The PHD and chromo domains regulate the ATPase activity of the human chromatin remodeler CHD4.

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-2ß) 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.

Solution structure and molecular interactions of lamin B receptor Tudor domain.

Lamin B receptor (LBR) is a polytopic protein of the nuclear envelope thought to connect the inner nuclear membrane with the underlying nuclear lamina and peripheral heterochromatin. To better understand the function of this protein, we have examined in detail its nucleoplasmic region, which is predicted to harbor a Tudor domain (LBR-TD). Structural analysis by multidimensional NMR spectroscopy establishes that LBR-TD indeed adopts a classical ß-barrel Tudor fold in solution, which, however, features an incomplete aromatic cage. Removal of LBR-TD renders LBR more mobile at the plane of the nuclear envelope, but the isolated module does not bind to nuclear lamins, heterochromatin proteins (MeCP2), and nucleosomes, nor does it associate with methylated Arg/Lys residues through its aromatic cage. Instead, LBR-TD exhibits tight and stoichiometric binding to the "histone-fold" region of unassembled, free histone H3, suggesting an interesting role in histone assembly. Consistent with such a role, robust binding to native nucleosomes is observed when LBR-TD is extended toward its carboxyl terminus, to include an area rich in Ser-Arg residues. The Ser-Arg region, alone or in combination with LBR-TD, binds both unassembled and assembled H3/H4 histones, suggesting that the TD/RS interface may operate as a "histone chaperone-like platform."

1H, 13C, and 15N assignment of the muscular LIM protein MLP/CRP3.

The family of CRP proteins comprises three members, which are composed of two LIM domains separated by a long linker of more than 50 residues. We determined the structure of the muscle variant, MLP (CRP3), by nuclear magnetic resonance and show that the two LIM domains are independent of each other.

The Ig doublet Z1Z2: a model system for the hybrid analysis of conformational dynamics in Ig tandems from titin.

Titin is a gigantic elastic filament that determines sarcomere ultrastructure and stretch response in vertebrate muscle. It folds into numerous Ig and FnIII domains connected in tandem. Data on interdomain arrangements and dynamics are essential for understanding the function of this filament. Here, we report a mechanistic analysis of the conformational dynamics of two Ig domains from the N terminus of titin, Z1Z2, by using X-ray crystallography, SAXS, NMR relaxation data, and residual dipolar couplings in combination. Z1Z2 preferentially adopts semiextended conformations in solution, with close-hinge arrangements representing low-probability states. Although interdomain contacts are not observed, the linker appears to acquire moderate rigidity via small, local hydrophobic interactions. Thus, Z1Z2 constitutes an adaptable modular system with restricted dynamics. We speculate that its preexistent conformation contributes to the selective recruitment of the binding partner telethonin onto the repetitive surface of the filament. The structural interconversion of four Z1Z2 conformers is analyzed.

Structure and phosphatidylinositol-(3,4)-bisphosphate binding of the C-terminal PH domain of human pleckstrin.

Pleckstrin is the major target of protein kinase C (PKC) in blood platelets. Its phosphorylation triggers responses that ultimately lead to platelet activation and blood clot formation. Pleckstrin consists of three domains: a pleckstrin homology (PH) domain at both termini and a central DEP (Dishevelled, Egl-1, Pleckstrin) domain. Here, we report the solution nuclear magnetic resonance (NMR) structure of the C-terminal PH domain (C-PH) of human pleckstrin-1. We show that this PH domain binds phosphatidylinositol-3,4-bisphosphate (PtdIns(3,4)P2) with high specificity in protein lipid overlay assays. Using NMR titration experiments and mutational analysis, residues involved in binding to PtdIns(3,4)P2 are identified. The binding site is formed by a patch of basic residues from the beta1 and beta2 strands and the beta1-beta2 loop. Since PtdIns(3,4)P2 is an important signaling molecule in platelets, our data suggest a C-PH dependent regulation of pleckstrin function in response to PtdIns(3,4)P2.