Serendipitous Discovery and X-Ray Structure of a Human Phosphate Binding Apolipoprotein
Total Page：16
File Type：pdf, Size：1020Kb
View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Structure 14, 601–609, March 2006 ª2006 Elsevier Ltd All rights reserved DOI 10.1016/j.str.2005.12.012 Serendipitous Discovery and X-Ray Structure of a Human Phosphate Binding Apolipoprotein Renaud Morales,1 Anne Berna,2 Philippe Carpentier,1 is the only known transporter capable of binding phos- Carlos Contreras-Martel,1 Fre´de´rique Renault,3 phate ions in human plasma and may become a new Murielle Nicodeme,4 Marie-Laure Chesne-Seck,6 predictor of or a potential therapeutic agent for phos- Franc¸ois Bernier,2 Je´roˆ me Dupuy,1 phate-related diseases such as atherosclerosis. Christine Schaeffer,7 He´le`ne Diemer,7 Alain Van-Dorsselaer,7 Juan C. Fontecilla-Camps,1 Introduction Patrick Masson,3 Daniel Rochu,3 and Eric Chabriere3,5,* Both cholesterol and HDL (high-density lipoprotein) 1 Laboratoire de Cristallogene` se et Cristallographie levels are commonly used as risk predictors of cardio- des Prote´ ines vascular disease (Gordon and Rifkind, 1989; Amann Institut de Biologie Structurale JP EBEL et al., 2003). HDL protects against atherosclerosis 38027 Grenoble mainly through the reverse cholesterol transport pro- France cess in which excess cholesterol is transferred from 2 Institut de Biologie Mole´ culaire des Plantes du CNRS peripheral tissues to the liver by the ATP binding cas- Universite´ Louis Pasteur sette ABCA1 transporter, which is defective in Tangier 67083 Strasbourg Cedex disease (Brooks-Wilson et al., 1999). Although HDLs France have been extensively studied, their metabolic role 3 Unite´ d’Enzymologie has not been completely elucidated yet. For instance, De´ partement de Toxicologie while the involvement of the calcium-dependent, HDL- Centre de Recherches du Service de Sante´ des Arme´ es associated paraoxonase 1 (PON1; aryldialkylphospha- 38702 La Tronche tase; EC 3.1.8.1) in atherosclerosis prevention is well France established (Watson et al., 1995; Shih et al., 1998), its 4 Laboratoire des Biosciences de l’Aliment physiological function remains unknown. PON1, named 5 Laboratoire de Cristallographie et Mode´ lisation des for its ability to hydrolyze the insecticide paraoxon, is Mate´ riaux Mine´ raux et Biologiques the subject of intensive research owing to its capacity Universite´ Henri Poincare´ -Nancy 1 to inactivate various organophosphorous compounds, 54506 Vandoeuvre-le` s-Nancy including nerve gases and pesticides, representing both France a terrorist threat and an environmental hazard. Albeit its 6 Laboratoire de Cristallographie Macromole´ culaire native activity is suggested to be lactonase (Khersonsky Institut de Biologie Structurale JP EBEL and Tawfik, 2005), PON1 is currently considered as an 38027 Grenoble enzyme with promiscuous functions. Accordingly, some France of the activities attributed to PON1 are for the moment 7 Laboratoire de Spectrome´ trie de Masse Bio-Organique largely debated. ECPM In preparing PON1 for functional studies, we have iso- 67087 Strasbourg lated another protein that copurifies with it. Here, we re- France port the discovery and X-ray structure of this protein, which is a human plasma solute binding protein (SBP) that binds phosphate and belongs to the sixth family Summary of SBPs (Tam and Saier, 1993). In prokaryotes, SBPs are associated with ABC transporters, a family of ubiqui- We report the serendipitous discovery of a human tous molecules that use ATP as an energy source by plasma phosphate binding protein (HPBP). This 38 which to carry a wide variety of molecules across mem- kDa protein is copurified with the enzyme paraoxo- branes (Higgins, 1992). ABC transporters may also play nase. Its X-ray structure is similar to the prokaryotic a central role in pathologies such as cystic fibrosis and phosphate solute binding proteins (SBPs) associated are involved in resistance to chemotherapeutic drugs with ATP binding cassette transmembrane trans- (Jones and George, 2002). These proteins are com- porters, though phosphate-SBPs have never been posed of two cytoplasmic nucleotide binding domains characterized or predicted from nucleic acid data- and two channel-forming transmembrane domains bases in eukaryotes. However, HPBP belongs to the (Chang and Roth, 2001). In both archaea and bacteria, family of ubiquitous eukaryotic proteins named interactions with extracytoplasmic SBPs largely con- DING, meaning that phosphate-SBPs are also wide- tribute to their efficiency and specificity. Prokaryotic spread in eukaryotes. The systematic absence of com- phosphate-SBPs are 38 kDa proteins, encoded by plete genes for eukaryotic phosphate-SBP from data- PstS genes (Surin et al., 1984), that bind inorganic phos- bases is intriguing, but the astonishing 90% phate ions with submicromolar affinity (Medveczky and sequence conservation between genes belonging to Rosenberg, 1971) and are mostly expressed during evolutionary distant species suggests that the corre- phosphate starvation (Gerdes and Rosenberg, 1974). sponding proteins play an important function. HPBP In gram-negative bacteria, these proteins are soluble and periplasmic, whereas in gram-positive bacteria, they are lipoproteins anchored to the outer face of the *Correspondence: [email protected] membrane (Young and Garbe, 1991). Structure 602 Figure 1. HPBP Structure (A) Ribbon representation of HPBP showing the two domains (blue and green) connected by a hinge (yellow), the phosphate molecule (red balls), and the two disulfide bridges (C113–C158 and C306–C359, orange sticks). The elongated protein structure exhibits two similar globular domains, each constituted by a five-stranded b sheet core (three parallel b strands followed by two antiparallel b strands) flanked by a helices. A hydropathy profile computed with the program DAS (Cserzo et al., 1997) indicates that HPBP is strongly hydrophobic between residues 168 and 194. This motif, located on the protein surface (H.S.), is involved in the packing of the crystallographic dimer. N and C represent the protein termini. (B) Stereoview of the Ca trace of HPBP. The orientation is the same as in (A). Every tenth a-carbon is labeled with its residue number. Rainbow coloring—from the N terminus to the C terminus—has been used. (C) Structural comparison of different known phophate-SBPs: HPBP is shown in blue, E. coli PstS protein is shown in yellow, and M. tuberculosis PstS1 is shown in green. The four HPBP-specific loops are indicated. Interestingly, eukaryotic ABC transporters do not rely mental Data, available with this article online). Due to ini- on SBPs. Proteins related to SBPs nevertheless exist in tial difficulties in obtaining heavy atom derivatives, the eukaryotes (Felder et al., 1999), the more relevant cases protein envelope was initially determined ab initio at being the glutamate receptors, the atrial natriuretic pep- 25 A˚ resolution (Fokine et al., 2003). Eventually, a uranyl tide receptors involved in blood pressure regulation, derivative was used to calculate an electron density map and the calcium-sensing G protein-coupled receptors, at 1.9 A˚ resolution and a polyalanine model was traced located in parathyroid and kidney cells. Anchored to automatically into this map (Figures 1A and 1B) (see Ex- the membrane with an extramembranar domain, they perimental Procedures). As the electron density did not act in a multimeric state as receptors. match the amino acid sequence of PON1, we carried out This article describes the discovery, characterization, structural comparisons with DALI (Holm and Sander, sequencing, and crystal structure at 1.9 A˚ resolution of 1994). This analysis revealed that our three-dimensional HPBP, a novel, to our knowledge, human HDL-associ- model (PDB ID code 2cap) displayed a fold very similar ated apolipoprotein, not predicted by any genomic da- to those of a phosphate binding protein from Escheri- tabase. This protein, structurally and functionally related chia coli (Luecke and Quiocho, 1990) and the PstS1 pro- to bacterial phosphate-SBPs, may be the first phos- tein from Mycobacterium tuberculosis, a major BCG phate transporter characterized in human plasma. A vaccine antigen (Vyas et al., 2003). Based on these ob- possible use of the protein in diagnosis or therapy of servations, the protein was named human phosphate atherosclerosis emerges from its capability of binding binding protein (HPBP). The complete amino acid se- phosphate, as well as a role for stabilizing functionally quence of HPBP was assigned from the electron density active conformation of PON1. map and later confirmed for about 75% of the protein by direct sequencing (Figure 2 and Experimental Proce- Results dures). HPBP consists of 376 residues with a predicted molecular mass of 38.4 kDa. X-Ray Structure of HPBP Superposition of the three-dimensional structures Crystals were obtained from an apparently pure PON1 gave rms deviations for a carbon atom positions be- solution (Contreras-Martel et al., 2006; see the Supple- tween HPBP and E. coli phosphate-SBP (216 atoms) A Human Phosphate Binding Apolipoprotein 603 Figure 2. Sequence Conservation between HPBP and SBPs or DING Proteins Alignment of the amino acid sequence of HPBP deduced from the crystal structure (residues confirmed by direct amino acid sequencing are in bold characters) with various SBPs and DING proteins by using the program ALIGN (Myers and Miller, 1988) Potato, potato DING protein (A.B. et al., unpublished data); L. major, L. major Friedlin almost complete DING protein sequence