Explore the vast range of titles available.

The kinetochore proteins CENP-N and CENP-C recognize the histone H3 variant CENP-A in the centromeric nucleosome. This ensures proper kinetochore assembly and accurate segregation of chromosomes. Chittori et al. describe the cryo-electron microscopy structure of the human CENP-A nucleosome-CENP-N complex. The interaction of CENP-N with CENP-A and the nucleosomal DNA together ensure specific and stable centromeric nucleosome recognition. Mutational analyses using both human and Xenopus CENP-A and CENP-N proteins suggest that the proteins have coevolved to preserve the interacting surfaces. Science , this issue p. 339 Cryo–electron microscopy reveals mechanisms of centromeric nucleosome recognition and initial assembly steps of the kinetochore complex. Accurate chromosome segregation requires the proper assembly of kinetochore proteins. A key step in this process is the recognition of the histone H3 variant CENP-A in the centromeric nucleosome by the kinetochore protein CENP-N. We report cryo–electron microscopy (cryo-EM), biophysical, biochemical, and cell biological studies of the interaction between the CENP-A nucleosome and CENP-N. We show that human CENP-N confers binding specificity through interactions with the L1 loop of CENP-A, stabilized by electrostatic interactions with the nucleosomal DNA. Mutational analyses demonstrate analogous interactions in Xenopus , which are further supported by residue-swapping experiments involving the L1 loop of CENP-A. Our results are consistent with the coevolution of CENP-N and CENP-A and establish the structural basis for recognition of the CENP-A nucleosome to enable kinetochore assembly and centromeric chromatin organization.

Linker H1 histones facilitate formation of higher-order chromatin structures and play important roles in various cell functions. Despite several decades of effort, the structural basis of how H1 interacts with the nucleosome remains elusive. Here, we investigated Drosophila H1 in complex with the nucleosome, using solution nuclear magnetic resonance spectroscopy and other biophysical methods. We found that the globular domain of H1 bridges the nucleosome core and one 10-base pair linker DNA asymmetrically, with its α3 helix facing the nucleosomal DNA near the dyad axis. Two short regions in the C-terminal tail of H1 and the C-terminal tail of one of the two H2A histones are also involved in the formation of the H1–nucleosome complex. Our results lead to a residue-specific structural model for the globular domain of the Drosophila H1 in complex with the nucleosome, which is different from all previous experiment-based models and has implications for chromatin dynamics in vivo.

Chromosome segregation during mitosis requires assembly of the kinetochore complex at the centromere. Kinetochore assembly depends on specific recognition of the histone variant CENP-A in the centromeric nucleosome by centromere protein C (CENP-C). We have defined the determinants of this recognition mechanism and discovered that CENP-C binds a hydrophobic region in the CENP-A tail and docks onto the acidic patch of histone H2A and H2B. We further found that the more broadly conserved CENP-C motif uses the same mechanism for CENP-A nucleosome recognition. Our findings reveal a conserved mechanism for protein recruitment to centromeres and a histone recognition mode whereby a disordered peptide binds the histone tail through hydrophobic interactions facilitated by nucleosome docking.

Chromatin structure and function are regulated by numerous proteins through specific binding to nucleosomes. The structural basis of many of these interactions is unknown, as in the case of the high mobility group nucleosomal (HMGN) protein family that regulates various chromatin functions, including transcription. Here, we report the architecture of the HMGN2-nucleosome complex determined by a combination of methyl-transverse relaxation optimized nuclear magnetic resonance spectroscopy (methyl-TROSY) and mutational analysis. We found that HMGN2 binds to both the acidic patch in the H2A-H2B dimer and to nucleosomal DNA near the entry/exit point, \"stapling\" the histone core and the DNA. These results provide insight into how HMGNs regulate chromatin structure through interfering with the binding of linker histone H1 to the nucleosome as well as a structural basis of how phosphorylation induces dissociation of HMGNs from chromatin during mitosis. Importantly, our approach is generally applicable to the study of nucleosome-binding interactions in chromatin.

The crystal structure of Drosophila melanogaster YL1 in complex with an H2A.Z–H2B dimer exposes a selective recognition mechanism distinct from those of other H2A.Z chaperones and suggests a hierarchical transfer mechanism mediating H2A.Z deposition. Histone variant H2A.Z, a universal mark of dynamic nucleosomes flanking gene promoters and enhancers, is incorporated into chromatin by SRCAP (SWR1), an ATP-dependent, multicomponent chromatin-remodeling complex. The YL1 (Swc2) subunit of SRCAP (SWR1) plays an essential role in H2A.Z recognition, but how it achieves this has been unclear. Here, we report the crystal structure of the H2A.Z-binding domain of Drosophila melanogaster YL1 (dYL1-Z) in complex with an H2A.Z–H2B dimer at 1.9-Å resolution. The dYL1-Z domain adopts a new whip-like structure that wraps over H2A.Z–H2B, and preferential recognition is largely conferred by three residues in loop 2, the hyperacidic patch and the extended αC helix of H2A.Z. Importantly, this domain is essential for deposition of budding yeast H2A.Z in vivo and SRCAP (SWR1)-catalyzed histone H2A.Z replacement in vitro . Our studies distinguish YL1-Z from known H2A.Z chaperones and suggest a hierarchical mechanism based on increasing binding affinity facilitating H2A.Z transfer from SRCAP (SWR1) to the nucleosome.

Histone recognition on the centromere Centromeres, regions on the chromosome that are essential for accurate chromosome segregation, contain unique chromatin that is marked by a histone H3 variant termed CenH3 or CENP-A. The simple centromeres of budding yeast provide an attractive system for investigating centromere biology, including the pathway of CenH3 deposition and the architecture of the centromeric nucleosome. The chaperone Scm3 is required in budding yeast for the deposition of CenH3 (called Cse4) at centromeres. Zhou et al . present the nuclear magnetic resonance structure of Cse4 and histone H4 complexed with Scm3, and outline the structural basis for the recognition of Cse4 by Scm3. They propose a model for Scm3 function as a chaperone that has implications for the assembly of centromeric nucleosomes. The centromere is a unique chromosomal locus that ensures accurate segregation of chromosomes during cell division by directing the assembly of a multiprotein complex, the kinetochore 1 . The centromere is marked by a conserved variant of conventional histone H3 termed CenH3 or CENP-A (ref. 2 ). A conserved motif of CenH3, the CATD, defined by loop 1 and helix 2 of the histone fold, is necessary and sufficient for specifying centromere functions of CenH3 (refs 3 , 4 ). The structural basis of this specification is of particular interest. Yeast Scm3 and human HJURP are conserved non-histone proteins that interact physically with the (CenH3–H4) 2 heterotetramer and are required for the deposition of CenH3 at centromeres in vivo 5 , 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 . Here we have elucidated the structural basis for recognition of budding yeast ( Saccharomyces cerevisiae ) CenH3 (called Cse4) by Scm3. We solved the structure of the Cse4-binding domain (CBD) of Scm3 in complex with Cse4 and H4 in a single chain model. An α-helix and an irregular loop at the conserved amino terminus and a shorter α-helix at the carboxy terminus of Scm3(CBD) wraps around the Cse4–H4 dimer. Four Cse4-specific residues in the N-terminal region of helix 2 are sufficient for specific recognition by conserved and functionally important residues in the N-terminal helix of Scm3 through formation of a hydrophobic cluster. Scm3(CBD) induces major conformational changes and sterically occludes DNA-binding sites in the structure of Cse4 and H4. These findings have implications for the assembly and architecture of the centromeric nucleosome.

Using native-state hydrogen-exchange-directed protein engineering and multidimensional NMR, we determined the high-resolution structure (rms deviation, 1.1 Å) for an intermediate of the four-helix bundle protein: Rd-apocytochrome b562. The intermediate has the N-terminal helix and a part of the C-terminal helix unfolded. In earlier studies, we also solved the structures of two other folding intermediates for the same protein: one with the N-terminal helix alone unfolded and the other with a reorganized hydrophobic core. Together, these structures provide a description of a protein folding pathway with multiple intermediates at atomic resolution. The two general features for the intermediates are (i) native-like backbone topology and (ii) nonnative side-chain interactions. These results have implications for important issues in protein folding studies, including large-scale conformation search, φ-value analysis, and computer simulations.

Werner syndrome (WS) is an autosomal recessive disease that results in premature aging. Mutations in the WS gene (WRN) result in a loss of expression of the WRN protein and predispose WS patients to accelerated aging. As a helicase and a nuclease, WRN is unique among the five human RecQ helicase family members and is capable of multiple functions involved in DNA replication, repair, recombination, and telomere maintenance. A 144-residue fragment of WRN was previously determined to be a multifunctional DNA- and protein-binding domain (DPBD) that interacts with structure-specific DNA and a variety of DNA-processing proteins. In addition, DPBD functions as a nucleolar targeting sequence of WRN. The solution structure of the DPBD, the first of a WRN fragment, has been solved by NMR. DPBD consists of a winged helix-like motif and an unstructured C-terminal region of ≈20 aa. The putative DNA-binding surface of DPBD has been identified by using known structural and biochemical data. Based on the structural data and on the biochemical data, we suggest a surface on the DPBD for interacting with other proteins. In this structural model, a single winged helix domain binds to both DNA and other proteins. Furthermore, we propose that DPBD functions as a regulatory domain to regulate the enzymatic activity of WRN and to direct cellular localization of WRN through protein-protein interaction.

The NMR structure of the H2A.Z-H2B histone chaperone, Chz1, reveals electrostatic interactions between Chz1 and the histone pair via a long, irregular chain with two capping helices, and, based on a model, the possibility that Chz1 has a more active role in histone replacement is suggested. The NMR structure of budding yeast chaperone Chz1 complexed with histones H2A.Z-H2B has been determined. Chz1 forms a long irregular chain capped by two short α-helices, and uses both positively and negatively charged residues to stabilize the histone dimer. A molecular model that docks Chz1 onto the nucleosome has implications for its potential functions.