EF-hand Pair Research Articles

NMR resonance assignment of the N-terminal GTPase domain of human Miro2 Bound to GTP

Miro2 and Miro1 are mitochondrial-associated proteins critical for regulating mitochondrial movement within the cell. Both Miro1 and Miro2 have roles in promoting neuron function, but recently Miro2 has been shown to have additional roles in response to nutrient starvation in tumor cells. Miro1 and 2 consist of two small GTPase domains flanking a pair of EF-hands. The N-terminal GTPase (nGTPase) domain is responsible for initiating mitochondrial trafficking and interactions with GCN1 in prostate cancer. The crystal structure of Miro1 nGTPase bound to GTP has been solved. However, no structural data is available for the nGTPase domain of Miro2. To better understand the similarities and differences in the functions of Miro1 and Miro2, we have initiated structural studies of Miro2. Here we report the backbone NMR chemical shift assignments of a 22 KDa construct of the nGTPase domain of Miro2 bound to GTP that includes residues 1–180 of the full-length protein. We affirm that the overall secondary structure of this complex closely resembles that of Miro1 nGTPase bound to GTP. Minor variations in the overall structures can be attributed to crystal packing interactions in the structure of Miro1. These NMR studies will form the foundation for future work identifying the specific interaction sites between Miro2 and its cellular binding partners.

Interplay between hevin, SPARC, and MDGAs: Modulators of neurexin-neuroligin transsynaptic bridges

Summary Hevin is secreted by astrocytes and its synaptogenic effects are antagonized by the related protein, SPARC. Hevin stabilizes neurexin-neuroligin transsynaptic bridges in vivo. A third protein, membrane-tethered MDGA, blocks these bridges. Here, we reveal the molecular underpinnings of a regulatory network formed by this trio of proteins. The hevin FS-EC structure differs from SPARC, in that the EC domain appears rearranged around a conserved core. The FS domain is structurally conserved and it houses nanomolar affinity binding sites for neurexin and neuroligin. SPARC also binds neurexin and neuroligin, competing with hevin, so its antagonist action is rooted in its shortened N-terminal region. Strikingly, the hevin FS domain competes with MDGA for an overlapping binding site on neuroligin, while the hevin EC domain binds the extracellular matrix protein collagen (like SPARC), so that this trio of proteins can regulate neurexin-neuroligin transsynaptic bridges and also extracellular matrix interactions, impacting synapse formation and ultimately neural circuits.

Calmodulin-Cork Model of Gap Junction Channel Gating-One Molecule, Two Mechanisms.

The Calmodulin-Cork gating model is based on evidence for the direct role of calmodulin (CaM) in channel gating. Indeed, chemical gating of cell-to-cell channels is sensitive to nanomolar cytosolic calcium concentrations [Ca2+]i. Calmodulin inhibitors and inhibition of CaM expression prevent chemical gating. CaMCC, a CaM mutant with higher Ca2+-sensitivity greatly increases chemical gating sensitivity (in CaMCC the NH2-terminal EF-hand pair (res. 9–76) is replaced by the COOH-terminal pair (res. 82–148). Calmodulin colocalizes with connexins. Connexins have high-affinity CaM binding sites. Several connexin mutants paired to wild-type connexins have a high gating sensitivity that is eliminated by inhibition of CaM expression. Repeated transjunctional voltage (Vj) pulses slowly and progressively close a large number of channels by the chemical/slow gate (CaM lobe). At the single-channel level, the chemical/slow gate closes and opens slowly with on-off fluctuations. The model proposes two types of CaM-driven gating: “Ca-CaM-Cork” and “CaM-Cork”. In the first, gating involves Ca2+-induced CaM-activation. In the second, gating takes place without [Ca2+]i rise. The Ca-CaM-Cork gating is only reversed by a return of [Ca2+]i to resting values, while the CaM-Cork gating is reversed by Vj positive at the gated side.

Arrhythmia mutations in calmodulin can disrupt cooperativity of Ca2+ binding and cause misfolding.

Mutations in the calmodulin protein (CaM) are associated with arrhythmia syndromes. This study focuses on understanding the structural characteristics of CaM disease mutants and their interactions with the voltage-gated calcium channel CaV 1.2. Arrhythmia mutations in CaM can lead to loss of Ca2+ binding, uncoupling of Ca2+ binding cooperativity, misfolding of the EF-hands and altered affinity for the calcium channel. These results help us to understand how different CaM mutants have distinct effects on structure and interactions with protein targets to cause disease. Calmodulinopathies are life-threatening arrhythmia syndromes that arise from mutations in calmodulin (CaM), a calcium sensing protein whose sequence is completely conserved across all vertebrates. These mutations have been shown to interfere with the function of cardiac ion channels, including the voltage-gated Ca2+ channel CaV 1.2 and the ryanodine receptor (RyR2), in a mutation-specific manner. The ability of different CaM disease mutations to discriminate between these channels has been enigmatic. We present crystal structures of several C-terminal lobe mutants and an N-terminal lobe mutant in complex with the CaV 1.2 IQ domain, in conjunction with binding assays and complementary structural biology techniques. One mutation (D130G) causes a pathological conformation, with complete separation of EF-hands within the C-lobe and loss of Ca2+ binding in EF-hand 4. Another variant (Q136P) has severely reduced affinity for the IQ domain, and shows changes in the CD spectra under Ca2+ -saturating conditions when unbound to the IQ domain. Ca2+ binding to a pair of EF-hands normally proceeds with very high cooperativity, but we found that N98S CaM can adopt different conformations with either one or two Ca2+ ions bound to the C-lobe, possibly disrupting the cooperativity. An N-lobe variant (N54I), which causes severe stress-induced arrhythmia, does not show any major changes in complex with the IQ domain, providing a structural basis for why this mutant does not affect function of CaV 1.2. These findings show that different CaM mutants have distinct effects on both the CaM structure and interactions with protein targets, and act via distinct pathological mechanisms to cause disease.

The crystal structure of MICU2 provides insight into Ca2+ binding and MICU1-MICU2 heterodimer formation.

The mitochondrial calcium uniporter (MCU) complex mediates the uptake of Ca2+ into mitochondria. Its activity is regulated by a heterodimer of MICU1 and MICU2, two EF-hand-containing proteins that act as the main gatekeeper of the uniporter. Herein we report the crystal structure of human MICU2 at 1.96Å resolution. Our structure reveals a dimeric architecture of MICU2, in which each monomer adopts the canonical two-lobe structure with a pair of EF-hands in each lobe. Both Ca2+ -bound and Ca2+ -free EF-hands are observed in our structure. Moreover, we characterize the interaction sites within the MICU2 homodimer, as well as the MICU1-MICU2 heterodimer in both Ca2+ -free and Ca2+ -bound conditions. Glu242 in MICU1 and Arg352 in MICU2 are crucial for apo heterodimer formation, while Phe383 in MICU1 and Glu196 in MICU2 significantly contribute to the interaction in the Ca2+ -bound state. Based on our structural and biochemical analyses, we propose a model for MICU1-MICU2 heterodimer formation and its conformational transition from apo to a more compact Ca2+ -bound state, which expands our understanding of this co-regulatory mechanism critical for MCU's mitochondrial calcium uptake function.

CRACR2a is a calcium-activated dynein adaptor protein that regulates endocytic traffic.

Cytoplasmic dynein is a minus end-directed microtubule motor that transports intracellular cargoes. Transport is initiated by coiled-coil adaptors that (a) join dynein and its cofactor dynactin into a motile complex and (b) interact with a cargo-bound receptor, which is frequently a Rab GTPase on an organelle. Here, we report two novel dynein adaptors, CRACR2a and Rab45, that have a coiled-coil adaptor domain, a pair of EF-hands, and a Rab GTPase fused into a single polypeptide. CRACR2a-mediated, but not Rab45-mediated, dynein motility is activated by calcium in vitro. In Jurkat T cells, elevation of intracellular calcium activates CRACR2a-mediated dynein transport. We further found that T cell receptor activation induces the formation of CRACR2a puncta at the plasma membrane, which initially associate with the actin cortex and subsequently detach and travel along microtubules, suggestive of an endocytic process. These results provide the first examples of Rab GTPases that directly act as dynein adaptors and implicate CRACR2a-dynein in calcium-regulated endocytic trafficking.

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.

Structural Consequences of Calmodulin EF Hand Mutations.

Calmodulin (CaM) is a cytosolic Ca2+-binding protein that serves as a control element for many enzymes. It consists of two globular domains, each containing two EF hand pairs capable of binding Ca2+, joined by a flexible central linker region. CaM is able to bind and activate its target proteins in the Ca2+-replete and Ca2+-deplete forms. To study the Ca2+-dependent/independent properties of binding and activation of target proteins by CaM, CaM constructs with Ca2+-binding disrupting mutations of Asp to Ala at position one of each EF hand have been used. These CaM mutant proteins are deficient in binding Ca2+ in either the N-lobe EF hands (CaM12), C-lobe EF hands (CaM34), or all four EF hands (CaM1234). To investigate potential structural changes these mutations may cause, we performed detailed NMR studies of CaM12, CaM34, and CaM1234 including determining the solution structure of CaM1234. We then investigated if these CaM mutants affected the interaction of CaM with a target protein known to interact with apoCaM by determining the solution structure of CaM34 bound to the iNOS CaM binding domain peptide. The structures provide direct structural evidence of changes that are present in these Ca2+-deficient CaM mutants and show these mutations increase the hydrophobic exposed surface and decrease the electronegative surface potential throughout each lobe of CaM. These Ca2+-deficient CaM mutants may not be a true representation of apoCaM and may not allow for native-like interactions of apoCaM with its target proteins.

The calmodulin-like proteins AtCML4 and AtCML5 are single-pass membrane proteins targeted to the endomembrane system by an N-terminal signal anchor sequence.

Calmodulins (CaMs) are important mediators of Ca(2+) signals that are found ubiquitously in all eukaryotic organisms. Plants contain a unique family of calmodulin-like proteins (CMLs) that exhibit greater sequence variance compared to canonical CaMs. The Arabidopsis thaliana proteins AtCML4 and AtCML5 are members of CML subfamily VII and possess a CaM domain comprising the characteristic double pair of EF-hands, but they are distinguished from other members of this subfamily and from canonical CaMs by an N-terminal extension of their amino acid sequence. Transient expression of yellow fluorescent protein-tagged AtCML4 and AtCML5 under a 35S-promoter in Nicotiana benthamiana leaf cells revealed a spherical fluorescence pattern. This pattern was confirmed by transient expression in Arabidopsis protoplasts under the native promoter. Co-localization analyses with various endomembrane marker proteins suggest that AtCML4 and AtCML5 are localized to vesicular structures in the interphase between Golgi and the endosomal system. Further studies revealed AtCML5 to be a single-pass membrane protein that is targeted into the endomembrane system by an N-terminal signal anchor sequence. Self-assembly green fluorescent protein and protease protection assays support a topology with the CaM domain exposed to the cytosolic surface and not the lumen of the vesicles, indicating that AtCML5 could sense Ca(2+) signals in the cytosol. Phylogenetic analysis suggests that AtCML4 and AtCML5 are closely related paralogues originating from a duplication event within the Brassicaceae family. CML4/5-like proteins seem to be universally present in eudicots but are absent in some monocots. Together these results show that CML4/5-like proteins represent a flowering plant-specific subfamily of CMLs with a potential function in vesicle transport within the plant endomembrane system.

Chemical shift assignments of calmodulin constructs with EF hand mutations.

Calmodulin (CaM) is a ubiquitous cytosolic Ca(2+)-binding protein able to bind and regulate hundreds of different proteins. It consists of two globular domains joined by a flexible central linker region. Each one of these domains contains two EF hand pairs capable of binding to Ca(2+). Upon Ca(2+) binding CaM undergoes a conformational change exposing hydrophobic patches that interact with its intracellular target proteins. CaM is able to bind to target proteins in the Ca(2+)-replete and Ca(2+)-deplete forms. To study the Ca(2+)-dependent/independent properties of binding and activation of target proteins by CaM, CaM constructs with Ca(2+) binding disrupting mutations of Asp to Ala at position one of each EF hand have been used. One target protein of CaM is nitric oxide synthase, which catalyzes the production of nitric oxide. At elevated Ca(2+) concentrations, CaM binds to neuronal NOS and endothelial NOS, making them the Ca(2+)-dependent NOS enzymes. In contrast, inducible NOS is transcriptionally regulated in vivo and binds to CaM at basal levels of Ca(2+). Here we report the NMR backbone and sidechain resonance assignments of C-lobe Ca(2+)-replete and deplete CaM12, N-lobe Ca(2+)-replete and deplete CaM34, CaM1234 in the absence of Ca(2+) and N-lobe Ca(2+)-replete CaM34 with the iNOS CaM-binding domain peptide.

Abstract 4173: Discovery at the interface: novel anticancer agents targeting human estrogen receptor/S100 interactions

Abstract Estrogen-triggered nuclear transcription factor, estrogen receptor (ER) is expressed in about 70% of breast cancer. ER mediates various cellular signaling events including the genomic regulation of target genes that affect reproduction, development, and general metabolism. The antiestrogen tamoxifen is currently administered to patients in order to induce regression of the tumor growth. However, upon continued administration, breast cancer patients develop resistance to tamoxifen. In addition, calcium binding proteins such as, calmodulin (CaM) which are significantly overexpressed in cancer cells, can activate transcription of target genes by directly binding to ER in lieu of estrogen. Calmodulin antagonists (w7 and melatonin) have been shown to significantly inhibit human estrogen receptor alpha (hERalpha) transcriptional activity. In search of a novel anti-cancer agent, we generated a library of interaction profiles of human ER-EF hand protein complexes and identified S100B and S100P (high sequence and structure homology with CaM) as potential targets. Furthermore, S100P is shown to mediate tamoxifen-acquired resistance and cell migration capacity in MCF-7 breast cancer cells. Our objective is to develop peptidomimetics and small molecules to block ER-S100 interactions using an integrated proteomics-assisted drug discovery approach. Briefly, using the fold information and contact regions obtained from the FT-ICR mass spectrometry, 3D structures of ER-S100 protein complexes were reconstructed. These structures of ER best represent the conformational state sensed by the specific interacting partner. ER-S100 complex is distinct from CaM. The lack of the connective peptidic region between the EF hand pairs in S100 attributed to less interaction coverage than CaM. The identified ER conformation-sensing regions of the interacting EF hand proteins were used to develop a library of top peptidomimetics and potential small molecule sensors targeting the interfaces of ER-S100 interactions. We have evaluated their ability to block specific ER-S100 interactions in a high throughput fluorescent polarization based screen, to inhibit cancer cell proliferation and ER-mediated transcription of target genes. We believe that the ensemble of ER-EF hand protein complexes generated by our integrated proteomics-assisted protein interaction profiling will shed light on the lingering issue of hormone independent activation of ER at the molecular level and novel therapeutics targeting ER-S100 interactions. Citation Format: David H. Lee, Bethany K. Asare, Matthew L. Hudson, Rajendram V. Rajnarayanan. Discovery at the interface: novel anticancer agents targeting human estrogen receptor/S100 interactions. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4173. doi:10.1158/1538-7445.AM2014-4173

Structural coupling of the EF hand and C‐terminal GTPase domains in the mitochondrial protein Miro

Miro is a highly conserved calcium-binding GTPase at the regulatory nexus of mitochondrial transport and autophagy. Here we present crystal structures comprising the tandem EF hand and carboxy terminal GTPase (cGTPase) domains of Drosophila Miro. The structures reveal two previously unidentified 'hidden' EF hands, each paired with a canonical EF hand. Each EF hand pair is bound to a helix that structurally mimics an EF hand ligand. A key nucleotide-sensing element and a Pink1 phosphorylation site both lie within an extensive EF hand-cGTPase interface. Our results indicate structural mechanisms for calcium, nucleotide and phosphorylation-dependent regulation of mitochondrial function by Miro.

Abstract 2891: Comparative analyses of human estrogen receptor- EF-hand protein complexes: molecular basis for hormone independent activation.

Abstract Estrogen Receptor (ER) belongs to the nuclear receptor super family of ligand-triggered transcription factors. ER is present in about 70% of breast cancers and plays a critical role in the biology of breast carcinoma and improves prognosis. Estrogen binds to ER and mediates a cascade of cellular signaling events. It has been shown that several proteins including calcium binding proteins with EF-hand motifs, which control cellular calcium and calcium mediated signaling pathways, including calmodulin (CaM) could bind to unliganded ER and activate ER. CaM has four EF-hand motifs that change conformation upon binding Ca2+ ions. Ca2+-CaM complex binds to the target proteins and initiates various signaling cascades. Each EF-hand motif contains two alpha helices connected by a 12-residue loop. Both X-ray and NMR studies show that the alpha helices of the EF-hand motifs change their position relative to each other, forming an almost perpendicular conformation. This allows calmodulin to increase its binding affinity for a number of target proteins. CaM forms a compact globular conformation by bending its central helix upon binding its target peptides. S100, a soluble protein that is recently reported to interact with ER has only two EF-hand motifs. S100 protein is overexpressed in several cancers and shares a high degree of sequence homology with calmodulin. Methods: Protein-protein interactions of CaM, S100 with ER alpha were simulated using HADDOCK and HEX. Docking results were analyzed in UCSF Chimera and integrated with data from in vitro proteomics experiments to determine the conformations of ER bound EF-hand protein complexes. Proteomic experiments consisted of chemical crosslinking of the corresponding protein complexes followed by tryptic digestion, analyzing the resulting peptide peaks using FTICR- Mass Spectrometry. Results/Discussion: Using the fold information and contact regions obtained from the Mass spectrometry, 3D structures of ER-EF hand protein complexes were reconstructed. These structures of ER best represent the conformational state sensed by the specific interacting partner. ER-S100 complex is distinct from CaM. The lack of the connective peptidic region between the EF-hand pairs in S100 attributed to less interaction coverage indicative of only inducing a partial agonist-like conformation. We believe that the ensemble of ER-EF hand protein complexes generated by our integrated proteomics-assisted protein interaction profiling will shed light on the lingering issue of hormone independent activation of ER at the molecular level. Acknowledgements: This research was supported via funds from the New York State, National Institutes of Health (5P20MD002725), through the NCMHD -RIMI program (RVR) Citation Format: David H. Lee, Bethany K. Asare, Suchitra Singh, Rajendram V. Rajnarayanan. Comparative analyses of human estrogen receptor- EF-hand protein complexes: molecular basis for hormone independent activation. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2891. doi:10.1158/1538-7445.AM2013-2891

Mapping the Binding and Calmodulin-Dependent Activation of Nitric Oxide Synthase Isozymes

Calmodulin (CaM) is a ubiquitous cytosolic Ca 2+ -binding protein that can bind and activate numerous intracellular target proteins including the nitric oxide synthase (NOS) enzymes. There are three different isoforms of NOS found in mammals - neuronal NOS (nNOS), endothelial NOS (eNOS), and inducible NOS (iNOS) - with all three isozymes capable of producing the free radical, nitric oxide (•NO). At elevated cellular Ca 2+ concentrations, CaM is able to bind and activate nNOS and eNOS. In contrast, the iNOS isozyme is transcriptionally regulated and binds to CaM in the absence of Ca 2+ . To further investigate the differences in the association of CaM to the Ca 2+ -dependent and Ca 2+ - independent NOS isoforms, a variety of CaM mutants were employed. These included CaM-troponin C chimeras, CaM EF hand pair proteins, CaM mutants incapable of binding to Ca 2+ , methionine oxidized CaM mutants, phosphomimetic CaM mutants and central linker CaM mutants. By characterizing these CaM proteins using a variety of biochemical techniques, we have mapped the binding and the CaM-dependent activation of the NOS enzymes to study their effect on the different electron transfer steps within the NOS enzymes. In addition, fluorescence and Forster resonance energy transfer (FRET) were used to monitor the orientation and conformation of CaM when bound to the NOS peptides and enzymes. Combining these cumulative results, a working model describing the CaM-dependent regulation of iNOS is proposed.

Follistatin-like 1 Suppresses Sensory Afferent Transmission by Activating Na+,K+-ATPase

Excitatory synaptic transmission is modulated by inhibitory neurotransmitters and neuromodulators. We found that the synaptic transmission of somatic sensory afferents can be rapidly regulated by a presynaptically secreted protein, follistatin-like 1 (FSTL1), which serves as a direct activator of Na(+),K(+)-ATPase (NKA). The FSTL1 protein is highly expressed in small-diameter neurons of the dorsal root ganglion (DRG). It is transported to axon terminals via small translucent vesicles and secreted in both spontaneous and depolarization-induced manners. Biochemical assays showed that FSTL1 binds to the α1 subunit of NKA and elevates NKA activity. Extracellular FSTL1 induced membrane hyperpolarization in cultured cells and inhibited afferent synaptic transmission in spinal cord slices by activating NKA. Genetic deletion of FSTL1 in small DRG neurons of mice resulted in enhanced afferent synaptic transmission and sensory hypersensitivity, which could be reduced by intrathecally applied FSTL1 protein. Thus, FSTL1-dependent activation of NKA regulates the threshold of somatic sensation.

Regulation and Function of the RasGRP Family of Ras Activators in Blood Cells

Ras guanyl nucleotide releasing proteins (RasGRPs) are guanyl nucleotide exchange factors that activate Ras and related GTPases such as Rap. Like Sos proteins, RasGRPs have a catalytic region composed of a Ras exchange motif (REM) and a CDC25 domain. RasGRPs also possess a pair of atypical EF hands that may bind calcium in vivo and a C1 domain resembling the diacylglycerol (DAG)-binding domain of protein kinase C. DAG directly activates RasGRPs by a membrane recruitment mechanism as well as indirectly by PKC-mediated phosphorylation. RasGRPs are prominently expressed in blood cells. RasGRP1 acts downstream of TCR, while RasGRP1 and RasGRP3 both act downstream of BCR. Together, they regulate Ras in adaptive immune cells. RasGRP2, through Rap, plays a role in controlling platelet adhesion, while RasGRP4 controls Ras activation in mast cells. RasGRP malfunction likely contributes to autoimmunity and may contribute to blood malignancies. RasGRPs might prove to be viable drug targets. The intracellular site of RasGRP action and the relationship between RasGRPs and other Ras regulatory mechanisms are subjects of lively debate.

Study of the Cooperativity of Calcium Binding in Calbindin D9k using 2D Replica-Exchange Umbrella Sampling

Department of Biochemistry and Molecular Biology, The University of Chicago, IL 60637 Argonne National Laboratory, Argonne, IL 60439 Calbindin D9k, a small protein possessing a pair of EF-hands that binds two calcium ions in a cooperative fashion, is chosen as a model system for studying the molecular mechanism of cooperativity.1 Binding is characterized in terms of a potential of mean force (PMF) as a function two variables: the distance r between the ion, and the binding pocket, and the root-mean-square deviation (RMSD) of the conformation of the EF-hand relative to its ion-bound structure. The PMF is calculated using a novel two-dimensional replica-exchange molecular dynamics (MD) umbrella sampling scheme, which is developed and implemented in the program CHARMM to increase the configuration space sampling. Using 2048 replicas on Blue Gene/P, the 2D-PMF/REMD calculation for the binding the second calcium ion converges within 800 ps, with an exchange probability of 30%. In contrast, standard umbrella sampling PMF/MD simulations without replica-exchange did not converge, even after 1.2 ns. The absolute binding free energy of a second ion to a singly occupied calbindin calculated from the 2D-PMF following the statistical mechanical formulation of noncovalent association2 is −9.4 kcal/mol, in excellent agreement with the experimental value3. The 2D-PMF/REMD simulations will be extended to provide important information about the molecular basis of calcium binding cooperativity. Footnotes 1 Marchand S. and Roux B. Protein Struct. Funct. Genet. 1998, 33, 265-284. 2 Woo H. and Roux B., PNAS, 2005, 19, 6825. 3 Linse S. et al. Biochemistry 1991, 30, 154.

Calcium-Dependent Protein Kinase CPK21 Functions in Abiotic Stress Response in Arabidopsis thaliana

Calcium-dependent protein kinases (CDPKs) comprise a family of plant serine/threonine protein kinases in which the calcium sensing domain and the kinase effector domain are combined within one molecule. So far, a biological function in abiotic stress signaling has only been reported for few CDPK isoforms, whereas the underlying biochemical mechanism for these CDPKs is still mainly unknown. Here, we show that CPK21 from Arabidopsis thaliana is biochemically activated in vivo in response to hyperosmotic stress. Loss-of-function seedlings of cpk21 are more tolerant to hyperosmotic stress and mutant plants show increased stress responses with respect to marker gene expression and metabolite accumulation. In transgenic Arabidopsis complementation lines in the cpk21 mutant background, in which either CPK21 wild-type, or a full-length enzyme variant carrying an amino-acid substitution were stably expressed, stress responsitivity was restored by CPK21 but not with the kinase inactive variant. The biochemical characterization of in planta synthesized and purified CPK21 protein revealed that within the calcium-binding domain, N-terminal EF1- and EF2-motifs compared to C-terminal EF3- and EF4-motifs differ in their contribution to calcium-regulated kinase activity, suggesting a crucial role for the N-terminal EF-hand pair. Our data provide evidence for CPK21 contributing in abiotic stress signaling and suggest that the N-terminal EF-hand pair is a calcium-sensing determinant controlling specificity of CPK21 function.

Structure of the N-terminal Regulatory Domain of a Plant NADPH Oxidase and Its Functional Implications

Plant NADPH oxidases (Rboh, for respiratory burst oxidase homolog) produce reactive oxygen species that are key regulators of various cellular events including plant innate immunity. Rbohs possess a highly conserved cytoplasmic N-terminal region containing two EF-hand motifs that regulate Rboh activity. Rice (Oryza sativa) RbohB (OsRbohB) is regulated by the direct binding of a small GTPase (Rac1) to this regulatory region as well as by Ca(2+) binding to the EF-hands. Here, we present the atomic structure of the N-terminal region of OsRbohB. The structure reveals that OsRbohB forms a unique dimer stabilized by swapping the EF-hand motifs. We identified two additional EF-hand-like motifs that were not predicted from sequence data so far. These EF-hand-like motifs together with the swapped EF-hands form a structure similar to that found in calcineurin B. We observed conformational changes mediated by Ca(2+) binding to only one EF-hand. Structure-based in vitro pulldown assays and NMR titration experiments defined the OsRac1 binding interface within the coiled-coil region created by swapping the EF-hands. In addition, we demonstrate a direct intramolecular interaction between the N and C terminus, and that the complete N-terminal cytoplasmic region is required for this interaction. The structural features and intramolecular interactions characterized here might be common elements shared by Rbohs that contribute to the regulation of reactive oxygen species production.

Structural And Mechanistic Insights Into Stim1-mediated Initiation Of Store Operated Calcium Entry.

Stromal interaction molecule-1 (STIM1) activates store operated Ca2+ entry (SOCE) in response to diminished luminal Ca2+ levels. We have recently determined the solution structure of the Ca2+−sensing region of STIM1 consisting of the EF-hand and sterile α motif (SAM) domains (EF-SAM) (Stathopulos et al. Cell Oct 3rd issue, 2008). The canonical EF-hand is paired with a previously unidentified EF-hand. Together, the EF-hand pair mediates mutually indispensable hydrophobic interactions between the EF-hand and SAM domains. Structurally critical mutations in the canonical EF-hand, ‘hidden’ EF-hand or SAM domain disrupt Ca2+ sensitivity in oligomerization via destabilization of the entire EF-SAM entity. In mammalian cells, EF-SAM destabilization mutations within full-length STIM1 induce punctae formation and activate SOCE independent of luminal Ca2+. We provide atomic resolution insight into the molecular basis for STIM1-mediated SOCE initiation and show that the folded/unfolded state of the Ca2+ sensing region of STIM is crucial to SOCE regulation. (Supported by CIHR and CFI).