The crystal structure of Arabidopsis VSP1 reveals the plant class C-like phosphatase structure of the DDDD superfamily of phosphohydrolases

Abstract

Arabidopsis thaliana 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.

Figure 1. Electron map of VSP1.

Figure 2. Overall structure of VSP1 and sequence alignment of homologues.

(A) Dimeric structure of VSP1. (B) Monomeric structure of VSP1. The core domain and cap-like domain are separated by a line. (C) Sequence aligment of VSP1 with other plant VSPs or plant acid phosphatases. Abbreviation: VSPα, soybean VSPα (AAA33937); VSPβ, VSPβ (AAA34020); PSP, pod storage protein of French Bean (BAA23563); soybeanAP, soybean root nodule phosphatase (CAA11075); tomatoAP, tomato acid phosphatase 1 (CAA39370); atAPlike, Acid phosphatase-like protein of Arabidopsis thaliana (AAM14241); VSP1, vegetative storage protein Vsp1 of Arabidopsis thaliana (CAC08252).

Figure 3. Comparison between VSP1 and P4 in active sites and dimer patterns.

(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.

Figure 4. Structural comparison of VSP1 and P4.

(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.