Crystallization and preliminary X-ray diffraction analysis of human phosphate-binding protein

Abstract

Human phosphate-binding protein (HPBP) was serendipitously discovered by crystallization and X-ray crystallography. HPBP belongs to a eukaryotic protein family named DING that is systematically absent from the genomic database. This apoprotein of 38 kDa copurifies with the HDL-associated apoprotein paraoxonase (PON1) and binds inorganic phosphate. HPBP is the first identified transporter capable of binding phosphate ions in human plasma. Thus, it may be regarded as a predictor of phosphate-related diseases such as atherosclerosis. In addition, HPBP may be a potential therapeutic protein for the treatment of such diseases. Here, the purification, detergent-exchange protocol and crystallization conditions that led to the discovery of HPBP are reported. crystallization communications Acta Cryst. (2006). F62, 67-69 Contreras-Martel et al. Human phosphate-binding protein 69

Stabilization of the active form(s) of human paraoxonase by human phosphate-binding protein

“…Unfortunately, this huge heterogeneity is unfavourable for easily studying the function of PON1. For instance, using apparently pure human PON1 in order to solve its three-dimensional structure, we obtained crystals [16] and solved a structure not matching with the amino acid sequence of PON1. This serendipitously discovered novel protein was termed HPBP [17], and it was hypothesized to be a stabilizing partner of PON1 [18].…”

Tandem purification of two HDL-associated partner proteins in human plasma, paraoxonase (PON1) and phosphate binding protein (HPBP) using hydroxyapatite chromatography

“…Unfortunately, the presence of this component systematically hindered detection of HPBP (∼39 kDa). A batch of human PON1 purified according to these criterions led to diffractable crystals of HPBP [11,12]. N-terminal amino acid sequence determination was performed afterwards on crystal supernatant and re-dissolved crystal.…”

“…In order to solve the 3D structure of human PON1, we used apparently pure and homogeneous enzyme preparations purified following the Gan's protocol [9]. Crystals were obtained from such preparations, but unfortunately the solved structure did not match with the amino-acid sequence of PON1 [11,12].…”

Tandem use of X‐ray crystallography and mass spectrometry to obtain ab initio the complete and exact amino acids sequence of HPBP, a human 38‐kDa apolipoprotein

“…In human, different works report DING proteins identification. There are the Genestein‐binding protein from human carcinoma cells,3 the Hirudin‐binding protein from human fibroblasts,6 the human synovial stimulatory protein (SSP) from synovial liquid,14 the Crystal Adhesion Inhibitor (CAI) from human kidney cells,5 and the Human Phosphate Binding Protein (HPBP)15, 16 from human plasma. The Genestein‐binding protein and the Hirudin‐binding protein are not enough characterized and sequenced to conclude whether their biological activities are due to different human DING protein or not.…”