FRET-based in vivo Ca2+ imaging by a new calmodulin-GFP fusion molecule.

Intracellular Ca2+ acts as a second messenger that regulates numerous physiological cellular phenomena including development, differentiation and apoptosis. Cameleons, a class of fluorescent indicators for Ca2+ based on green fluorescent proteins (GFPs) and calmodulin (CaM), have proven to be a useful tool in measuring free Ca2+ concentrations in living cells. Traditional cameleons, however, have a small dynamic range of fluorescence resonance energy transfer (FRET), making subtle changes in Ca2+ concentrations difficult to detect and study in some cells and organelles. Using the NMR structure of CaM bound to the CaM binding peptide derived from CaM-dependent kinase kinase (CKKp), we have rationally designed a new cameleon that displays a two-fold increase in the FRET dynamic range within the physiologically significant range of cytoplasmic Ca2+ concentration of 0.05-1 microM.

Target-induced conformational adaptation of calmodulin revealed by the crystal structure of a complex with nematode Ca(2+)/calmodulin-dependent kinase kinase peptide.

Calmodulin (CaM) is a ubiquitous calcium (Ca(2+)) sensor which binds and regulates protein serine/threonine kinases along with many other proteins in a Ca(2+)-dependent manner. For this multi-functionality, conformational plasticity is essential; however, the nature and magnitude of CaM's plasticity still remains largely undetermined. Here, we present the 1.8 A resolution crystal structure of Ca(2+)/CaM, complexed with the 27-residue synthetic peptide corresponding to the CaM-binding domain of the nematode Caenorhabditis elegans Ca(2+)/CaM-dependent kinase kinase (CaMKK). The peptide bound in this crystal structure is a homologue of the previously NMR-derived complex with rat CaMKK, but benefits from improved structural resolution. Careful comparison of the present structure to previous crystal structures of CaM complexed with unrelated peptides derived from myosin light chain kinase and CaM kinase II, allow a quantitative analysis of the differences in the relative orientation of the N and C-terminal domains of CaM, defined as a screw axis rotation angle ranging from 156 degrees to 196 degrees. The principal differences in CaM interaction with various peptides are associated with the N-terminal domain of CaM. Unlike the C-terminal domain, which remains unchanged internally, the N-terminal domain of CaM displays significant differences in the EF-hand helix orientation between this and other CaM structures. Three hydrogen bonds between CaM and the peptide (E87-R336, E87-T339 and K75-T339) along with two salt bridges (E11-R349 and E114-K334) are the most probable determinants for the binding direction of the CaMKK peptide to CaM.

Alternate routes to conformational specificity in a Greek key beta barrel protein.

The N-terminal domain of protein S, a Greek key calcium-binding protein from Myxococcus xanthus, forms an atypical molten globule in the calcium-free state. The structure of this state is characterized by significant conformational fluctuations, which are localized to a subdomain that is not contiguous along the polypeptide chain. The conformational instability of this subdomain appears to arise from repulsive electrostatic interactions of four acidic side chains that are clustered together but are removed from the calcium-binding sites. This domain can be induced to form a native-like state through two different routes, calcium binding or reduction of pH. Acid-induced folding stabilizes the locally unfolded subdomain by selectively removing repulsive interactions without significantly affecting global stability. In contrast, calcium binding appears to increase local stability indirectly by causing global stabilization.

Calcium-regulated DNA binding and oligomerization of the neuronal calcium-sensing protein, calsenilin/DREAM/KChIP3.

Calsenilin/DREAM/KChIP3, a member of the recoverin branch of the EF-hand superfamily, interacts with presenilins, serves as a calcium-regulated transcriptional repressor, and interacts with A-type potassium channels. Here we report physicochemical characterization of calcium binding, oligomerization, and DNA binding of human calsenilin/DREAM/KChIP3. Equilibrium Ca(2+) binding measurements indicate that the protein binds 3 Ca(2+) with a dissociation constant of 14 microM and a Hill coefficient of 0.7. Dynamic light scattering and size exclusion chromatography show that the Ca(2+)-bound protein exists as a dimer at protein concentrations lower than 150 microM and forms a tetramer at concentrations above 200 microM. The Ca(2+)-free protein is a tetramer in the concentration range 20-450 microM. Isothermal titration calorimetry and dynamic light scattering indicate that the Ca(2+)-free protein tetramer binds endothermically (DeltaH = +25 kcal/mol) to four molecules of DNA derived from the downstream regulatory element (DRE) of either the prodynorphin or c-fos genes. One DRE molecule binds tightly to the protein with a dissociation constant (K(d)) of 75 nM, and the other three bind more weakly (K(d) = 640 nM). No significant DNA binding was observed for the Ca(2+)-bound protein. The N-terminal protein fragment (residues 1-70) binds nonspecifically to DRE in a Ca(2+)-independent manner, whereas a C-terminal fragment containing the four EF-hands (residues 65-256) binds DRE (K(d) = 200 nM) in a Ca(2+)-regulated and sequence-specific fashion. The C-terminal fragment is a tetramer in the Ca(2+)-free state and dissociates into dimers at saturating Ca(2+) levels.

Calexcitin B is a new member of the sarcoplasmic calcium-binding protein family.

Calexcitin (CE) is a calcium sensor protein that has been implicated in associative learning. The CE gene was previously cloned from the long-finned squid, Loligo pealei, and the gene product was shown to bind GTP and modulate K(+) channels and ryanodine receptors in a Ca(2+)-dependent manner. We cloned a new gene from L. pealei, which encodes a CE-like protein, here named calexcitin B (CE(B)). CE(B) has 95% amino acid identity to the original form. Our sequence analyses indicate that CEs are homologous to the sarcoplasmic calcium-binding protein subfamily of the EF-hand superfamily. Far and near UV circular dichroism and nuclear magnetic resonance studies demonstrate that CE(B) binds Ca(2+) and undergoes a conformational change. CE(B) is phosphorylated by protein kinase C, but not by casein kinase II. CE(B) does not bind GTP. Western blot experiments using polyclonal antibodies generated against CE(B) showed that CE(B) is expressed in the L. pealei optic lobe. Taken together, the neuronal protein CE represents the first example of a Ca(2+) sensor in the sarcoplasmic calcium-binding protein family.

The use of FRET imaging microscopy to detect protein-protein interactions and protein conformational changes in vivo.

Intermolecular and intramolecular FRET between two spectrally overlapping green fluorescent protein variants fused to two different host proteins or at two different sites within the same protein offers a unique opportunity to monitor real-time protein-protein interactions or protein conformational changes. By using fluorescence digital imaging microscopy, one can visualize the location of green fluorescent proteins within a living cell and follow the time course of the changes in FRET corresponding to cellular events at a millisecond time resolution. The observation of such dynamic molecular events in vivo provides vital insight into the action of biological molecules.

A role of transcriptional activators as antirepressors for the autoinhibitory activity of TATA box binding of transcription factor IID.

The TATA box-binding activity of transcription factor IID (TFIID) is autoinhibited by the N-terminal domain of the Drosophila TATA box-binding protein- (TBP) associated factor 230/yeast TBP-associated factor 145 subunit, which binds to the TATA box-binding domain of TBP by mimicking the TATA box structure. Here, we propose a mechanism of transcriptional activation that involves antirepression of this autoinhibitory activity by transcriptional activators. Like the autoinhibitory domain of TFIID, various acidic activators interact with the TATA box-binding domain of TBP. Moreover, the autoinhibitory domain of TFIID, which is known to interact with only the TATA box-binding domain of TBP, acts as an activation domain when fused to the GAL4 DNA-binding domain, indicating that interaction with the TATA-binding domain of TBP is crucial for activation of transcription. In a reciprocal fashion, the acidic activation domains can function as the autoinhibitory domain when the latter is replaced by the former within TFIID. These results indicate that activation domains and the autoinhibitory domain of TFIID are interchangeable, supporting a role for transcriptional activators as antirepressors of the autoinhibitory activity of the TATA box binding of TFIID.

A monomeric histidine kinase derived from EnvZ, an Escherichia coli osmosensor.

Histidine kinases function as dimers. The kinase domain of the osmosensing histidine kinase EnvZ of Escherichia coli consists of two domains: domain A (67 residues) responsible for histidine phosphotransfer and dimerization, and domain B (161 residues) responsible for the catalytic and ATP-binding function. The individual structures of these two domains have been recently solved by NMR spectroscopy. Here, we demonstrate that an enzymatically functional monomeric histidine kinase can be constructed by fusing in tandem two domains A and one domain B to produce a single polypeptide (A-A-B). We show that this protein, EnvZc[AAB], is soluble and exists as a stable monomer. The autophosphorylation and OmpR kinase activities of the monomeric EnvZc[AAB] are similar to that of the wild-type EnvZ, while OmpR-binding and phosphatase functions are reduced. V8 protease digestion and mutational analyses indicate that His-243 of only the amino proximal domain A is phosphorylated. Based on these results, molecular models are proposed for the structures of EnvZc[AAB] and the kinase domain of EnvZ. The present results demonstrate for the first time the construction of a functional, monomeric histidine kinase, further structural studies of which may provide important insights into the structure-function relationships of histidine kinases.

Regulatory mechanism of Ca2+/calmodulin-dependent protein kinase kinase.

Ca(2+)/calmodulin-dependent protein kinase kinase (CaM-KK) is a novel member of the CaM kinase family, which specifically phosphorylates and activates CaM kinase I and IV. In this study, we characterized the CaM-binding peptide of alphaCaM-KK (residues 438-463), which suppressed the activity of constitutively active CaM-KK (84-434) in the absence of Ca(2+)/CaM but competitively with ATP. Truncation and site-directed mutagenesis of the CaM-binding region in CaM-KK reveal that Ile(441) is essential for autoinhibition of CaM-KK. Furthermore, CaM-KK chimera mutants containing the CaM-binding sequence of either myosin light chain kinases or CaM kinase II located C-terminal of Leu(440), exhibited enhanced Ca(2+)/CaM-independent activity (60% of total activity). Although the CaM-binding domains of myosin light chain kinases and CaM kinase II bind to the N- and C-terminal domains of CaM in the opposite orientation to CaM-KK (Osawa, M., Tokumitsu, H., Swindells, M. B., Kurihara, H., Orita, M., Shibanuma, T., Furuya, T., and Ikura, M. (1999) Nat. Struct. Biol. 6, 819-824), the chimeric CaM-KKs containing Ile(441) remained Ca(2+)/CaM-dependent. This result demonstrates that the orientation of the CaM binding is not critical for relief of CaM-KK autoinhibition. However, the requirement of Ile(441) for autoinhibition, which is located at the -3 position from the N-terminal anchoring residue (Trp(444)) to CaM, accounts for the opposite orientation of CaM binding of CaM-KK compared with other CaM kinases.

TFIIA-TAF regulatory interplay: NMR evidence for overlapping binding sites on TBP.

TATA box binding protein (TBP)-promoter interaction nucleates assembly of the RNA polymerase II transcription initiation complex. Transcription factor IIA (TFIIA) stabilizes the TBP-promoter complex whereas the N-terminal domain of the largest TAF(II) inhibits TBP-promoter interaction. We have mapped the interaction sites on TBP of Drosophila TAF(II)230 and yeast TFIIA (comprising two subunits, TOA1 and TOA2), using nuclear magnetic resonance (NMR), and also report structural evidence that subdomain II of the TAF(II)230 N-terminal inhibitory domain and TFIIA have overlapping binding sites on the convex surface of TBP. Together with previous mutational and biochemical data, our NMR results indicate that subdomain II augments subdomain I-mediated inhibition of TBP function by blocking TBP-TFIIA interaction.

Cadherins in embryonic and neural morphogenesis.

Cadherins not only maintain the structural integrity of cells and tissues but also control a wide array of cellular behaviours. They are instrumental for cell and tissue polarization, and they regulate cell movements such as cell sorting, cell migration and cell rearrangements. Cadherins may also contribute to neurite outgrowth and pathfinding, and to synaptic specificity and modulation in the central nervous system.

Calmodulin target database.

The intracellular calcium sensor protein calmodulin (CaM) interacts with a large number of proteins to regulate their biological functions in response to calcium stimulus. This molecular recognition process is diverse in its mechanism, but can be grouped into several classes based on structural and sequence information. We have developed a web-based database (http://calcium.uhnres.utoronto.ca/ctdb) for this family of proteins containing CaM binding sites or, as we propose to call it herein, CaM recruitment signaling (CRS) motifs. At present the CRS motif found in approximately 180 protein sequences in the databases can be divided into four subclasses, each subclass representing a distinct structural mode of molecular recognition involving CaM. The database can predict a putative CRS location within a given protein sequence, identify the subclass to which it may belong, and structural and biophysical parameters such as hydrophobicity, hydrophobic moment, and propensity for alpha-helix formation.

Diversity of conformational states and changes within the EF-hand protein superfamily.

The EF-hand motif, which assumes a helix-loop-helix structure normally responsible for Ca2+ binding, is found in a large number of functionally diverse Ca2+ binding proteins collectively known as the EF-hand protein superfamily. In many superfamily members, Ca2+ binding induces a conformational change in the EF-hand motif, leading to the activation or inactivation of target proteins. In calmodulin and troponin C, this is described as a change from the closed conformational state in the absence of Ca2+ to the open conformational state in its presence. It is now clear from structures of other EF-hand proteins that this "closed-to-open" conformational transition is not the sole model for EF-hand protein structural response to Ca2+. More complex modes of conformational change are observed in EF-hand proteins that interact with a covalently attached acyl group (e.g., recoverin) and in those that dimerize (e.g., S100B, calpain). In fact, EF-hand proteins display a multitude of unique conformational states, together constituting a conformational continuum. Using a quantitative 3D approach termed vector geometry mapping (VGM), we discuss this tertiary structural diversity of EF-hand proteins and its correlation with target recognition.

A novel target recognition revealed by calmodulin in complex with Ca2+-calmodulin-dependent kinase kinase.

The structure of calcium-bound calmodulin (Ca2+/CaM) complexed with a 26-residue peptide, corresponding to the CaM-binding domain of rat Ca2+/CaM-dependent protein kinase kinase (CaMKK), has been determined by NMR spectroscopy. In this complex, the CaMKK peptide forms a fold comprising an alpha-helix and a hairpin-like loop whose C-terminus folds back on itself. The binding orientation of this CaMKK peptide by the two CaM domains is opposite to that observed in all other CaM-target complexes determined so far. The N- and C-terminal hydrophobic pockets of Ca2+/CaM anchor Trp 444 and Phe 459 of the CaMKK peptide, respectively. This 14-residue separation between two key hydrophobic groups is also unique among previously determined CaM complexes. The present structure represents a new and distinct class of Ca2+/CaM target recognition that may be shared by other Ca2+/CaM-stimulated proteins.

Solution structure of the homodimeric core domain of Escherichia coli histidine kinase EnvZ.

Escherichia coli osmosensor EnvZ is a protein histidine kinase that plays a central role in osmoregulation, a cellular adaptation process involving the His-Asp phosphorelay signal transduction system. Dimerization of the transmembrane protein is essential for its autophosphorylation and phosphorelay signal transduction functions. Here we present the NMR-derived structure of the homodimeric core domain (residues 223-289) of EnvZ that includes His 243, the site of autophosphorylation and phosphate transfer reactions. The structure comprises a four-helix bundle formed by two identical helix-turn-helix subunits, revealing the molecular assembly of two active sites within the dimeric kinase.