Background: The NuRD complex controls gene expression through altering chromatin structure.Results: The MTA1-RbAp48 structure shows how the RbAp46/p48 histone chaperones are recruited to NuRD.Conclusion: The MTA subunits act as scaffolds for NuRD complex assembly.Significance: The MTA/RbAp48 interaction prevents binding of histone H4, which is crucial for understanding the role of the RbAp46/p48 chaperones in the complex.

We have recently shown that the C-type lectin-like receptor, CLEC-2, is expressed on platelets and that it mediates powerful platelet aggregation by the snake venom toxin rhodocytin. In addition, we have provided indirect evidence for an endogenous ligand for CLEC-2 in renal cells expressing HIV-1. This putative ligand facilitates transmission of HIV through its incorporation into the viral envelope and binding to CLEC-2 on platelets. The aim of the present study was to identify the ligand on these cells which binds to CLEC-2 on platelets. Recombinant CLEC-2 exhibits specific binding to HEK-293T (human embryonic kidney) cells in which the HIV can be grown. Furthermore, HEK-293T cells activate both platelets and CLEC-2-transfected DT-40 B-cells. The transmembrane protein podoplanin was identified on HEK-293T cells and was demonstrated to mediate both binding of HEK-293T cells to CLEC-2 and HEK-293T cell activation of CLEC-2-transfected DT-40 B-cells. Podoplanin is expressed on renal cells (podocytes). Furthermore, a direct interaction between CLEC-2 and podoplanin was confirmed using surface plasmon resonance and was shown to be independent of glycosylation of CLEC-2. The interaction has an affinity of 24.5+/-3.7 microM. The present study identifies podoplanin as a ligand for CLEC-2 on renal cells.

The human C-type lectin-like molecule CLEC-2 is expressed on the surface of platelets and signaling through CLEC-2 causes platelet activation and aggregation. CLEC-2 is a receptor for the platelet-aggregating snake venom protein rhodocytin. It is also a newly identified co-receptor for human immunodeficiency virus type 1 (HIV-1). An endogenous ligand has not yet been identified. We have solved the crystal structure of the extracellular domain of CLEC-2 to 1.6-Å resolution, and identified the key structural features involved in ligand binding. A semi-helical loop region and flanking residues dominate the surface that is available for ligand binding. The precise distribution of hydrophobic and electrostatic features in this loop will determine the nature of any endogenous ligand with which it can interact. Major ligand-induced conformational change in CLEC-2 is unlikely as its overall fold is compact and robust. However, ligand binding could induce a tilt of a 3-10 helical portion of the long loop region. Mutational analysis and surface plasmon resonance binding studies support these observations. This study provides a framework for understanding the effects of rhodocytin venom binding on CLEC-2 and for understanding the nature of likely endogenous ligands and will provide a basis for rational design of drugs to block ligand binding.Platelet activation at sites of vascular injury is critical for primary hemostasis, but can also trigger arterial thrombosis in vascular disease. The C-type lectin-like protein CLEC-2 was recently shown to be expressed on platelets and signaling through CLEC-2 is sufficient to mediate platelet aggregation (1). CLEC-2 was identified by sequence similarity to C-type lectin-like molecules with immune functions, such as the immunoreceptor NKG2D (2). The gene encoding CLEC-2 is located in the human natural killer (NK) 2 complex on chromosome 12, along with the C-type lectin-like receptors NKG2D, LOX-1, and Dectin-1 (3). CLEC-2 is a 32-kDa type II transmembrane receptor, and transcripts have been identified in immune cells of myeloid origin, including monocytes, dendritic cells, and granulocytes, and in liver (2). CLEC-2 has been shown to be a receptor on the surface of platelets for the snake venom toxin rhodocytin, which is produced by the Malayan pit viper Calloselasma rhodostoma (1, 4). An endogenous CLEC-2 ligand has not yet been identified. Binding of rhodocytin to CLEC-2 leads to phosphorylation of a single tyrosine residue in a YXXL motif in the intracellular domain of CLEC-2. This phosphorylation promotes the binding of spleen tyrosine kinase (Syk), further downstream tyrosine phosphorylation events and the activation of PLC␥2 (1). These signaling events result in platelet aggregation.The ability of rhodocytin or CLEC-2-specific antibodies to trigger platelet aggregation in the absence of other stimuli indicates the potency with which CLEC-2 can modulate platelet activity (1). Therefore, CLEC-2 is a potential therapeutic target in thrombotic cardiovascular disease. In addition, snak...

The NuRD (nucleosome remodeling and deacetylase) complex serves as a crucial epigenetic regulator of cell differentiation, proliferation, and hematopoietic development by coupling the deacetylation and demethylation of histones, nucleosome mobilization, and the recruitment of transcription factors. The core nucleosome remodeling function of the mammalian NuRD complex is executed by the helicase-domain-containing ATPase CHD4 (Mi-2β) subunit, which also contains N-terminal plant homeodomain (PHD) and chromo domains. The mode of regulation of chromatin remodeling by CHD4 is not well understood, nor is the role of its PHD and chromo domains. Here, we use small-angle X-ray scattering, nucleosome binding ATPase and remodeling assays, limited proteolysis, cross-linking, and tandem mass spectrometry to propose a three-dimensional structural model describing the overall shape and domain interactions of CHD4 and discuss the relevance of these for regulating the remodeling of chromatin by the NuRD complex.