex with a histone H4 peptide. Our data show the amino (N)-terminal AutoN domain contains two inhibitory elements, which collectively bind the second RecA-like domain (core2), holding the enzyme in an inactive conformation. The H4 peptide binds to the core2 domain coincident with one of the AutoN-binding sites, explaining the ISWI activation by H4. The H4-binding surface is conserved in Snf2 and functions beyond AutoN regulation. The C-terminal NegC domain is involved in binding to the core2 domain and functions as an allosteric element for ISWI to respond to the extranucleosomal DNA length.

Crystal structure of a nuclear actin ternary complex.

Actin polymerizes and forms filamentous structures (F-actin) in the cytoplasm of eukaryotic cells. It also exists in the nucleus and regulates various nucleic acid transactions, particularly through its incorporation into multiple chromatin-remodeling complexes. However, the specific structure of actin and the mechanisms that regulate its polymeric nature inside the nucleus remain unknown. Here, we report the crystal structure of nuclear actin (N-actin) complexed with actin-related protein 4 (Arp4) and the helicase-SANT-associated (HSA) domain of the chromatin remodeler Swr1. The inner face and barbed end of N-actin are sequestered by interactions with Arp4 and the HSA domain, respectively, which prevents N-actin from polymerization and binding to many actin regulators. The two major domains of N-actin are more twisted than those of globular actin (G-actin), and its nucleotide-binding pocket is occluded, freeing N-actin from binding to and regulation by ATP. These findings revealed the salient structural features of N-actin that distinguish it from its cytoplasmic counterpart and provide a rational basis for its functions and regulation inside the nucleus.

Involvement of budding yeast Rad5 in translesion DNA synthesis through physical interaction with Rev1.

DNA damage tolerance (DDT) is responsible for genomic stability and cell viability by bypassing the replication block. In Saccharomyces cerevisiae DDT employs two parallel branch pathways to bypass the DNA lesion, namely translesion DNA synthesis (TLS) and error-free lesion bypass, which are mediated by sequential modifications of PCNA. Rad5 has been placed in the error-free branch of DDT because it contains an E3 ligase domain required for PCNA polyubiquitination. Rad5 is a multi-functional protein and may also play a role in TLS, since it interacts with the TLS polymerase Rev1. In this study we mapped the Rev1-interaction domain in Rad5 to the amino acid resolution and demonstrated that Rad5 is indeed involved in TLS possibly through recruitment of Rev1. Genetic analyses show that the dual functions of Rad5 can be separated and reconstituted. Crystal structure analysis of the Rad5-Rev1 interaction reveals a consensus RFF motif in the Rad5 N-terminus that binds to a hydrophobic pocket within the C-terminal domain of Rev1 that is highly conserved in eukaryotes. This study indicates that Rad5 plays a critical role in pathway choice between TLS and error-free DDT.

Structure and control of the actin regulatory WAVE complex.

Members of the Wiskott-Aldrich syndrome protein (WASP) family control cytoskeletal dynamics by promoting actin filament nucleation with the Arp2/3 complex. The WASP relative WAVE regulates lamellipodia formation within a 400-kilodalton, hetero-pentameric WAVE regulatory complex (WRC). The WRC is inactive towards the Arp2/3 complex, but can be stimulated by the Rac GTPase, kinases and phosphatidylinositols. Here we report the 2.3-ångstrom crystal structure of the WRC and complementary mechanistic analyses. The structure shows that the activity-bearing VCA motif of WAVE is sequestered by a combination of intramolecular and intermolecular contacts within the WRC. Rac and kinases appear to destabilize a WRC element that is necessary for VCA sequestration, suggesting the way in which these signals stimulate WRC activity towards the Arp2/3 complex. The spatial proximity of the Rac binding site and the large basic surface of the WRC suggests how the GTPase and phospholipids could cooperatively recruit the complex to membranes.

Mechanism of homologous recombination from the RecA-ssDNA/dsDNA structures.

The RecA family of ATPases mediates homologous recombination, a reaction essential for maintaining genomic integrity and for generating genetic diversity. RecA, ATP and single-stranded DNA (ssDNA) form a helical filament that binds to double-stranded DNA (dsDNA), searches for homology, and then catalyses the exchange of the complementary strand, producing a new heteroduplex. Here we have solved the crystal structures of the Escherichia coli RecA-ssDNA and RecA-heteroduplex filaments. They show that ssDNA and ATP bind to RecA-RecA interfaces cooperatively, explaining the ATP dependency of DNA binding. The ATP gamma-phosphate is sensed across the RecA-RecA interface by two lysine residues that also stimulate ATP hydrolysis, providing a mechanism for DNA release. The DNA is underwound and stretched globally, but locally it adopts a B-DNA-like conformation that restricts the homology search to Watson-Crick-type base pairing. The complementary strand interacts primarily through base pairing, making heteroduplex formation strictly dependent on complementarity. The underwound, stretched filament conformation probably evolved to destabilize the donor duplex, freeing the complementary strand for homology sampling.

Structure of the ISW1a complex bound to the dinucleosome.

Nucleosomes are basic repeating units of chromatin and form regularly spaced arrays in cells. Chromatin remodelers alter the positions of nucleosomes and are vital in regulating chromatin organization and gene expression. Here we report the cryo-EM structure of chromatin remodeler ISW1a complex from Saccharomyces cerevisiae bound to the dinucleosome. Each subunit of the complex recognizes a different nucleosome. The motor subunit binds to the mobile nucleosome and recognizes the acidic patch through two arginine residues, while the DNA-binding module interacts with the entry DNA at the nucleosome edge. This nucleosome-binding mode provides the structural basis for linker DNA sensing of the motor. Notably, the Ioc3 subunit recognizes the disk face of the adjacent nucleosome through interacting with the H4 tail, the acidic patch and the nucleosomal DNA, which plays a role in the spacing activity in vitro and in nucleosome organization and cell fitness in vivo. Together, these findings support the nucleosome spacing activity of ISW1a and add a new mode of nucleosome remodeling in the context of a chromatin environment.

Effect of CO binding to P450 BM3 F393 mutants on electron density distribution in the heme cofactor.

Resonance Raman spectroscopy has been performed on a set of cytochrome P450 BM3 heme domains in which mutation of the highly conserved Phe393 induces significant variation in heme iron reduction potential. In previous work [Chen, Z., Ost, T.W.B., and Schelvis, J.P.M. (2004) Biochemistry 43, 1798-1808], a correlation between heme vinyl conformation and the heme iron reduction potential indicated a steric control by the protein over the distribution of electron density in the reduced heme cofactor. The current study aims to monitor changes in electron density on the ferrous heme cofactor following CO binding. In addition, ferric-NO complexes have been studied to investigate potential changes to the proximal Cys400 thiolate. We find that binding of CO to the ferrous heme domains results in a reorientation of the vinyl groups to a largely out-of-plane conformation, the extent of which correlates with the size of the residue at position 393. We conclude that FeII dπ back bonding to the CO ligand largely takes away the need for conjugation of the vinyl groups with the porphyrin ring to accommodate FeII dπ back bonding to the porphyrin ligand. The ferrous-CO and ferric-NO data are consistent with a small decrease in σ-electron donation from the proximal Cys400 thiolate in the F393A mutant and, to a lesser extent, the F393H mutant, potentially due to a small increase in hydrogen bonding to the proximal ligand. Phe393 seems strategically placed to preserve robust σ-electron donation to the heme iron and to fine-tune its electron density by limiting vinyl group rotation.

Structural basis of ALC1/CHD1L autoinhibition and the mechanism of activation by the nucleosome.

Chromatin remodeler ALC1 (amplification in liver cancer 1) is crucial for repairing damaged DNA. It is autoinhibited and activated by nucleosomal epitopes. However, the mechanisms by which ALC1 is regulated remain unclear. Here we report the crystal structure of human ALC1 and the cryoEM structure bound to the nucleosome. The structure shows the macro domain of ALC1 binds to lobe 2 of the ATPase motor, sequestering two elements for nucleosome recognition, explaining the autoinhibition mechanism of the enzyme. The H4 tail competes with the macro domain for lobe 2-binding, explaining the requirement for this nucleosomal epitope for ALC1 activation. A dual-arginine-anchor motif of ALC1 recognizes the acidic pocket of the nucleosome, which is critical for chromatin remodeling in vitro. Together, our findings illustrate the structures of ALC1 and shed light on its regulation mechanisms, paving the way for the discovery of drugs targeting ALC1 for the treatment of cancer.

Pulmonary capillary hemangiomatosis in Chinese patients without EIF2AK4 mutations.

BACKGROUND: Pulmonary capillary hemangiomatosis (PCH) is a very rare and refractory pulmonary vascular disease that causes pulmonary hypertension. Differentiation of PCH from idiopathic pulmonary arterial hypertension (iPAH) is essential because treatment and prognosis can vary greatly between these two diseases. CASE PRESENTATION: A 20-year-old female and a 33-year-old male both presented with progressive exertional dyspnea and cough. High-resolution computed tomography (HRCT) showed bilateral, diffuse, ill-defined centrilobular nodules of ground-glass opacity, without subpleural thickened septal lines or mediastinal lymphadenopathy. Both cases showed clinical and imaging features characteristic of pulmonary veno-occlusive disease (PVOD) or PCH. The entire EIF2AK4 coding sequence was detected with Sanger sequencing, and no pathogenic EIF2AK4 mutations were identified in either case. Video-assisted thoracoscopic surgery (VATS) was safely performed in both cases, and histopathological examinations of biopsies showed that both patients had PCH. CONCLUSION: Two patients presented with clinical and imaging characteristics suspicious for PVOD/PCH. Despite having no pathogenic EIF2AK4 mutations, both were diagnosed with PCH by VATS lung biopsies. The diagnostic distinction of PCH is important to prompt timely evaluations of patients who may need lung transplantations.

Structures of the ISWI-nucleosome complex reveal a conserved mechanism of chromatin remodeling.

Chromatin remodelers are diverse enzymes, and different models have been proposed to explain how these proteins work. Here we report the 3.3 Å-resolution cryogenic electron microscopy (cryo-EM) structures of Saccharomyces cerevisiae ISWI (ISW1) in complex with the nucleosome in adenosine diphosphate (ADP)-bound and ADP-BeFx-bound states. The data show that after nucleosome binding, ISW1 is activated by substantial rearrangement of the catalytic domains, with the regulatory AutoN domain packing the first RecA-like core and the NegC domain being disordered. The high-resolution structure reveals local DNA distortion and translocation induced by ISW1 in the ADP-bound state, which is essentially identical to that induced by the Snf2 chromatin remodeler, suggesting a common mechanism of DNA translocation. The histone core remains largely unperturbed, and prevention of histone distortion by crosslinking did not inhibit the activity of yeast ISW1 or its human homolog. Together, our findings suggest a general mechanism of chromatin remodeling involving local DNA distortion without notable histone deformation.