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Computational Analysis of Surface Properties of Ef-Hand Calcium Binding Proteins

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Computational Analysis of Surface Properties of Ef-Hand Calcium Binding Proteins BIOPHYSICS COMPUTATIONAL ANALYSIS OF SURFACE PROPERTIES OF EF-HAND CALCIUM BINDING PROTEINS DANA CRACIUN1, ADRIANA ISVORAN2 1Teacher Training Department, West University of Timisoara, 4 V.Pirvan, 300223 Timisoara, Romania, Email: [email protected] 2Department of Biology-Chemistry, West University of Timisoara, 16 Pestalozzi, 300316 Timisoara, Romania, Email: [email protected] Received August 14, 2013 Within present study we perform a computational analysis of the surface properties of the EF-hand calcium binding proteins (EFCaBPs), both at global and local levels. Among EFCaBPs there are calcium sensors involved in signal transduction processes and exhibiting extended spatial structures and calcium buffering proteins exhibiting compact structures. Structures superposition reflects higher structural similarity between extended forms, the compact ones being more divergent in good correlation with their sequence alignment. Surfaces of extended EFCaBPs present a smaller number of cavities but with larger volumes and areas than compact ones in correlation with their known biological functions. Surface electrostatic potential is higher for extended EFCaBPs, underlying the role of electrostatics repulsions in adopting their spatial structures and also the possible role in binding charged peptides. Key words: calcium binding proteins, surface, electrostatic potential. 1. INTRODUCTION Calcium ions are indispensable for the physiology of living cell being involved in many cellular processes. The key role of calcium ions strongly depends on a large number of proteins able to bind them, so-called calcium binding proteins, CaBPs [1]. The group of CaBPs is wide and heterogeneous. There are membrane intrinsic CaBPs acting as calcium transporters and involved in the control of calcium ions concentration, calcium buffers and calcium-modulated proteins involved in signal-transduction processes [2]. Calcium modulated proteins may be found both in extra- and intracellular environment and from structural point of view they may be divided in two families [3]: CaBPs containing EF-hand motifs and CaBPs lacking EF-hand motifs respectively. Rom. Journ. Phys., Vol. 59, Nos. 3–4, P. 339–345, Bucharest, 2014 340 Dana Craciun, Adriana Isvoran 2 Within this study we fix our attention on the EF-hand calcium binding proteins. The EF-hand motif is a structural domain containing a calcium binding loop twelve residues long flanked on both sides by an alpha-helix also twelve residues long [3].The biggest part of CaBPs enclose two domains, N- and C- terminal, each containing two EF-hand motifs and connected by a linker region [4]. Among EF-hand CaBPs there are calcium sensors involved in signal transduction processes and exhibiting open, extended spatial structures and calcium buffering and calcium transporting proteins exhibiting closed, compact structures [5]. The main structural difference between the two categories of EF-hand CaPBs [4] is displayed by the terminal domains linker region that is structured in a straight helix for the calcium sensors (as it is revealed in figure 1 for rabbit skeletal muscle troponin, black) and is unstructured for calcium buffers and calcium transporting proteins (figure 1 for bovine neurocalcin delta - grey). These structures have been retrieved from the Protein Data Bank [6]. Structural dissimilarity of the two categories of EF-hand CaBPs is reflected in their distinct biological functions. Fig. 1 – Superpostion of two structures of EFCaBPs: the extended form of rabbit skeletal muscle troponin in black (PDB code entry 1TN4) and the compact form of bovine neurocalcine delta in grey (PDB entry code 1REC). Tacking into account the involvement of CaBPs in many diseases and the fact that not all of them have determined spatial structures, it becomes important to predict their spatial organizations and their structural and functional properties, respectively. There are in specific literature a few studies concerning the physicochemical factors determining the extended or compact structure of the EF-hand CaBPs [7–9] revealing the involvement of electrostatics interactions [7–8] and the essential role 3 Computational analysis of surface properties of EF-hand calcium binding proteins 341 of the hydrophilicity of resides linking the domains [9]. Also, the concepts of the fractal geometry have been applied to investigate their global structural properties [10–11] illustrating distinct fractal properties of backbones of extended respective compact structures [10] and distinct scaling properties of radius of gyration and surface area of the two categories of EF-hand CaBPs [11] confirming the dissimilar mechanisms responsible for their global folding. Within present study we perform a computational analysis of the surface properties of the two categories of EF-hand CaBPs both at global and local levels and interpret them in terms of specific biological interactions. 2. METHODS Experimentally determined structures of proteins are archived in the Protein Data Bank [6] that we have used in order to retrieve the structural files of EFCaBPs. There are numerous structures of EFCaBPs deposited in PDB, but, with the exception of human centrin 2 (that has experimentally-determined structure only in the presence of a peptide) we have considered in our study only those structures obtained using X-ray diffraction technique and containing the entire protein and the bound calcium ions where it was the case. These proteins are presented in the Table 1. Table 1 The investigated proteins Protein PDB code Number of amino acids Conformation in structural file Human calmodulin 1CLL 143 Extended Chicken skeletal muscle 4TNC 159 Extended troponin C Rabbit skeletan muscle troponin 1TN4 157 Extended C Paramecium tetraurelia 1OSA 148 Extended calmodulin Potato calmodulin 1RFJ 148 Extended Human calmodulin-like protein 1GGZ 140 Extended Human centrin 2 2GGM 144 Extended Bovine recombinant neurocalcin 1BJF 181 Compact delta Rattus norvegicus calcineurin B 2CT9 195 Compact Bos Taurus recoverin 1REC 191 Compact Amphioxus sarcoplasmic 2SAS 185 Compact calcium-binding protein Nereis diversicolor sarcoplasmic 2SCP 174 Compact calcium-binding protein 342 Dana Craciun, Adriana Isvoran 4 Multiple sequence alignment of the considered proteins has been done using CLUSTALW [12], a free accessible on-line tool. In order to analyze the surface properties of considered proteins we have used a few computational tools: CHIMERA [13] for surface area calculation and structures superposition, PyMol [14] for electrostatic potential computation and CASTp [15] for cavities identification and characterization. The distribution of physico-chemical properties along the protein chain is obtained using free on-line available tool ProtScale [16]. 3. RESULTS AND DISCUSSIONS Sequence alignment of considered proteins reveals high sequence identity between extended EFCaBPs (more than 73%) and a low sequence identity between extended and compact ones (lower than 47%, data not shown), the calcium binding loops sequences being the most conserved in both extended and compact structures. This remark is also confirmed by structures superposition for the considered EFCaBPs. The superposition of extended structures reveals a high structural similarity between them, the structural motif that is almost identical in all these proteins being the domains linker region. For the compact EFCaBPs, the structure superposition indicates a small structural identity. Also, there is low structural identity between extended and compact EFCaBPs. The structural files of considered proteins and their relevant surface properties are presented in Table 2. Table 2 The surface properties of the considered proteins Volume of the Surface of biggest PDB Number Surface electrostatic potential biggest cavity cavity code of cavities [k T/e] (T=300˚K) [Å3] [Å2] b 1CLL 16 1941,4 581,8 -102.38÷102.38 4TNC 17 581,3 241,6 -102.38÷102.38 1TN4 15 489 296,5 -100.44÷100.44 1OSA 17 2135,1 621,2 -97.18÷97.18 1RFJ 14 255,7 180,9 -97.89÷97.89 1GGZ 20 274,7 148,4 -94.72÷94.72 2GGM 18 2258,8 1286,5 -106.19÷106.19 1BJF 24 484,6 323,7 -67.99÷67.99 2CT9 29 466,6 329,1 -80.37÷80.37 1REC 23 306,1 168,4 -80.48÷80.48 2SAS 19 1274,9 801,4 -78.74÷78.74 2SCP 36 259,6 271,7 -76.09÷76.09 5 Computational analysis of surface properties of EF-hand calcium binding proteins 343 Surfaces of extended EFCaBPs present a smaller number of cavities but with larger volumes and areas than compact ones, in good correlation with their known biological functions [1, 3]. Surface electrostatic potential is higher for extended EFCaBPs, underlying the role of electrostatics repulsions in adopting their spatial structures [7] and also their possible role in binding charged peptides. As these proteins contain distinct numbers of amino acids, in order to compare their surface areas we have considered the surface area per amino acid by dividing the total surface area to the amino acid number. Figure 2 shows that surface area per amino acid for extended forms (medium pattern) and compact forms (dense pattern) are distinct. Fig. 2 – Surface area per amino acid for extended forms (medium pattern) and compact forms (dense pattern). There is a statistically difference between the two means, (46.39±7.2 ) Å2 for extended forms and (54.46±6.5) Å2 for compact ones, reflected by t- and one-way ANOVA tests and underlying the distinct degree of density of their tertiary structures. It is already known that extended EFCABPs present a large solvent exposed hydrophobic surface
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