Abstract
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.
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.
Chromatin remodelers are RecA-like DNA translocases that couple to ATP hydrolysis to alter the position and composition of the nucleosome.The mechanism by which chromatin remodelers translocate DNA around nucleosomes has been the subject of debate, and several models have been proposed, including DNA twist, loop, and wave-ratchet-wave models.Recent structures of chromatin-remodeling enzymes, including Snf2, Chd1, ISWI, and Ino80, in complex with nucleosomes have shed light on a conserved mechanism of DNA translocation.By contrast, Swr1 binds at the canonical site on a nucleosome, but induces abnormal DNA distortion and does not possess DNA translocation activity.
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-BeF
-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.
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.
Short-chain fatty acids and their corresponding acyl-CoAs sit at the crossroads of metabolic pathways and play important roles in diverse cellular processes. They are also precursors for protein ...post-translational lysine acylation modifications. A noteworthy example is the newly identified lysine 2-hydroxyisobutyrylation （Khib） that is derived from 2-hydroxyisobutyrate and 2-hydroxyisobutyryl-CoA. Histone Khib has been shown to be associated with active gene expression in spermatogenic cells. However, the key elements that regulate this post-translational lysine acyla- tion pathway remain unknown. This has hindered characterization of the mechanisms by which this modification exerts its biological functions. Here we show that Esalp in budding yeast and its homologue Tip60 in human could add Khib to substrate proteins both in vitro and in vivo. In addition, we have identified HDAC2 and HDAC3 as the major enzymes to remove Khmb. Moreover, we report the first global profiling of Khib proteome in mammalian cells, identifying 6 548 Khb sites on 1 725 substrate proteins. Our study has thus discovered both the ＂writers＂ and ＂erasers＂ for histone Kh~b marks, and major Khib protein substrates. These results not only illustrate the landscape of this new lysine acylation pathway, but also open new avenues for studying diverse functions of cellular metabolites associated with this pathway.
The RSC complex remodels chromatin structure and regulates gene transcription. We used cryo-electron microscopy to determine the structure of yeast RSC bound to the nucleosome. RSC is delineated into ...the adenosine triphosphatase motor, the actin-related protein module, and the substrate recruitment module (SRM). RSC binds the nucleosome mainly through the motor, with the auxiliary subunit Sfh1 engaging the H2A-H2B acidic patch to enable nucleosome ejection. SRM is organized into three substrate-binding lobes poised to bind their respective nucleosomal epitopes. The relative orientations of the SRM and the motor on the nucleosome explain the directionality of DNA translocation and promoter nucleosome repositioning by RSC. Our findings shed light on RSC assembly and functionality, and they provide a framework to understand the mammalian homologs BAF/PBAF and the Sfh1 ortholog INI1/BAF47, which are frequently mutated in cancers.
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.
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•Many potential WRC ligands defined by a peptide motif (WIRS) were identified•Motif binds to a conserved WRC surface formed by Sra and Abi subunits•WIRS/WRC interaction regulates oogenesis in flies
A short peptide motif that binds to a conserved surface of the WAVE regulatory complex (WRC) has been identified in a large family of diverse membrane proteins. This interaction recruits the WRC to membranes, regulates the actin cytoskeleton, and is important during Drosophila development.
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.
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