Plant-specific HDT family histone deacetylases are nucleoplasmins.

Histone acetyltransferase (HAT)- and histone deacetylase (HDAC)-mediated histone acetylation and deacetylation regulate nucleosome dynamics and gene expression. HDACs are classified into different families, with HD-tuins or HDTs being specific to plants. HDTs show some sequence similarity to nucleoplasmins, the histone chaperones that aid in binding, storing, and loading H2A/H2B dimers to assemble nucleosomes. Here, we solved the crystal structure of the N-terminal domain (NTD) of all four HDTs (HDT1, HDT2, HDT3, and HDT4) from Arabidopsis (Arabidopsis thaliana). The NTDs form a nucleoplasmin fold, exist as pentamers in solution, and are resistant to protease treatment, high temperature, salt, and urea conditions. Structurally, HDTs do not form a decamer, unlike certain classical nucleoplasmins. The HDT-NTD requires an additional A2 acidic tract C-terminal to the nucleoplasmin domain for interaction with histone H3/H4 and H2A/H2B oligomers. We also report the in-solution structures of HDT2 pentamers in complex with histone oligomers. Our study provides a detailed structural and in vitro functional characterization of HDTs, revealing them to be nucleoplasmin family histone chaperones. The experimental confirmation that HDTs are nucleoplasmins may spark new interest in this enigmatic family of proteins.

AtFKBP53: a chimeric histone chaperone with functional nucleoplasmin and PPIase domains.

FKBP53 is one of the seven multi-domain FK506-binding proteins present in Arabidopsis thaliana, and it is known to get targeted to the nucleus. It has a conserved PPIase domain at the C-terminus and a highly charged N-terminal stretch, which has been reported to bind to histone H3 and perform the function of a histone chaperone. To better understand the molecular details of this PPIase with histone chaperoning activity, we have solved the crystal structures of its terminal domains and functionally characterized them. The C-terminal domain showed strong PPIase activity, no role in histone chaperoning and revealed a monomeric five-beta palm-like fold that wrapped over a helix, typical of an FK506-binding domain. The N-terminal domain had a pentameric nucleoplasmin-fold; making this the first report of a plant nucleoplasmin structure. Further characterization revealed the N-terminal nucleoplasmin domain to interact with H2A/H2B and H3/H4 histone oligomers, individually, as well as simultaneously, suggesting two different binding sites for H2A/H2B and H3/H4. The pentameric domain assists nucleosome assembly and forms a discrete complex with pre-formed nucleosomes; wherein two pentamers bind to a nucleosome.

Structural insights into the ability of nucleoplasmin to assemble and chaperone histone octamers for DNA deposition.

Nucleoplasmin (NP) is a pentameric histone chaperone that regulates the condensation state of chromatin in different cellular processes. We focus here on the interaction of NP with the histone octamer, showing that NP could bind sequentially the histone components to assemble an octamer-like particle, and crosslinked octamers with high affinity. The three-dimensional reconstruction of the NP/octamer complex generated by single-particle cryoelectron microscopy, revealed that several intrinsically disordered tail domains of two NP pentamers, facing each other through their distal face, encage the histone octamer in a nucleosome-like conformation and prevent its dissociation. Formation of this complex depended on post-translational modification and exposure of the acidic tract at the tail domain of NP. Finally, NP was capable of transferring the histone octamers to DNA in vitro, assembling nucleosomes. This activity may have biological relevance for processes in which the histone octamer must be rapidly removed from or deposited onto the DNA.

Oligomerization of Drosophila Nucleoplasmin-Like Protein is required for its centromere localization.

The evolutionarily conserved nucleoplasmin family of histone chaperones has two paralogues in Drosophila, named Nucleoplasmin-Like Protein (NLP) and Nucleophosmin (NPH). NLP localizes to the centromere, yet molecular underpinnings of this localization are unknown. Moreover, similar to homologues in other organisms, NLP forms a pentamer in vitro, but the biological significance of its oligomerization has not been explored. Here, we characterize the oligomers formed by NLP and NPH in vivo and find that oligomerization of NLP is required for its localization at the centromere. We can further show that oligomerization-deficient NLP is unable to bind the centromeric protein Hybrid Male Rescue (HMR), which in turn is required for targeting the NLP oligomer to the centromere. Finally, using super-resolution microscopy we find that NLP and HMR largely co-localize in domains that are immediately adjacent to, yet distinct from centromere domains defined by the centromeric histone dCENP-A.

On the link between conformational changes, ligand binding and heat capacity.

BACKGROUND: Conformational changes coupled to ligand binding constitute the structural and energetics basis underlying cooperativity, allostery and, in general, protein regulation. These conformational rearrangements are associated with heat capacity changes. ITC is a unique technique for studying binding interactions because of the simultaneous determination of the binding affinity and enthalpy, and for providing the best estimates of binding heat capacity changes. SCOPE OF REVIEW: Still controversial issues in ligand binding are the discrimination between the "conformational selection model" and the "induced fit model", and whether or not conformational changes lead to temperature dependent apparent binding heat capacities. The assessment of conformational changes associated with ligand binding by ITC is discussed. In addition, the "conformational selection" and "induced fit" models are reconciled, and discussed within the context of intrinsically (partially) unstructured proteins. MAJOR CONCLUSIONS: Conformational equilibrium is a major contribution to binding heat capacity changes. A simple model may explain both conformational selection and induced fit scenarios. A temperature-independent binding heat capacity does not necessarily indicate absence of conformational changes upon ligand binding. ITC provides information on the energetics of conformational changes associated with ligand binding (and other possible additional coupled equilibria). GENERAL SIGNIFICANCE: Preferential ligand binding to certain protein states leads to an equilibrium shift that is reflected in the coupling between ligand binding and additional equilibria. This represents the structural/energetic basis of the widespread dependence of ligand binding parameters on temperature, as well as pH, ionic strength and the concentration of other chemical species.

The pentameric nucleoplasmin fold is present in Drosophila FKBP39 and a large number of chromatin-related proteins.

Nucleoplasmin is a histone chaperone that consists of a pentameric N-terminal domain and an unstructured C-terminal tail. The pentameric core domain, a doughnut-like structure with a central pore, is only found in the nucleoplasmin family. Here, we report the first structure of a nucleoplasmin-like domain (NPL) from the unrelated Drosophila protein, FKBP39, and we present evidence that this protein associates with chromatin. Furthermore, we show that two other chromatin proteins, Arabidopsis thaliana histone deacetylase type 2 (HD2) and Saccharomyces cerevisiae Fpr4, share the NPL fold and form pentamers, or a dimer of pentamers in the case of HD2. Thus, we propose a new family of proteins that share the pentameric nucleoplasmin-like NPL domain and are found in protists, fungi, plants and animals.

The intrinsically disordered distal face of nucleoplasmin recognizes distinct oligomerization states of histones.

The role of Nucleoplasmin (NP) as a H2A-H2B histone chaperone has been extensively characterized. To understand its putative interaction with other histone ligands, we have characterized its ability to bind H3-H4 and histone octamers. We find that the chaperone forms distinct complexes with histones, which differ in the number of molecules that build the assembly and in their spatial distribution. When complexed with H3-H4 tetramers or histone octamers, two NP pentamers form an ellipsoidal particle with the histones located at the center of the assembly, in stark contrast with the NP/H2A-H2B complex that contains up to five histone dimers bound to one chaperone pentamer. This particular assembly relies on the ability of H3-H4 to form tetramers either in solution or as part of the octamer, and it is not observed when a variant of H3 (H3C110E), unable to form stable tetramers, is used instead of the wild-type protein. Our data also suggest that the distal face of the chaperone is involved in the interaction with distinct types of histones, as supported by electron microscopy analysis of the different NP/histone complexes. The use of the same structural region to accommodate all type of histones could favor histone exchange and nucleosome dynamics.

Crystal structure and function of human nucleoplasmin (npm2): a histone chaperone in oocytes and embryos.

Human Npm2 is an ortholog of Xenopus nucleoplasmin (Np), a chaperone that binds histones. We have determined the crystal structure of a truncated Npm2-core at 1.9 Å resolution and show that the N-terminal domains of Npm2 and Np form similar pentamers. This allowed us to model an Npm2 decamer which may be formed by hydrogen bonds between quasi-conserved residues in the interface between two pentamers. Interestingly, the Npm2 pentamer lacks a prototypical A1-acidic tract in each of its subunits. This feature may be responsible for the inability of Npm2-core to bind histones. However, Npm2 with a large acidic tract in its C-terminal tail (Npm2-A2) is able to bind histones and form large complexes. Fluorescence resonance energy transfer experiments and biochemical analysis of loop mutations support the premise that nucleoplasmins form decamers when they bind H2A-H2B dimers and H3-H4 tetramers simultaneously. In the absence of histone tetramers, these chaperones bind H2A-H2B dimers with a single pentamer forming the central hub. When taken together, our data provide insights into the mechanism of histone binding by nucleoplasmins.

The nuclear transport machinery recognizes nucleoplasmin-histone complexes.

The nuclear transport of the chromatin remodeling protein nucleoplasmin and chromatin building histones is mediated by importins. Nucleoplasmin (NP) contains a classical bipartite nuclear localization signal (NLS) that is recognized by the importin α/ß heterodimer, while histones present multiple NLS-like motifs that are recognized by importin ß family members for nuclear targeting. To explore the possibility of a cotransport of histones and their chaperone NP to the nucleus, we have analyzed the assembly of complexes of NP/histones with importins by means of fluorescence anisotropy, centrifugation in sucrose gradients, and isothermal titration calorimetry. Data show that importin α ΔIBB (a truncated form of importin α lacking the autoinhibitory N-terminal domain) and histones (linker, H5, and nucleosomal core, H2AH2B) can simultaneously bind to NP. Analysis of the binding energetics reveals an enthalpy-driven formation of high affinity ternary, NP/Δα/H5 and NP/Δα/H2AH2B, complexes. We find that different amount of importin α molecules can be loaded on NP/histone complexes dependent on the histone type, linker or core, and the amount of bound histones. We further demonstrate that NP/H5 complexes can also incorporate importin α/ß, thus forming quaternary NP/histones/α/ß complexes that might represent a putative coimport pathway for nuclear import of histones and their chaperone protein NP, enhancing the histone import efficiency.

NLP is a novel transcription regulator involved in VSG expression site control in Trypanosoma brucei.

Trypanosoma brucei mono-allelically expresses one of approximately 1500 variant surface glycoprotein (VSG) genes while multiplying in the mammalian bloodstream. The active VSG is transcribed by RNA polymerase I in one of approximately 15 telomeric VSG expression sites (ESs). T. brucei is unusual in controlling gene expression predominantly post-transcriptionally, and how ESs are mono-allelically controlled remains a mystery. Here we identify a novel transcription regulator, which resembles a nucleoplasmin-like protein (NLP) with an AT-hook motif. NLP is key for ES control in bloodstream form T. brucei, as NLP knockdown results in 45- to 65-fold derepression of the silent VSG221 ES. NLP is also involved in repression of transcription in the inactive VSG Basic Copy arrays, minichromosomes and procyclin loci. NLP is shown to be enriched on the 177- and 50-bp simple sequence repeats, the non-transcribed regions around rDNA and procyclin, and both active and silent ESs. Blocking NLP synthesis leads to downregulation of the active ES, indicating that NLP plays a role in regulating appropriate levels of transcription of ESs in both their active and silent state. Discovery of the unusual transcription regulator NLP provides new insight into the factors that are critical for ES control.

Recognition of nucleoplasmin by its nuclear transport receptor importin α/ß: insights into a complete import complex.

Nuclear import of the pentameric histone chaperone nucleoplasmin (NP) is mediated by importin α, which recognizes its nuclear localization sequence (NLS), and importin ß, which interacts with α and is in charge of the translocation of the NP/α/ß complex through the nuclear pore. Herein, we characterize the assembly of a functional transport complex formed by full-length NP with importin α/ß. Isothermal titration calorimetry (ITC) was used to analyze the thermodynamics of the interactions of importin α with ß, α with NP, and the α/ß heterodimer with NP. Our data show that binding of both importin α and α/ß to NP is governed by a favorable enthalpic contribution and that NP can accommodate up to five importin molecules per NP pentamer. Phosphomimicking mutations of NP, which render the protein active in histone chaperoning, do not modulate the interaction with importin. Using small-angle X-ray scattering, we model the α/ß heterodimer, NP/α, and NP/α/ß solution structures, which reveal a glimpse of a complete nuclear import complex with an oligomeric cargo protein. The set of alternative models, equally well fitting the scattering data, yields asymmetric elongated particles that might represent consecutive geometries the complex can adopt when stepping through the nuclear pore.

Nucleoplasmin binds histone H2A-H2B dimers through its distal face.

Nucleoplasmin (NP) is a pentameric chaperone that regulates the condensation state of chromatin extracting specific basic proteins from sperm chromatin and depositing H2A-H2B histone dimers. It has been proposed that histones could bind to either the lateral or distal face of the pentameric structure. Here, we combine different biochemical and biophysical techniques to show that natural, hyperphosphorylated NP can bind five H2A-H2B dimers and that the amount of bound ligand depends on the overall charge (phosphorylation level) of the chaperone. Three-dimensional reconstruction of NP/H2A-H2B complex carried out by electron microscopy reveals that histones interact with the chaperone distal face. Limited proteolysis and mass spectrometry indicate that the interaction results in protection of the histone fold and most of the H2A and H2B C-terminal tails. This structural information can help to understand the function of NP as a histone chaperone.

Genetically encoded photocontrol of protein localization in mammalian cells.

Precise photochemical control of protein function can be achieved through the site-specific introduction of caging groups. Chemical and enzymatic methods, including in vitro translation and chemical ligation, have been used to photocage proteins in vitro. These methods have been extended to allow the introduction of caged proteins into cells by permeabilization or microinjection, but cellular delivery remains challenging. Since lysine residues are key determinants for nuclear localization sequences, the target of key post-translational modifications (including ubiquitination, methylation, and acetylation), and key residues in many important enzyme active sites, we were interested in photocaging lysine to control protein localization, post-translational modification, and enzymatic activity. Photochemical control of these important functions mediated by lysine residues in proteins has not previously been demonstrated in living cells. Here we synthesized 1 and evolved a pyrrolysyl-tRNA synthetase/tRNA pair to genetically encode the incorporation of this amino acid in response to an amber codon in mammalian cells. To exemplify the utility of this amino acid, we caged the nuclear localization sequences (NLSs) of nucleoplasmin and the tumor suppressor p53 in human cells, thus mislocalizing the proteins in the cytosol. We triggered protein nuclear import with a pulse of light, allowing us to directly quantify the kinetics of nuclear import.

NPM3, a member of the nucleophosmin/nucleoplasmin family, enhances activator-dependent transcription.

The chromatin is comprised of repeating subunits that make up the nucleosome which is composed of an octamer of histones: H3, H4, H2A, and H2B. The replication-dependent and -independent nucleosome assembly occurs in an ordered fashion and is aided by cellular proteins such as histone chaperones and chromatin remodelers. Previously, we found that the histone chaperone NPM1 activates transcription from the chromatin template. Here we report that NPM3, a member of the nucleophosmin/nucleoplasmin family, lacks intrinsic histone chaperone activity, inhibits histone assembly activity of NPM1 in vitro, and dramatically enhances transcription in a cellular system.

Acetylation of transition protein 2 (TP2) by KAT3B (p300) alters its DNA condensation property and interaction with putative histone chaperone NPM3.

The hallmark of mammalian spermiogenesis is the dramatic chromatin remodeling process wherein the nucleosomal histones are replaced by the transition proteins TP1, TP2, and TP4. Subsequently these transition proteins are replaced by the protamines P1 and P2. Hyperacetylation of histone H4 is linked to their replacement by transition proteins. Here we report that TP2 is acetylated in vivo as detected by anti-acetylated lysine antibody and mass spectrometric analysis. Further, recombinant TP2 is acetylated in vitro by acetyltransferase KAT3B (p300) more efficiently than by KAT2B (PCAF). In vivo p300 was demonstrated to acetylate TP2. p300 acetylates TP2 in its C-terminal domain, which is highly basic in nature and possesses chromatin-condensing properties. Mass spectrometric analysis showed that p300 acetylates four lysine residues in the C-terminal domain of TP2. Acetylation of TP2 by p300 leads to significant reduction in its DNA condensation property as studied by circular dichroism and atomic force microscopy analysis. TP2 also interacts with a putative histone chaperone, NPM3, wherein expression is elevated in haploid spermatids. Interestingly, acetylation of TP2 impedes its interaction with NPM3. Thus, acetylation of TP2 adds a new dimension to its role in the dynamic reorganization of chromatin during mammalian spermiogenesis.