Centromeres are specified epigenetically, and the histone H3 variant, CENP-A, is assembled into the chromatin of all active centromeres1. Divergence from H3 raises the possibility that CENP-A generates unique chromatin features to physically mark centromere location. The crystal structure of the sub-nucleosomal heterotetramer reported here reveals three distinguishing properties encoded by the residues that comprise the CENP-A Targeting Domain (CATD2): (1) a CENP-A/CENP-A interface that is substantially rotated relative to the H3/H3 interface, (2) a protruding loop L1 of the opposite charge as on H3, and (3) strong hydrophobic contacts that rigidify the CENP-A/H4 interface. Residues involved in the CENP-A/CENP-A rotation are required for efficient incorporation into centromeric chromatin suggesting specificity for an unconventional nucleosome shape. DNA topological analysis indicates that CENP-A-containing nucleosomes are nonetheless octameric with conventional left-handed DNA wrapping, in contrast to other recent proposals3-6. Our results indicate rather that CENP-A marks centromere location by restructuring the nucleosome from within its folded histone core.

The centromere is the chromosomal locus that ensures fidelity in genome transmission at cell division. Centromere protein A (CENP-A) is a histone H3 variant that specifies centromere location independently of DNA sequence. Conflicting evidence has emerged regarding the histone composition and stoichiometry of CENP-A nucleosomes. Here we show that the predominant form of the CENP-A particle at human centromeres is an octameric nucleosome. CENP-A nucleosomes are very highly phased on α-satellite 171 bp monomers at normal centromeres, and also display strong positioning at neocentromeres. At either type of functional centromere, CENP-A nucleosomes exhibit similar DNA wrapping behavior as octameric CENP-A nucleosomes reconstituted with recombinant components, having looser DNA termini than those on their conventional counterparts containing canonical H3. Thus, the fundamental unit of the chromatin that epigenetically specifies centromere location in mammals is an octameric nucleosome with loose termini.

Summary During mitosis, the macromolecular kinetochore complex assembles on the centromere to orchestrate chromosome segregation. The properties and architecture of the 16-subunit Constitutive Centromere-Associated Network (CCAN) that allow it to build a robust platform for kinetochore assembly are poorly understood. Here, we use inducible CRISPR knockouts and biochemical reconstitutions to define the interactions between the human CCAN proteins. We find that the CCAN does not assemble as a linear hierarchy, and instead, each sub-complex requires multiple non-redundant interactions for its localization to centromeres and the structural integrity of the overall assembly. We demonstrate that the CENP-L-N complex plays a crucial role at the core of this assembly through interactions with CENP-C and CENP-H-I-K-M. Finally, we show that the CCAN is remodeled over the cell cycle such that sub-complexes depend on their interactions differentially. Thus, an interdependent meshwork within the CCAN underlies the centromere specificity and stability of the kinetochore.

Inheritance of each chromosome depends upon its centromere. A histone H3 variant, CENP-A, is essential for epigenetically marking centromere location. We find that CENP-A is quantitatively retained at the centromere upon which it is initially assembled. CENP-C binds to CENP-A nucleosomes and is a prime candidate to stabilize centromeric chromatin. Using purified components, we find that CENP-C reshapes the octameric histone core of CENP-A nucleosomes, rigidifies both surface and internal nucleosome structure, and modulates terminal DNA to match the loose wrap that is found on native CENP-A nucleosomes at functional human centromeres. Thus, CENP-C affects nucleosome shape and dynamics in a manner analogous to allosteric regulation of enzymes. CENP-C depletion leads to rapid removal of CENP-A from centromeres, indicating their collaboration in maintaining centromere identity.

Viral proteins mimic host protein structure and function to redirect cellular processes and subvert innate defenses1. Small basic proteins compact and regulate both viral and cellular DNA genomes. Nucleosomes are the repeating units of cellular chromatin and play an important role in innate immune responses2. Viral encoded core basic proteins compact viral genomes but their impact on host chromatin structure and function remains unexplored. Adenoviruses encode a highly basic protein called protein VII that resembles cellular histones3. Although protein VII binds viral DNA and is incorporated with viral genomes into virus particles4,5, it is unknown whether protein VII impacts cellular chromatin. Our observation that protein VII alters cellular chromatin led us to hypothesize that this impacts antiviral responses during adenovirus infection. We found that protein VII forms complexes with nucleosomes and limits DNA accessibility. We identified post-translational modifications on protein VII that are responsible for chromatin localization. Furthermore, proteomic analysis demonstrated that protein VII is sufficient to alter protein composition of host chromatin. We found that protein VII is necessary and sufficient for retention in chromatin of members of the high-mobility group protein B family (HMGB1, HMGB2, and HMGB3). HMGB1 is actively released in response to inflammatory stimuli and functions as a danger signal to activate immune responses6,7. We showed that protein VII can directly bind HMGB1 in vitro and further demonstrated that protein VII expression in mouse lungs is sufficient to decrease inflammation-induced HMGB1 content and neutrophil recruitment in the bronchoalveolar lavage fluid. Together our in vitro and in vivo results show that protein VII sequesters HMGB1 and can prevent its release. This study uncovers a viral strategy in which nucleosome binding is exploited to control extracellular immune signaling.

Summary Centromeres are defined by the presence of chromatin containing the histone H3 variant, CENP-A, whose assembly into nucleosomes requires the chromatin assembly factor HJURP. We find that while surface-exposed residues in the CENP-A targeting domain (CATD) are the primary sequence determinants for HJURP recognition, buried CATD residues that generate rigidity with H4 are also required for efficient incorporation into centromeres. HJURP contact points adjacent to the CATD on the CENP-A surface are not used for binding specificity but rather to transmit stability broadly throughout the histone fold domains of both CENP-A and H4. Further, an intact CENP-A/CENP-A interface is a requirement for stable chromatin incorporation immediately upon HJURP-mediated assembly. These data offer insight into the mechanism by which HJURP discriminates CENP-A from bulk histone complexes and chaperones CENP-A/H4 for a substantial portion of the cell cycle prior to mediating chromatin assembly at the centromere.

Choline kinase, responsible for the phosphorylation of choline to phosphocholine as the first step of the CDP-choline pathway for the biosynthesis of phosphatidylcholine, has been recognized as a new target for anticancer therapy. Crystal structures of human choline kinase in its apo, ADP and phosphocholine-bound complexes, respectively, reveal the molecular details of the substrate binding sites. ATP binds in a cavity where residues from both the N and C-terminal lobes contribute to form a cleft, while the choline-binding site constitutes a deep hydrophobic groove in the C-terminal domain with a rim composed of negatively charged residues. Upon binding of choline, the enzyme undergoes conformational changes independently affecting the N-terminal domain and the ATP-binding loop. From this structural analysis and comparison with other kinases, and from mutagenesis data on the homologous Caenorhabditis elegans choline kinase, a model of the ternary ADP.phosphocholine complex was built that reveals the molecular basis for the phosphoryl transfer activity of this enzyme.

Guanylate kinase (GMPK) is a nucleoside monophosphate kinase that catalyzes the reversible phosphoryl transfer from ATP to GMP to yield ADP and GDP. In addition to phosphorylating GMP, antiviral prodrugs such as acyclovir, ganciclovir, and carbovir and anticancer prodrugs such as the thiopurines are dependent on GMPK for their activation. Hence, structural information on mammalian GMPK could play a role in the design of improved antiviral and antineoplastic agents. Here we present the structure of the mouse enzyme in an abortive complex with the nucleotides ADP and GMP, refined at 2.1 Å resolution with a final crystallographic R factor of 0.19 (R free ‫؍‬ 0.23). Guanylate kinase is a member of the nucleoside monophosphate (NMP) kinase family, a family of enzymes that despite having a low primary structure identity share a similar fold, which consists of three structurally distinct regions termed the CORE, LID, and NMP-binding regions. Previous studies on the yeast enzyme have shown that these parts move as rigid bodies upon substrate binding. It has been proposed that consecutive binding of substrates leads to "closing" of the active site bringing the NMP-binding and LID regions closer to each other and to the CORE region. Our structure, which is the first of any guanylate kinase with both substrates bound, supports this hypothesis. It also reveals the binding site of ATP and implicates arginines 44, 137, and 148 (in addition to the invariant P-loop lysine) as candidates for catalyzing the chemical step of the phosphoryl transfer.Guanylate kinase (GMPK, 1 ATP:GMP phosphotransferase, EC 2.7.4.8) is a critical enzyme for the biosynthesis of GTP and dGTP by catalyzing the phosphoryl transfer from ATP to (d)GMP resulting in ADP and (d)GDP (1, 2). GMPK also plays an important role in the recycling of the second messenger cGMP (3). In addition to these physiological roles, GMPK is essential for the activation of prodrugs used for the treatment of cancers and viral infections (4, 5). Therefore, it is medically important to elucidate its enzymatic mechanism and the structural basis for its nucleotide specificity. Our current structural understanding of this enzyme is derived from the apo-and GMP-bound structures of the yeast GMPK (6) and from analogy to other nucleoside monophosphate (NMP) kinases (7).It has been shown that the induced fit mechanism (8) plays an important role in NMP kinases, of which adenylate kinase is the best characterized (9 -11). NMP kinases catalyze phosphoryl transfer by binding both donor and acceptor nucleotides to form a ternary complex. Comparison of the crystal structures of nucleotide-free adenylate kinase to the one in which a single substrate is bound (AMP or ATP) and to the complex in which both substrates are present revealed the conformational changes that occur along the reaction coordinate: from an open unbound enzyme via a partially closed intermediate in which a single substrate is present to the fully closed form in the presence of both substrates. These substrate-induced conform...