Structural and functional diversity of EF‐hand proteins: Evolutionary perspectives

We have classified 865 sequences of EF‐hand proteins from five proteomes into 156 subfamilies. These subfamilies were put into six groups. Evolutionary relationships among subfamilies and groups were analyzed from the inferred ancestral sequence for each subfamily. CTER, CPV, and PEF groups arose from a common EF‐lobe (pair of adjacent EF‐hands). They have two or more EF‐lobes; the relative positions of their EF‐lobes differ from each other. Comparisons of the ancestral sequences and the inferred structures of the EF‐lobes of these groups indicate that the mutual positions of EF‐lobes were established soon after divergence of an EF‐lobe for each group and before the duplication and fusion of EF‐lobe gene(s). These ancestral sequences reveal that some subfamilies in low similarity and isolated groups did not evolve from the EF‐lobe precursor, even if their conformations are similar to the canonical EF‐hand. This is an example of convergent evolution.     

X‐ray structure of Danio rerio secretagogin: A hexa‐EF‐hand calcium sensor

Many essential physiological processes are regulated by the modulation of calcium concentration in the cell. The EF‐hand proteins represent a superfamily of calcium‐binding proteins involved in calcium signaling and homeostasis. Secretagogin is a hexa‐EF‐hand protein that is highly expressed in pancreatic islet of Langerhans and neuroendocrine cells and may play a role in the trafficking of secretory granules. We present the X‐ray structure of Danio rerio secretagogin, which is 73% identical to human secretagogin, in calcium‐free form at 2.1‐Å resolution. Secretagogin consists of the three globular domains each of which contains a pair of EF‐hand motifs. The domains are arranged into a V‐shaped molecule with a distinct groove formed at the interface of the domains. Comparison of the secretagogin structure with the solution structure of calcium‐loaded calbindin D_28K revealed a striking difference in the spatial arrangement of their domains, which involves ～180° rotation of the first globular domain with respect to the module formed by the remaining domains. Proteins 2009. © 2009 Wiley‐Liss, Inc.     

Analysis of the movements of helices in EF‐hands

We have developed a method to place an EF‐lobe in a coordinate system that recognizes the similarity of its two EF‐hand domains as well as their relationship by a pseudo‐two fold axis, z. The x‐axis connects the center of mass, calculated from α‐carbons of helices E1 and F1, with the center of mass of E2 and F2. The resulting coordinate system is intrinsic to each EF‐lobe and requires no comparison with other EF‐lobes. It has provided an intuitive and informative way to compare EF‐lobes and especially those changes associated with calcium and/or target binding. We analyzed the EF‐lobes of calmodulin and of other subfamilies with four EF‐hands. We have rationalized a complex pattern of changes of conformation associated with calcium coordination and effector binding as observed in different subfamilies of EF‐hand proteins. Proteins 2012. © 2012 Wiley Periodicals, Inc.     

Structural differences among subfamilies of EF‐hand proteins—A view from the pseudo two‐fold symmetry axis

We have analyzed the conformations of EF‐lobes, adjacent pairs of EF‐hand domains, in a coordinate system based on the approximate two‐fold (z) axis that relates the two EF‐hands. Two parameters ‐ dE(ø), the azimuthal angle between the y‐axis and the projection of the offset vector to helix E onto the yz‐plane, and δdF(ø), the difference angle between the two helices (F1 and F2) of odd and even domains—characterize the openness of a single EF‐hand domain and of an EF‐lobe, respectively. We describe and compare values of dE(ø) and of δdF(ø) for EF‐hand proteins of five subfamilies—CTER, CPV, S100, PARV, CALP—in calci‐ and apo‐ forms, with and without bound target proteins. Each subfamily has characteristic changes associated with binding calcium and/or target proteins. Proteins 2014; 82:2915–2924. © 2014 Wiley Periodicals, Inc.     

Infrared study of synthetic peptide analogues of the calcium‐binding site III of troponin C: The role of helix F of an EF‐hand motif

The EF‐hand motif (helix–loop–helix) is a Ca²⁺‐binding domain that is common among many intracellular Ca²⁺‐binding proteins. We applied Fourier‐transform infrared spectroscopy to study the synthetic peptide analogues of site III of rabbit skeletal muscle troponin C (helix E–loop–helix F). The 17‐residue peptides corresponding to loop–helix F (DRDADGYIDAEELAEIF), where one residue is substituted by the D ‐type amino acid, were investigated to disturb the α‐helical conformation of helix F systematically. These D ‐type‐substituted peptides showed no band at about 1555 cm^?1 even in the Ca²⁺‐loaded state although the native peptide (L ‐type only) showed a band at about 1555 cm^?1 in the Ca²⁺‐loaded state, which is assigned to the side‐chain COO^? group of Glu at the 12th position, serving as the ligand for Ca²⁺ in the bidentate coordination mode. Therefore, helix F is vital to the interaction between the Ca²⁺ and the side‐chain COO^? group of Glu at the 12th position. Implications of the COO^? antisymmetric stretch and the amide‐I′ of the synthetic peptide analogues of the Ca²⁺‐binding sites are discussed. © 2012 Wiley Periodicals, Inc. Biopolymers 99: 342–347, 2013.     

Detection of membrane protein–protein interaction in planta based on dual‐intein‐coupled tripartite split‐GFP association

Despite the great interest in identifying protein–protein interactions (PPIs) in biological systems, only a few attempts have been made at large‐scale PPI screening in planta. Unlike biochemical assays, bimolecular fluorescence complementation allows visualization of transient and weak PPIs in vivo at subcellular resolution. However, when the non‐fluorescent fragments are highly expressed, spontaneous and irreversible self‐assembly of the split halves can easily generate false positives. The recently developed tripartite split‐GFP system was shown to be a reliable PPI reporter in mammalian and yeast cells. In this study, we adapted this methodology, in combination with the β‐estradiol‐inducible expression cassette, for the detection of membrane PPIs in planta. Using a transient expression assay by agroinfiltration of Nicotiana benthamiana leaves, we demonstrate the utility of the tripartite split‐GFP association in plant cells and affirm that the tripartite split‐GFP system yields no spurious background signal even with abundant fusion proteins readily accessible to the compartments of interaction. By validating a few of the Arabidopsis PPIs, including the membrane PPIs implicated in phosphate homeostasis, we proved the fidelity of this assay for detection of PPIs in various cellular compartments in planta. Moreover, the technique combining the tripartite split‐GFP association and dual‐intein‐mediated cleavage of polyprotein precursor is feasible in stably transformed Arabidopsis plants. Our results provide a proof‐of‐concept implementation of the tripartite split‐GFP system as a potential tool for membrane PPI screens in planta.     

Metal binding affinity and structural properties of calmodulin‐like protein 14 from Arabidopsis thaliana

In addition to the well‐known Ca²⁺ sensor calmodulin, plants possess many calmodulin‐like proteins (CMLs) that are predicted to have specific roles in the cell. Herein, we described the biochemical and biophysical characterization of recombinant Arabidopsis thaliana CML14. We applied isothermal titration calorimetry to analyze the energetics of Ca²⁺ and Mg²⁺ binding to CML14, and nuclear magnetic resonance spectroscopy, together with intrinsic and ANS‐based fluorescence, to evaluate the structural effects of metal binding and metal‐induced conformational changes. Furthermore, differential scanning calorimetry and limited proteolysis were used to characterize protein thermal and local stability. Our data demonstrate that CML14 binds one Ca²⁺ ion with micromolar affinity (K_d ～ 12 µM) and the presence of 10 mM Mg²⁺ decreases the Ca²⁺ affinity by ～5‐fold. Although binding of Ca²⁺ to CML14 increases protein stability, it does not result in a more hydrophobic protein surface and does not induce the large conformational rearrangement typical of Ca²⁺ sensors, but causes only localized structural changes in the unique functional EF‐hand. Our data, together with a molecular modelling prediction, provide interesting insights into the biochemical properties of Arabidopsis CML14 and may be useful to direct additional studies aimed at understanding its physiological role.     

Structure‐guided design of a reversible fluorogenic reporter of protein‐protein interactions

A reversible green fluorogenic protein‐fragment complementation assay was developed based on the crystal structure of UnaG, a recently discovered fluorescent protein. In living mammalian cells, the nonfluorescent fragments complemented and rapidly became fluorescent upon rapamycin‐induced FKBP and Frb protein interaction, and lost fluorescence when the protein interaction was inhibited. This reversible fluorogenic reporter, named uPPI [UnaG‐based protein‐protein interaction (PPI) reporter], uses bilirubin (BR) as the chromophore and requires no exogenous cofactor. BR is an endogenous molecule in mammalian cells and is not fluorescent by itself. uPPI may have many potential applications in visualizing spatiotemporal dynamics of PPIs.     

Solution NMR structures of the C‐domain of Tetrahymena cytoskeletal protein Tcb2 reveal distinct calcium‐induced structural rearrangements

Tcb2 is a calcium‐binding protein that localizes to the membrane‐associated skeleton of the ciliated protozoan Tetrahymena thermophila with hypothesized roles in ciliary movement, cell cortex signaling, and pronuclear exchange. Tcb2 has also been implicated in a unique calcium‐triggered, ATP‐independent type of contractility exhibited by filamentous networks isolated from the Tetrahymena cytoskeleton. To gain insight into Tcb2's structure‐function relationship and contractile properties, we determined solution NMR structures of its C‐terminal domain in the calcium‐free and calcium‐bound states. The overall architecture is similar to other calcium‐binding proteins, with paired EF‐hand calcium‐binding motifs. Comparison of the two structures reveals that Tcb2‐C's calcium‐induced conformational transition differs from the prototypical calcium sensor calmodulin, suggesting that the two proteins play distinct functional roles in Tetrahymena and likely have different mechanisms of target recognition. Future studies of the full‐length protein and the identification of Tcb2 cellular targets will help establish the molecular basis of Tcb2 function and its unique contractile properties. Proteins 2016; 84:1748–1756. © 2016 Wiley Periodicals, Inc.     

Solution structures of polcalcin Phl p 7 in three ligation states: Apo‐, hemi‐Mg2+‐bound,and fully Ca2+‐bound

Polcalcins are small EF‐hand proteins believed to assist in regulating pollen‐tube growth. Phl p 7, from timothy grass (Phleum pratense), crystallizes as a domain‐swapped dimer at low pH. This study describes the solution structures of the recombinant protein in buffered saline at pH 6.0, containing either 5.0 mM EDTA, 5.0 mM Mg²⁺, or 100 μM Ca²⁺. Phl p 7 is monomeric in all three ligation states. In the apo‐form, both EF‐hand motifs reside in the closed conformation, with roughly antiparallel N‐ and C‐terminal helical segments. In 5.0 mM Mg²⁺, the divalent ion is bound by EF‐hand 2, perturbing interhelical angles and imposing more regular helical structure. The structure of Ca²⁺‐bound Phl p 7 resembles that previously reported for Bet v 4—likewise exposing apolar surface to the solvent. Occluded in the apo‐ and Mg²⁺‐bound forms, this surface presumably provides the docking site for Phl p 7 targets. Unlike Bet v 4, EF‐hand 2 in Phl p 7 includes five potential anionic ligands, due to replacement of the consensus serine residue at –x (residue 55 in Phl p 7) with aspartate. In the Phl p 7 crystal structure, D55 functions as a helix cap for helix D. In solution, however, D55 apparently serves as a ligand to the bound Ca²⁺. When Mg²⁺ resides in site 2, the D55 carboxylate withdraws to a distance consistent with a role as an outer‐sphere ligand. ¹⁵N relaxation data, collected at 600 MHz, indicate that backbone mobility is limited in all three ligation states. Proteins 2013. © 2012 Wiley Periodicals, Inc.     

Reconstitution of purified membrane protein dimers in lipid nanodiscs with defined stoichiometry and orientation using a split GFP tether

Many membrane proteins function as dimers or larger oligomers, including transporters, channels, certain signaling receptors, and adhesion molecules. In some cases, the interactions between individual proteins may be weak and/or dependent on specific lipids, such that detergent solubilization used for biochemical and structural studies disrupts functional oligomerization. Solubilized membrane protein oligomers can be captured in lipid nanodiscs, but this is an inefficient process that can produce stoichiometrically and topologically heterogeneous preparations. Here, we describe a technique to obtain purified homogeneous membrane protein dimers in nanodiscs using a split GFP (sGFP) tether. Complementary sGFP tags associate to tether the coexpressed dimers and control both stoichiometry and orientation within the nanodiscs, as assessed by quantitative Western blotting and negative-stain EM. The sGFP tether confers several advantages over other methods: it is highly stable in solution and in SDS-PAGE, which facilitates screening of dimer expression and purification by fluorescence, and also provides a dimer-specific purification handle for use with GFP nanobody–conjugated resin. We used this method to purify a Frizzled-4 homodimer and a Frizzled-4/low-density lipoprotein receptor–related protein 6 heterodimer in nanodiscs. These examples demonstrate the utility and flexibility of this method, which enables subsequent mechanistic molecular and structural studies of membrane protein pairs.     

Selectable high‐yield recombinant protein production in human cells using a GFP/YFP nanobody affinity support

Recombinant protein expression systems that produce high yields of pure proteins and multi‐protein complexes are essential to meet the needs of biologists, biochemists, and structural biologists using X‐ray crystallography and cryo‐electron microscopy. An ideal expression system for recombinant human proteins is cultured human cells where the correct translation and chaperone machinery are present. However, compared to bacterial expression systems, human cell cultures present several technical challenges to their use as an expression system. We developed a method that utilizes a YFP fusion‐tag to generate recombinant proteins using suspension‐cultured HEK293F cells. YFP is a dual‐function tag that enables direct visualization and fluorescence‐based selection of high expressing clones for and rapid purification using a high‐stringency, high‐affinity anti‐GFP/YFP nanobody support. We demonstrate the utility of this system by expressing two large human proteins, TOP2α (340 KDa dimer) and a TOP2β catalytic core (260 KDa dimer). This robustly and reproducibly yields >10 mg/L liter of cell culture using transient expression or 2.5 mg/L using stable expression.     

Role of the Lectin VIP36 in Post‐ER Quality Control of Human α1‐Antitrypsin

The leguminous‐type (L‐type) lectin VIP36 localizes to the Golgi apparatus and cycles early in the secretory pathway. In vitro, VIP36 binds high‐mannose glycans with a pH optimum of 6.5, a value similar to the luminal pH of the Golgi apparatus. Although the sugar‐binding properties of VIP36 in vitro have been characterized in detail, the function of VIP36 in the intact cell remains unclear as no convincing glycoprotein cargo has been identified. Here, we used yellow fluorescent protein (YFP) fragment complementation to identify luminal interaction partners of VIP36. By screening a human liver cDNA library, we identified the glycoprotein α1‐antitrypsin (α1‐AT) as a cargo of VIP36. The VIP36/α1‐AT complex localized to Golgi and endoplasmic reticulum (ER). In the living cell, VIP36 bound exclusively to the high‐mannose form of α1‐AT. The binding was increased when complex glycosylation was prevented by kifunensine and abolished when the glycosylation sites of α1‐AT were inactivated by mutagenesis. Silencing VIP36 accelerated α1‐AT transport, arguing against a role of VIP36 in anterograde traffic. The complex formed by VIP36 and α1‐AT in the Golgi recycled back to the ER. The combined data are most consistent with a function of VIP36 in post‐ER quality control of α1‐AT.     

Interactions between the budding yeast IQGAP homologue Iqg1p and its targets revealed by a split-EGFP bimolecular fluorescence complementation assay

A split-EGFP based bimolecular fluorescence complementation (BiFC) assay has been used to detect interactions between the Saccharomyces cerevisiae cytoskeletal scaffolding protein Iqg1p and three targets: myosin essential light chain (Mlc1p), calmodulin (Cmd1p) and the small GTPase Cdc42p. The format of the BiFC assay used ensures that the proteins are expressed at wild type levels thereby avoiding artefacts due to overexpression. This is the first direct in vivo detection of these interactions; in each case, the complex is localised to discrete regions of the yeast cytoplasm. The labelling with EGFP fragments results in changes in growth kinetics, cell size and budding frequency. This is partly due to the reassembled EGFP locking the complexes into essentially permanent interactions. The consequences of this for Iqg1p interactions and BiFC assays in general are discussed.     

Thermal green protein,an extremely stable,nonaggregating fluorescent protein created by structure‐guided surface engineering

In this article, we describe the engineering and X‐ray crystal structure of Thermal Green Protein (TGP), an extremely stable, highly soluble, non‐aggregating green fluorescent protein. TGP is a soluble variant of the fluorescent protein eCGP123, which despite being highly stable, has proven to be aggregation‐prone. The X‐ray crystal structure of eCGP123, also determined within the context of this paper, was used to carry out rational surface engineering to improve its solubility, leading to TGP. The approach involved simultaneously eliminating crystal lattice contacts while increasing the overall negative charge of the protein. Despite intentional disruption of lattice contacts and introduction of high entropy glutamate side chains, TGP crystallized readily in a number of different conditions and the X‐ray crystal structure of TGP was determined to 1.9 Å resolution. The structural reasons for the enhanced stability of TGP and eCGP123 are discussed. We demonstrate the utility of using TGP as a fusion partner in various assays and significantly, in amyloid assays in which the standard fluorescent protein, EGFP, is undesirable because of aberrant oligomerization. Proteins 2015; 83:1225–1237. © 2014 Wiley Periodicals, Inc.