X-linked intellectual disability mutations alter the conformational state of a histone demethylase's sensing of chromatin

The H3K4me3 chromatin modification, a hallmark of promoters of actively transcribed genes, is dynamically removed by the KDM5 family of histone demethylases. The KDM5 demethylases have a number of accessory domains, two of which, ARID and PHD1, lie within the catalytic domain. KDM5C, which has a unique role in neural development, harbors a number of mutations adjacent to its accessory domains that cause X-linked intellectual disability (XLID). The roles of these accessory domains remain unknown, limiting an understanding of how XLID mutations affect KDM5C activity. We find that while the ARID and PHD1 domains are required for efficient nucleosome demethylation, the PHD1 domain alone has an inhibitory role in KDM5C catalysis. We further find that binding of the H3 tail to PHD1 is coupled to the recognition of linker DNA by KDM5C. Our data suggests a model in which the PHD1 domain regulates DNA recognition by the ARID domain based on available substrate cues. In this model, recognition of distinct chromatin features is coupled to a conformational rearrangement of the ARID and PHD1 domains, which in turn modulates the positioning of the catalytic domain for efficient nucleosome demethylation. Importantly, we find that XLID mutations adjacent to the ARID and PHD1 domains alter the conformational state of these domains to enhance DNA binding. This results in the loss of specificity in chromatin recognition by KDM5C and renders catalytic activity sensitive to inhibition by linker DNA. Our findings suggest a unifying model by which XLID mutations alter chromatin recognition and enable euchromatin-specific dysregulation of demethylation by KDM5C.

Coordinated conformational changes in the V1 complex during V-ATPase reversible dissociation

Vacuolar-type ATPases (V-ATPases) are rotary enzymes that acidify intracellular compartments in eukaryotic cells. These multi-subunit complexes consist of a cytoplasmic V1 region that hydrolyzes ATP and a membrane-embedded VO region that transports protons. V-ATPase activity is regulated by reversible dissociation of the two regions, with the isolated V1 and VO complexes becoming autoinhibited upon disassembly and subunit C subsequently detaching from V1. In yeast, assembly of the V1 and VO regions is mediated by the RAVE complex through an unknown mechanism. We used cryoEM of yeast V-ATPase to determine structures of the intact enzyme, the dissociated but complete V1 complex, and the V1 complex lacking subunit C. Upon separation, V1 undergoes a dramatic conformational rearrangement, with its rotational state becoming incompatible for reassembly with VO. Loss of subunit C allows V1 to match the rotational state of VO, suggesting how RAVE could reassemble V1 and VO by recruiting subunit C.

Use of additives to regulate solute aggregation and direct conformational polymorph nucleation of pimelic acid

Understanding the nucleation pathway and achieving regulation to produce the desired crystals are mutually beneficial. The authors previously proposed a nucleation pathway of conformational polymorphs in which solvation and solute self-assembly could affect the result of the conformational rearrangement and further nucleation outcomes. Based on this, herein α,ω-alkanedicarboxylic acids (DAn, where n represents the number of carbon atoms in the molecule, n = 2–6, 8–11) were designed as homologous additives to interfere with the self-assembly of pimelic acid (DA7) to further induce the form II compound, which differs from form I only in conformation. Interestingly, longer-chain additives (DA6–11) have a stronger form II-inducing ability than short-chain ones (DA2–4). In addition, an apparent gradient of the degree of interference with solute self-assembly, consistent with form II-inducing ability, was detected by infrared and nuclear magnetic resonance spectroscopy. The calculated molecular electrostatic potential charges also clearly indicate that additive–solute electrostatic interactions gradually increase with increasing carbon chain length of the additives, reaching a maximum value with DA6–11. This novel use of additives demonstrates a direct link between solute aggregation and conformational polymorph nucleation.

The flagellar motor of Vibrio alginolyticus undergoes major structural remodeling during rotational switching

The bacterial flagellar motor switches rotational direction between counterclockwise (CCW) and clockwise (CW) to direct the migration of the cell. The cytoplasmic ring (C-ring) of the motor, which is composed of FliG, FliM, and FliN, is known for controlling the rotational sense of the flagellum. However, the mechanism underlying rotational switching remains elusive. Here, we deployed cryo-electron tomography to visualize the C-ring in two rotational biased mutants in Vibrio alginolyticus. We determined the C-ring molecular architectures, providing novel insights into the mechanism of rotational switching. We report that the C-ring maintained 34-fold symmetry in both rotational senses, and the protein composition remained constant. The two structures show FliG conformational changes elicit a large conformational rearrangement of the rotor complex that coincides with rotational switching of the flagellum. FliM and FliN form a stable spiral-shaped base of the C-ring, likely stabilizing the C-ring during the conformational remodeling.

Architecture of the AP2/clathrin coat on the membranes of clathrin-coated vesicles

Clathrin-mediated endocytosis (CME) is crucial for modulating the protein composition of a cell’s plasma membrane. Clathrin forms a cage-like, polyhedral outer scaffold around a vesicle, to which cargo-selecting clathrin adaptors are attached. Adaptor protein complex (AP2) is the key adaptor in CME. Crystallography has shown AP2 to adopt a range of conformations. Here, we used cryo–electron microscopy, tomography, and subtomogram averaging to determine structures, interactions, and arrangements of clathrin and AP2 at the key steps of coat assembly, from AP2 in solution to membrane-assembled clathrin-coated vesicles (CCVs). AP2 binds cargo and PtdIns(4,5)P2 (phosphatidylinositol 4,5-bisphosphate)–containing membranes via multiple interfaces, undergoing conformational rearrangement from its cytosolic state. The binding mode of AP2 β2 appendage into the clathrin lattice in CCVs and buds implies how the adaptor structurally modulates coat curvature and coat disassembly.