Biochemical and structural characterization of a novel arginine kinase from the spider Polybetes pythagoricus

Abstract

Energy buffering systems are key for homeostasis during variations in energy supply. Spiders are the most important predators for insects and therefore key in terrestrial ecosystems. From biomedical interest, spiders are important for their venoms and as a source of potent allergens, such as arginine kinase (AK, EC 2.7.3.3). AK is an enzyme crucial for energy metabolism, keeping the pool of phosphagens in invertebrates, and also an allergen for humans. In this work, we studied AK from the Argentininan spider Polybetes pythagoricus (PpAK), from its complementary DNA to the crystal structure. The PpAK cDNA from muscle was cloned, and it is comprised of 1068 nucleotides that encode a 384-amino acids protein, similar to other invertebrate AKs. The apparent Michaelis-Menten kinetic constant (K_m ) was 1.7 mM with a k_cat of 75 s^-1. Two crystal structures are presented, the apoPvAK and PpAK bound to arginine, both in the open conformation with the active site lid (residues 310-320) completely disordered. The guanidino group binding site in the apo structure appears to be organized to accept the arginine substrate. Finally, these results contribute to knowledge of mechanistic details of the function of arginine kinase.

Keywords: Allergen; Arachnida; Arginine kinase; Arthropoda; Crystal structure; Open conformation; Phosphagen; Polybetes pytagoricus; Spider; cDNA cloning.

Figure 1. Sequence alignment.

Aligment of PpAK of spider P. pythagoricus amino acid sequences with AK of: Stegodyphus mimosarum (KFM68792.1), Limulus polyphemus (NP_001301013.1), Dermatophagoides farinae (AAP57094.1), Penaeus monodon (AGV55412.1), Litopenaeus vannamei (ABY57915.1), Neocaridina denticulata (BAH56609.1), Metapenaeus ensis (ACA51932.1), Dermatophagoides pteronyssinus (ACD50950.1). Arginine and ADP-binding residues in AK from Limulus crystal structure (Zhou et al., 1998) are indicated in yellow, respectively; highly conserved residues D62 and R193 (Suzuki et al., 2000) in green, and guanidino group substrate-specific residues (Edmiston et al., 2001; Tanaka & Suzuki, 2004) in red.

Figure 2. Structural alignment.

Superposition of crystallographic structures of free-ligand (grey) and arginine binary complex (yellow) PpAK. The arginine substrate is shown as spheres colored by atom type (green for carbon, blue for nitrogen and red for oxygen) in the hinge between N-terminal and C-terminal domains.

Figure 3. Arginine binding-site of PpAK.

Arginine substrate (ball-sticks colored by atom type) is stabilized by the guanidino group binding loop (Gly64 and Val65), Cys 271 and Glu225. All amino acids are presented as cylinders colored by atom type (Arg-PpAK) and in dark-grey (apo-PpAK). Hydrogen bonds are shown as a dotted line. (A) Electron density 2Fo-Fc map of arginine is displayed as a blue mesh with a 1.5 σ contour level. (B) Polder omit density map of arginine is displayed as a blue mesh with a 3 σ contour level.