Unfolding Pathway in Red Kidney Bean Acid Phosphatase Is Dependent on Ligand Binding

Anil G Cashikar,Nalam Madhusudhana Rao

doi:10.1074/jbc.271.9.4741

Abstract

Structural basis for ligand-induced protein stabilization was investigated in the case of an acid phosphatase (red kidney bean purple acid phosphatase (KBPAP)) from red kidney bean. Phosphate, a physiological ligand, increases the stability against solvent denaturation by 3.5 kcal/mol. Generality of phosphate stabilization was shown by similar effects with other KBPAP ligands viz. adenosine 5'-O-(thiotriphosphate), a nonhydrolyzable ligand, and arsenate, an inhibitor. The dissociation constant of phosphate obtained from denaturation curves matches with the dissociation constant estimated by conventional methods. The guanidinium chloride-mediated denaturation of KBPAP was monitored by several structural and functional parameters viz. activity, tryptophan fluorescence, 8-anilinonaphthalene 1-sulfonic acid binding, circular dichroism, and size exclusion chromatography, in the presence and absence of 10 mm phosphate. In the presence of phosphate, profiles of all the parameters shift to a higher guanidinium chloride concentration. Noncoincidence of these profiles in the absence of phosphate indicates multistate unfolding pathway for KBPAP; however, in the presence of phosphate, KBPAP unfolds with a single intermediate. Based on the crystal structure, we propose that the Arg258 may have an important role to play in stabilization mediated by phosphate.

Highlights

Acid phosphatase from red kidney beans (KBPAP)1 is a homodimeric glycoprotein with a subunit molecular mass of 55 kDa [12,13,14,15]
KBPAP is a homodimer with an intersubunit disulfide bond at Cys345, and an intact disulfide is necessary for native conformation and activity [13]
In the absence of phosphate, the D1/2 decreased to 1.6 M Guanidinium chloride (GdmCl), while in the presence of phosphate the D1/2 remained unchanged (Fig. 1C and Table I)

Summary

Introduction

Acid phosphatase from red kidney beans (KBPAP) is a homodimeric glycoprotein with a subunit molecular mass of 55 kDa [12,13,14,15]. Based on amino acid sequence alignment, it was shown that the protein has ␤␣␤␣␤ motif and has same amino acids ligating the metal ions as in uteroferrin, its mammalian counterpart [16]. The N-terminal 120-amino acid stretch was absent in uteroferrin and does not have any active site or metal ion-ligating amino acid residues [16]. We observed significant enhancement in the stability of KBPAP toward solvent and heat denaturation in the presence of phosphate. In this communication, we report data on the influence of phosphate binding on the denaturation pathway of KBPAP and demonstrate that phosphate stabilizes KBPAP to denaturation and alters the unfolding pathway

Methods

Results

Conclusion