Chemoreceptor arrays are supramolecular transmembrane machines of unknown structure that allow bacteria to sense their surroundings and respond by chemotaxis. We have combined X-ray crystallography of purified proteins with electron cryotomography of native arrays inside cells to reveal the arrangement of the component transmembrane receptors, histidine kinases (CheA) and CheW coupling proteins. Trimers of receptor dimers lie at the vertices of a hexagonal lattice in a "two-facing-two" configuration surrounding a ring of alternating CheA regulatory domains (P5) and CheW couplers. Whereas the CheA kinase domains (P4) project downward below the ring, the CheA dimerization domains (P3) link neighboring rings to form an extended, stable array. This highly interconnected protein architecture underlies the remarkable sensitivity and cooperative nature of transmembrane signaling in bacterial chemotaxis.protein structure | hybrid methods | two-component systems C hemotactic bacteria sense their surrounding conditions through an array of transmembrane chemoreceptors (methylaccepting chemotaxis proteins, or MCPs), which are found with histidine kinases (CheA) and couplers (CheW) in polar clusters (1-3) and along the sides of cells (4, 5). Repellents and attractants bind to the periplasmic domains of the MCPs either directly (6, 7) or via periplasmic binding proteins (8). The status of the binding domain is transmitted along the length of the receptors through the transmembrane region, across one or more HAMP (histidine kinases, adenyl cyclases, MCPs, and some phosphatases) domain (s), and down the coiled-coil cytoplasmic signaling domain where they ultimately regulate the activity of the histidine kinase CheA located at the receptors' cytoplasmic tips (1-3, 9). CheA is a large, five-domain (P1-P5) protein. P1 contains the substrate histidine, P2 is the docking site for the response regulator CheY, P3 is the dimerization domain, P4 binds ATP and is the kinase, and P5 binds CheW. P1, P2, and P3 are connected to each other by flexible linkers (1, 2). Crystal structures of all domains from Thermotoga maritima CheA are already available (10-13).In the model system Escherichia coli, the addition of attractants or removal of repellents results in kinase inactivation, causing the flagella to rotate counterclockwise. In that case, the multiple flagella form one large bundle that propels the cells smoothly forward and the cells "run." In contrast, addition of repellents or removal of attractants activates CheA, which autophosphorylates and then transfers the phosphoryl group to the second messenger CheY, which in turn binds to the flagellar motors and changes the direction of flagellar rotation to clockwise (CW). This switch results in disassembly of the flagellar bundle and causes the cells to "tumble" (14). CheA also regulates the activity of the receptor-modifying enzyme CheB (a methylesterase), which together with CheR (a methyltransferase) controls the methylation state of residues in the MCP adaptation region (1). Methylation...

Neutrophils activated by the formyl peptide f-Met-Leu-Phe transiently accumulate a small subset of highly polar inositol lipids. A similar family of lipids also appear in many other cells in response to a range of growth factors and activated oncogenes, and are presumed to be the direct or indirect products of 3-phosphatidylinositol kinase. The structures of these lipids are shown to be phosphatidylinositol 3-phosphate, phosphatidylinositol-(3,4)bisphosphate and phosphatidylinositol-(3,4,5)trisphosphate, and we present evidence that in intact neutrophils a phosphatidyl-inositol-(4,5)bisphosphate-3-kinase seems to be the focal point through which agonists stimulate the formation of 3-phosphorylated inositol lipids.

Most nucleosomes that package eukaryotic DNA are assembled during DNA replication, but chromatin structure is routinely disrupted in active regions of the genome. Replication-independent nucleosome replacement using the H3.3 histone variant efficiently repackages these regions, but how histones are recruited to these sites is unknown. Here, we use an inducible system that produces nucleosome-depleted chromatin at the Hsp70 genes in Drosophila to define steps in the mechanism of nucleosome replacement. We find that the Xnp chromatin remodeler and the Hira histone chaperone independently bind nucleosome-depleted chromatin. Surprisingly, these two factors are only displaced when new nucleosomes are assembled. H3.3 deposition assays reveal that Xnp and Hira are required for efficient nucleosome replacement, and double-mutants are lethal. We propose that Xnp and Hira recognize exposed DNA and serve as a binding platform for the efficient recruitment of H3.3 predeposition complexes to chromatin gaps. These results uncover the mechanisms by which eukaryotic cells actively prevent the exposure of DNA in the nucleus.nucleosome assembly | transcription D NA in the eukaryotic nucleus is associated with histone proteins to form nucleosomes, the fundamental units of chromatin. Most nucleosomes are assembled during DNA replication, but chromatin structure is routinely disrupted in active regions of the genome. These regions are repackaged by replication-independent (RI) nucleosome replacement using the H3.3 histone variant (1, 2). This process results in the enrichment of the H3.3 histone variant at all sites where nucleosomes are unstable or disrupted (3, 4). How H3.3 is delivered to dynamic chromatin sites is unknown. However, biochemical isolation of predeposition complexes has identified shared and distinctive assembly factors that associate with the H3 and H3.3 histones and mediate the replicationcoupled or RI assembly of nucleosomes, respectively (4-6). These factors include histone chaperones and chromatin remodelers that are important for new nucleosome assembly, and might potentially target histones to active chromatin regions. However, mutants in some of these factors have surprisingly limited phenotypes. The Hira chaperone promotes H3.3 deposition at genes (4) but is only essential for H3.3 deposition on sperm chromatin during fertilization (7,8). In Drosophila the ATRX/XNP remodeler homolog Xnp colocalizes with H3.3 in somatic cells, but is not essential (9). In mammals, ATRX/XNP promotes H3.3 deposition only at telomeres and some heterochromatic sequences (4, 6, 10). These results have raised the possibilities that H3.3 assembly factors are redundant or that additional factors involved in the deposition of this histone variant exist. Loss of H3.3 itself can be compensated in somatic cells by the major H3 histone, suggesting that assembly of any nucleosome suffices (11).In this work we use an inducible system that produces nucleosome-depleted chromatin at the Hsp70 genes in Drosophila to study the mechanism of nuc...

Background: Major histocompatibility complex class II molecules are structurally and functionally heterogeneous. Results: Combined mutagenesis and structural studies establish a role for pairing between conserved transmembrane (TM) GXXXG dimerization motifs in determining class II conformation. Conclusion: Differential pairing of highly conserved TM domain dimerization motifs contributes to class II structure and function. Significance: Global conformation contributes to the function of peptide-class II complexes.

Polycomb-mediated chromatin repression modulates gene expression during development in metazoans. Binding of multiple sequence-specific factors at discrete Polycomb response elements (PREs) is thought to recruit repressive complexes that spread across an extended chromatin domain. To dissect the structure of PREs, we applied high-resolution mapping of nonhistone chromatin proteins in native chromatin of Drosophila cells. Analysis of occupied sites reveal interactions between transcription factors that stabilize Polycomb anchoring to DNA, and implicate the general transcription factor ADF1 as a novel PRE component. By comparing two Drosophila cell lines with differential chromatin states, we provide evidence that repression is accomplished by enhanced Polycomb recruitment both to PREs and to target promoters of repressed genes. These results suggest that the stability of multifactor complexes at promoters and regulatory elements is a crucial aspect of developmentally regulated gene expression.