Ellard K et al. (2016), Expression and purification of the full murine ...

XB-ART-54082

Biochem Biophys Rep 2016 Mar 31;6:165-171. doi: 10.1016/j.bbrep.2016.04.002.

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Expression and purification of the full murine NPM2 and study of its interaction with protamines and histones.

Ellard K , Serpa JJ , Petrotchenko EV , Borchers CH , Ausió J .

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Mouse nucleoplasmin M.NPM2 was recombinantly expressed and the protein consisting of the complete sequence was purified and characterized. Similar to its Xenopus laevis X.NPM2 counterpart, the protein forms stable pentameric complexes and exhibits an almost undistinguishable hydrodynamic ionic strength-dependent unfolding behavior. The interaction of N.PM2 with histones and mouse P1/P2 protamines revealed that these chromosomal proteins bind preferentially to the distal part of the nucleoplasmin pentamer. Moreover, the present work highlights the critical role played by histones H2B and H4 in the association of the histone H2A-H2B dimers and histone octamer with nucleoplasmin.

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Species referenced: Xenopus laevis
Genes referenced: h2ac21 h2bc21 npm1 npm2 npm3

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	Fig. 1. Characterization of the protein interacting partners. (A) Primary structure alignment of mouse NPM2 and NPM3 (M.NPM2/MNPM3) in comparison to Xenopus NPM2 (X.NPM2) and cartoon representation highlighting the position of the three acidic tracts (A1, A2 and A3) and the nuclear localization signal (NLS). The arrow indicates the beta-stranded region. (B) SDS-PAGE analysis of M.NPM2 before (B-) and after boiling (B+) the sample, in comparison to a protein (PM) and a chicken histone marker (CM). M: monomer; P: pentamer. (C) Isolation (1) and purification (2) of the mouse testes sperm nuclear basic proteins (SNBPs) and histone octamers (3). (D) Amino acid sequence of mouse protamines P1 and P2, Reference Sequences: P02319 and NP_032959, respectively.
	Fig. 2. Native PAGE of a titration of histone octamers (A) and mouse protamines P1/P2 (B) with increasing amounts of M.NPM2. ). In this type of analysis, NPM2: histone/protamine complexes display a complex ‘shift’ and are unable to enter the gel. (C) Amino acid sequence of mouse NPM2 and core histones H2A, H2B and H4, with interaction sites determined through CXMS data (Supp. Table 1a) highlighted in red. Three peptide sequences in NPM2 represent strong cross-linking candidates with sequences in H2A, H2B and H4 (highlighted in red). Gallus gallus H2A NCBI Reference Sequence: AAC60008.1, G. gallus H2B NCBI Reference Sequence: AAC60000.1, G. gallus H4 NCBI Reference Sequence: NP_001032932.1. (D) Amino acid sequence of mouse NPM2, P1 and P2, with possible interaction sites determined through CXMS data (Supp. Table 1a) highlighted in red. Three peptide sequences within M.NPM2 represent possible cross-linking candidates. M. musculus NPM2 NCBI Reference Sequence: NP_851990.2; M. musculus P1 NCBI Reference Sequence: NP_038665.1; M. musculus P2 NCBI Reference Sequence: P07978.1.
	Fig. 3. (A) Sedimentation velocity analysis of X.NPM2 and M.NPM2 at different sodium chloride concentrations. (A) Plots of the relative sample concentration vs. the sedimentation coefficient at three different NaCl concentrations (120, 240, and 480 mM). Data was obtained using the histogram envelope analysis from the UltraScan software [31]. (B) Sodium chloride dependence of the sedimentation coefficient of X.NPM2 (open circles) and M.NPM2 (black circles). S20,w=sedimentation coefficient corrected to standard conditions (water and 20 °C). S=Svedberg units.
	Fig. 4. Schematic representation and 3D structure of mouse NPM2 with highlighted chromosomal protein interaction sites as determined by CXMS. The intensity of the interactions is depicted in red (see Supp. Table 1). (A) Schematic representation of the secondary structure organization of the NPM2 monomer. Acidic tracts are highlighted in black boxes. Note: Acidic tract A1 denotes the region where the A1 tract is seen in other species (but not in mouse) (see Fig.1). Beta barrels β1-β8 are also shown by wide arrows and intra-sheet hydrogen bonds are represented by dotted black lines. (B) Tertiary structure of the M.NPM2 monomer created using PyMOL. (C) Tertiary structure organization of the M.NPM2 pentamer created using PyMOL.

References [+] :

Arnan, Interaction of nucleoplasmin with core histones. 2003, Pubmed, Xenbase