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

<div><p>Proteins with EF-hand calcium-binding motifs are essential for many cellular processes, but are also associated with cancer, autism, cardiac arrhythmias, and Alzheimer's, skeletal muscle and neuronal diseases. Functionally, all EF-hand proteins are divided into two groups: (1) calcium sensors, which function to translate the signal to various responses; and (2) calcium buffers, which control the level of free Ca²⁺ ions in the cytoplasm. The borderline between the two groups is not clear, and many proteins cannot be described as definitive buffers or sensors. Here, we describe two highly-conserved structural motifs found in all known different families of the EF-hand proteins. The two motifs provide a supporting scaffold for the DxDxDG calcium binding loop and contribute to the hydrophobic core of the EF hand domain. The motifs allow more precise identification of calcium buffers and calcium sensors. Based on the characteristics of the two motifs, we could classify individual EF-hand domains into five groups: (1) Open static; (2) Closed static; (3) Local dynamic; (4) Dynamic; and (5) Local static EF-hand domains.</p></div

Effects of calcium binding on the conformation of all EF-hand domains, whose structures are known in the <i>apo</i>-form and with bound Ca²⁺ ions, and target protein ligands (where they exist).

<p>The RMSD values between the <i>apo</i>-form and the protein with bound Ca²⁺ ions and the target ligand, are calculated using the back-bone atoms of the amino acids of the clusters, and separately, using all heavy atoms of the same amino acids. The RMSD data are shown for the superposition of clusters I and II separately (groups A–C), and for superposition of the two clusters, cluster I/cluster II, simultaneously (groups D and E).</p><p>*While groups A–C coincide between <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109287#pone-0109287-t003" target="_blank">Tables 3</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109287#pone-0109287-t004" target="_blank">4</a>, the RMSD calculations (this table) between clusters I and II were made only for the protein structures where the conformation of cluster II does not change upon calcium binding, and thus, proteins from group C were not included in D and E. This was done in order to observe only the inter-cluster conformational change.</p><p>Effects of calcium binding on the conformation of all EF-hand domains, whose structures are known in the <i>apo</i>-form and with bound Ca²⁺ ions, and target protein ligands (where they exist).</p

List of eleven non-redundant, representative calcium-bound X-ray and NMR protein complexes, which represent eleven different families of EF-hand domains.

<p>All of the structures share the same fold (Fold: EF Hand-like) and belong to the same EF-hand fold superfamily (Superfamily: EF-hand) (from SCOP <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109287#pone.0109287-Andreeva1" target="_blank">[48]</a>).</p><p>List of eleven non-redundant, representative calcium-bound X-ray and NMR protein complexes, which represent eleven different families of EF-hand domains.</p

Five distinguishable groups of EF-hand domains based on the degree of structural rearrangements within clusters I and II, and the inter-cluster interactions that take place upon calcium binding: (1) Open Static (open domain conformation and no conformational changes); (2) Closed Static (closed domain conformation and no conformational changes); (3) Local Dynamic (simultaneous conformational changes in clusters I and II and the entire domain); (4) Dynamic (only global conformational changes, but not in clusters I and II); and (5) Local Static (stable open domain conformation, conformational changes only in clusters I and II, but not the entire domain).

<p>Domain level conformational changes, are shown by the small (“closed” domains) and large (“open” domains) distance between clusters I and II (triangles, ellipses and inverted triangles). Domain types (3) and (4) do undergo domain opening, while domain types (1), (2) and (5) do not. The conformation of the domains of type (2) remains “closed”, while the conformation the domains of types (1) and (5) remains open. Local conformational changes in clusters I and II are shown by normal triangles (compact conformation of cluster I), inverted triangles (compact conformation of cluster II), and ellipses (less compact, more open conformation of cluster II). The conformation of cluster I does not change in all of the known structures and is the same in buffers and sensors, such as in calbindin 9K, calmodulin and troponin C. The conformation of cluster II does change from being less compact to more compact in the domains of types (3) and (5). In group (4), EF-hand domains undergo domain opening, but the conformations of the conserved clusters I and II remain intact.</p

Example interactions within cluster I.

<p>(A) and (B) illustrate two types of interactions between the flanking α-helices I and IV (cluster I in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109287#pone-0109287-g001" target="_blank">Figure 1</a>). The interactions occur <i>via</i> amino acids at positions -X+1, X-4 and -Z+1, which are also shown as black circles in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109287#pone-0109287-g001" target="_blank">Figures 1A and 1C</a> and highlighted in black in the alignment in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109287#pone-0109287-g001" target="_blank">Figure 1B</a>. Because in cluster I, positions X-4 and -Z+1 are purely aromatic (Φ) in all EF-hand representative structures, cluster I is called aromatic. (C) Contains the description of interactions for the eleven EF-hand fold representatives. The pattern “(-X+1)_helixIV^<u>CH-π</u> Φ(X-4)_helixI<u>^CH-π</u> Φ(-Z+1)_helixIV<u>^{weak HB}</u> (-X+1)_helixIV” indicates a circular interaction, where a side-chain atom of the –X+1 residue from the flanking α-helix IV forms a CH-π interaction with the ring of the X-4 aromatic amino acid from helix I, which, in turn, forms a CH-π interaction with the ring of the –Z+1 aromatic amino acid from helix IV, which interacts with the initial –X+1 residue from the flanking α-helix IV by means of a weak CH-O hydrogen bond.</p

Single calcium binding EF-hand domains with known <i>apo</i>- and <i>holo</i>-form structures.

<p>Summarized areas of interacting surfaces among the amino acids within cluster I and cluster II (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109287#pone-0109287-g001" target="_blank">Figure 1</a>), and the interacting area between the two clusters (I/II) are shown. NI, no interaction between the clusters.</p><p>Single calcium binding EF-hand domains with known <i>apo</i>- and <i>holo</i>-form structures.</p

Early Chordate Origin of the Vertebrate Integrin αI Domains

<div><p>Half of the 18 human integrins α subunits have an inserted αI domain yet none have been observed in species that have diverged prior to the appearance of the urochordates (ascidians). The urochordate integrin αI domains are not human orthologues but paralogues, but orthologues of human αI domains extend throughout later-diverging vertebrates and are observed in the bony fish with duplicate isoforms. Here, we report evidence for orthologues of human integrins with αI domains in the agnathostomes (jawless vertebrates) and later diverging species. Sequence comparisons, phylogenetic analyses and molecular modeling show that one nearly full-length sequence from lamprey and two additional fragments include the entire integrin αI domain region, have the hallmarks of collagen-binding integrin αI domains, and we show that the corresponding recombinant proteins recognize the collagen GFOGER motifs in a metal dependent manner, unlike the α1I domain of the ascidian <i>C. intestinalis</i>. The presence of a functional collagen receptor integrin αI domain supports the origin of orthologues of the human integrins with αI domains prior to the earliest diverging extant vertebrates, a domain that has been conserved and diversified throughout the vertebrate lineage.</p></div

Summary of integrin evolution across a broad range of species: αI domain specialization, as seen in humans, is a vertebrate invention.

<p>Individual domains having the same fold class as integrin component domains (i.e. β propeller, immunoglobulin fold, epidermal growth factor fold, vWFA) are observed already in prokaryotes but the earliest diverging sets of identifiable integrin subunits have been observed in the choanozoan <i>C. owczarzaki</i>, a single-cell eukaryote. The number of α and β subunits expands with increasing organismal complexity with 18 α and 8 β subunits forming up to 24 heterodimers in humans. Integrins undergo considerable functional diversification with the introduction of the αI domain in some α subunits. Tunicates like <i>C. intestinalis</i> and <i>H. roretzi</i> are the earliest diverging organisms where integrins with αI domains have been identified, but they are not direct vertebrate orthologues as they form a distinct clade. αI domain containing fragments can be detected in the lamprey <i>P. marinus</i> and possibly the hagfish <i>E. burgeri</i>; both are extant representatives of the first vertebrates. The lamprey fragments share characteristic features in common with the human collagen-binding αI domain group and they bind different mammalian collagens at MIDAS; four shark sequences are orthologues of the corresponding human α subunits, three collagen binding and one from the leukocyte clade, and duplicate isoforms are observed in observed in bony fish e.g. <i>D. rerio</i>, <i>C. carpio</i> and <i>O. niloticus</i>.</p

Binding of Pma_f αI domains to rat collagen I as a function of the concentration of Pma_f αI.

<p>(A–C) Binding affinities of Pma_f αI domains to rat collagen I were estimated by fitting binding data using a hyperbolic function, which is identical to Hill's equation when h = 1. BSA was used as a control.</p

Key features of the integrin αI domain.

<p>(A) Alignment of representative sequences, including the three sea lamprey fragments, one short EST fragment derived from the inshore hagfish genome, and four sequences from the elephant shark genome (highlighted in bold). The residues DxSxS…D…T of MIDAS (in bold) function to bind directly or via water molecules to the metal ion where natural ligands bind via a glutamate residue. The sequence ESH (bold) is characteristic of collagen-binding αI domains; the αC helix (bold) is a distinctive hallmark of the collagen receptor α subunits. The intrinsic glutamate ligand (bold) of the αI domain binds to MIDAS of the βI-like domain in integrins that have the inserted αI domain. Structure of the α2I domain without (B) (PDB code: 1AOX; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112064#pone.0112064-Emsley1" target="_blank">[14]</a>) and with (C) bound GFOGER tripeptide (PDB code: 1DZI; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112064#pone.0112064-Emsley2" target="_blank">[15]</a>). The peptide binds to the metal (yellow sphere) at MIDAS via glutamate E11 of the peptide. Consequently, the αC helix unravels and the α6 helix lengthens.</p