The Crystal Structure of <em>Arabidopsis</em> VSP1 Reveals the Plant Class C-Like Phosphatase Structure of the DDDD Superfamily of Phosphohydrolases

<div><p><em>Arabidopsis thaliana</em> vegetative storage proteins, VSP1 and VSP2, are acid phosphatases and belong to the haloacid dehalogenase (HAD) superfamily. In addition to their potential nutrient storage function, they were thought to be involved in plant defense and flower development. To gain insights into the architecture of the protein and obtain clues about its function, we have tested their substrate specificity and solved the structure of VSP1. The acid phosphatase activities of these two enzymes require divalent metal such as magnesium ion. Conversely, the activity of these two enzymes is inhibited by vanadate and molybdate, but is resistant to inorganic phosphate. Both VSP1 and VSP2 did not exhibit remarkable activities to any physiological substrates tested. In the current study, we presented the crystal structure of recombinant VSP1 at 1.8 Å resolution via the selenomethionine single-wavelength anomalous diffraction (SAD). Specifically, an α-helical cap domain on the top of the α/β core domain is found to be involved in dimerization. In addition, despite of the low sequence similarity between VSP1 and other HAD enzymes, the core domain of VSP1 containing conserved active site and catalytic machinery displays a classic haloacid dehalogenase fold. Furthermore, we found that VSP1 is distinguished from bacterial class C acid phosphatase P4 by several structural features. To our knowledge, this is the first study to reveal the crystal structure of plant vegetative storage proteins.</p> </div

Electron map of VSP1.

<p>Electron map of VSP1.</p

Statistics of Data Reduction and Structure Refinement.

a<p>Data for the highest resolution bin is in parentheses.</p>b<p><i>R</i>_merge = Σ|Ii−Im|/ΣIi, where Ii is the intensity of the measured reflection and Im is the mean intensity of all symmetry-related reflections.</p>c<p><i>R</i>_work = Σ| |Fobs|−|Fcalc| |/Σ|Fobs|, where Fobs and Fcalc are observed and calculated structure factors, respectively. <i>R</i>_free = Σ_T| |Fobs|−|Fcalc| |/Σ_T|Fobs|, where T denotes a test data set of about 5% of the total reflections randomly chosen and set aside prior to refinement.</p>d<p>RMSD = root-mean-square deviation.</p

Relative activities of VSP1 and VSP2 toward different substrates.

<p>ND, not detectable.</p><p>Relative activities are expressed as the percentage of the activity with <i>p</i>NPP. The results are the average of the values determined in triplicates and the respective standard error is constantly lower than 10%.</p

Structural comparison of VSP1 and P4.

<p>(A) Topology of VSP1. (B) Topology of P4. (C) Superposition of VSP1 and P4 (stereo view). In (A) (B) (C), an additional α helix in P4 (yellow) is marked by a rectangle. Longer N-terminus in VSP1 (cyan) is colored blue, while longer C-terminus in P4 is colored magenta. Structural elements of P4 in (B) are labeled based on the structural alignment with VSP1. (D) NMN binding with P4. NMN is shown in sticks model. Important residues of P4 interacting with NMN are labeled. (E) Superpose VSP1 to P4 while NMN is modeled at the same site as in P4. Corresponding residues in VSP1 are labeled. NMN is shown in sticks model.</p

Kinetic parameters for VSP1 protein with different divalent metal ions.

<p>Data represent the means±SD from three independent assays.</p

Comparison between VSP1 and P4 in active sites and dimer patterns.

<p>(A) Residues in the catalytic site of VSP1. Magnesium ions and water molecules are colored by green and red, respectively. (B) Residues in the catalytic site of P4. Magnesium ion and water molecules are colored by green and red, respectively. (C) Hydrophobic core between two VSP1 monomers. Two VSP1 monomers are colored magenta and cyan, respectively. (D) Hydrogen bonds between two VSP1 monomers. Two VSP1 monomers are colored magenta and cyan, respectively. (E) Interaction pattern of VSP1 dimer. One monomer is coloured magenta, while the N-terminal helices of the other monomer are coloured cyan. (F) Interaction pattern of P4 dimer. One monomer is colored yellow, while the N-terminal helices of the other monomer are colored green. The magenta VSP1 monomer in (E) and the yellow P4 monomer in (F) are aligned.</p

Association rate, dissociation rate, and equilibrium dissociation constants of Trn1 and wild-type and mutant FUS-NLS.

<p>The 12 ALS mutations are organized in the order of decreasing affinity.</p>a<p>The relative affinity is defined as <i>K</i>_D of WT (M) divided by <i>K</i>_D of the FUS-NLS mutants <i>(</i>M<i>).</i></p><p>The correlation coefficient χ² value is a statistical measure of how closely the fitted curve fits the experimental data <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047056#pone.0047056-Imasaki1" target="_blank">[15]</a> (see Methods).</p

The structure of the FUS-NLS/Trn1 complex.

<p>(A) The overall structure. (B) The 2<i>F_o</i> − <i>F_c</i> composite omit electron density map around the FUS-NLS fragment (residues 508–526) contoured at 1.0 σ (gray mesh). The Trn1 and the FUS-NLS are shown in cyan and yellow, respectively. (C) The superimposition of residues 508–526 of FUS-NLS (yellow; PDB code: 4FQ3) with the corresponding regions from hnRNP A1-NLS (blue; PDB code: 2H4M), hnRNP D-NLS (grey; PDB code: 2Z5N), hnRNP M-NLS (magenta; PDB code: 2OT8), and TAP-NLS (cyan; PDB code: 2Z5K). The α-helix is unique in FUS-NLS whereas no specific secondary structure was found in the other structures.</p

Subcellular localization of WT and mutant FUS.

<p>GFP-tagged WT full-length human FUS or different ALS mutants (S513P, G515C, R521G, R522G and P525L) were transfected into N2a cells. The cells were fixed in 4% paraformaldehyde and permeabilized by 0.1% Triton X-100 24 hours after transfection. The nuclei were stained by 4′,6-diamidino-2-phenylindole (DAPI). The coverslips were mounted and images were acquired using an Olympus confocal microscope (Olympus Fluoview, Ver.1.7c).</p