Computational protocol: Two Structural Motifs within Canonical EF-Hand Calcium-Binding Domains Identify Five Different Classes of Calcium Buffers and Sensors

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Protocol publication

[…] Currently, there are eleven known calcium binding consensus motifs , whose sequence-variation profiles are established within the PROSITE database . The EF-hand motif is currently found in more than 71 non-redundant representative domains (see Homologous Superfamily EF-hand (1.10.238.10), the CATH database ). We have extracted and analyzed more than 280 (all) crystal structures and 130 NMR structures currently listed in the Protein Data Bank (PDB) that contain one or several domains with the “EF hand-like” fold belonging to the “EF-hand” structural superfamily, according to the SCOP database (SCOP; Fold: EF hand-like; Superfamily: EF-hand) .From 410 available X-ray and NMR structures of EF-hand domains, we had to create three different datasets: (1) a representative non-redundant dataset of different EF-hand domains to scan for structural motifs, i.e. clusters of amino acids and interactions, which are conserved across different structural families of EF-hand proteins; (2) a dataset to study effects of calcium binding on the domain conformation of double calcium binding EF-hand motifs, which would include all known pairs of EF-hand domains, whose three-dimensional structures are known with and without two bound calcium atoms; and (3) a similar dataset to study calcium binding effects on single calcium binding EF-hand motifs. For the first task of searching for conserved amino acid clusters and interactions, we had to manually choose a set of best resolution representative structures from the 11 different structural families of EF-hand proteins, given in SCOP . Where a family contained only NMR structures, a most represented and complete NMR structure was chosen (fold families 7 and 11, ). The search for structural motifs and creation of the first dataset involved a semi-manual pairwise and multiple global and local structural comparisons of the 11 representative structures using the Accelrys Discovery Studio molecular modeling environment (www.accelrys.com). Because of semi-manual all-against-all structural comparisons and analysis of local structural similarities, we stayed at the structural family level (11 structures), and did not go below, to the level of individual protein domains (51 structures). Beyond the 51 structures of individual protein domains, the rest of the 410 structures included either orthologs of the same proteins from the different species or structures of various mutants, which could be discarded from analysis. For the second and third tasks to study effects of calcium binding on the domain conformation, the creation of two different datasets was straightforward. We manually analyzed all 410 structures of EF-hand domains and the corresponding literature to select all double and single calcium binding EF-hand domains, whose three-dimensional structures are known with and without bound calcium and possibly other ligands.All types of structural superpositions and RMSD calculations based on back-bone and all atoms other than hydrogen, including (1) the superposition of the entire Odd and Even EF-hand motifs of EF-hand containing proteins; and (2) local structural superpositions of clusters I and II, were done using the SuperPose superposition server and the Accelrys Discovery Studio molecular modeling environment (www.accelrys.com).Calculation of atomic contacts and interacting surface areas was done using the Contacts of Structural Units (CSU) software, which is based on the surface complementarity approach developed by Sobolev et al. . The “Detailed Analysis” procedure within the CSU software was used to calculate bond and surface parameters for all amino acids of the clusters individually.Geometric parameters to assign CH-π interactions were chosen to satisfy criteria given in Brandl et al. . If X designates the center of an aromatic ring, then the C-X distance must be ≤4.5 Å; the C-H-X angle must be greater than 120°; and the dHp-X projection distance must be ≤1.2 Å . We used an additional criteria, d(H-X) ≤3.5 Å, to ensure that the CH group points directly to the center of the π-ring (Table S2 in ).The criteria to assign weak hydrogen bonds were taken from Derewenda et al. . Firstly, the C-H-O angle ζ must be greater than 90°. Secondly, an electronegative atom must be located adjacent to the carbon atom, such that the acidity of hydrogen atoms attached to the carbon atom increases, and consequently, the carbon atom could be a hydrogen bond donor. Thirdly, the C-O distance must be ≤4.0 Å and the H-O distance must be ≤3.0 Å. Two distance criteria d(N-O) ≤3.7 Å and d(H-O) ≤2.7 Å were used for the conventional hydrogen NH-O bonds; angular criteria, as described above, were also imposed.All geometrical calculations (i.e., angles, torsion angles and distances) were made using the Accelrys Discovery Studio molecular modeling environment. Color figures in this manuscript were produced with MOLSCRIPT and Raster3D . […]

Pipeline specifications

Software tools SuperPose, MolScript, Raster3D
Databases PROSITE
Applications Drug design, Protein structure analysis
Diseases Neoplasms
Chemicals Calcium