Congmin Li

The wheat and barley vernalization gene VRN3 is an orthologue of FT

Winter wheat and barley varieties require an extended exposure to low temperatures to accelerate flowering (vernalization), whereas spring varieties do not have this requirement. In this study, we show that in these species, the vernalization gene VRN3 is linked completely to a gene similar to Arabidopsis FLOWERING LOCUS T (FT). FT induction in the leaves results in a transmissible signal that promotes flowering. Transcript levels of the barley and wheat orthologues, designated as HvFT and TaFT, respectively, are significantly higher in plants homozygous for the dominant Vrn3 alleles (early flowering) than in plants homozygous for the recessive vrn3 alleles (late flowering). In wheat, the dominant Vrn3 allele is associated with the insertion of a retroelement in the TaFT promoter, whereas in barley, mutations in the HvFT first intron differentiate plants with dominant and recessive VRN3 alleles. Winter wheat plants transformed with the TaFT allele carrying the promoter retroelement insertion flowered significantly earlier than nontransgenic plants, supporting the identity between TaFT and VRN-B3. Statistical analyses of flowering times confirmed the presence of significant interactions between vernalization and FT allelic classes in both wheat and barley (P < 0.0001). These interactions were supported further by the observed up-regulation of HvFT transcript levels by vernalization in barley winter plants (P = 0.002). These results confirmed that the wheat and barley FT genes are responsible for natural allelic variation in vernalization requirement, providing additional sources of adaptive diversity to these economically important crops.

Structural and functional conservation of key domains in InsP3 and ryanodine receptors.

Inositol-1,4,5-trisphosphate receptors (InsP3Rs) and ryanodine receptors (RyRs) are tetrameric intracellular Ca2+ channels. In each of these receptor families, the pore, which is formed by carboxy-terminal transmembrane domains, is regulated by signals that are detected by large cytosolic structures. InsP3R gating is initiated by InsP3 binding to the InsP3-binding core (IBC, residues 224–604 of InsP3R1) and it requires the suppressor domain (SD, residues 1–223 of InsP3R1). Here we present structures of the amino-terminal region (NT, residues 1–604) of rat InsP3R1 with (3.6 Å) and without (3.0 Å) InsP3 bound. The arrangement of the three NT domains, SD, IBC-β and IBC-α, identifies two discrete interfaces (α and β) between the IBC and SD. Similar interfaces occur between equivalent domains (A, B and C) in RyR1 (ref. 9). The orientations of the three domains when docked into a tetrameric structure of InsP3R and of the ABC domains docked into RyR are remarkably similar. The importance of the α-interface for activation of InsP3R and RyR is confirmed by mutagenesis and, for RyR, by disease-causing mutations. Binding of InsP3 causes partial closure of the clam-like IBC, disrupting the β-interface and pulling the SD towards the IBC. This reorients an exposed SD loop (‘hotspot’ (HS) loop) that is essential for InsP3R activation. The loop is conserved in RyR and includes mutations that are associated with malignant hyperthermia and central core disease. The HS loop interacts with an adjacent NT, suggesting that activation re-arranges inter-subunit interactions. The A domain of RyR functionally replaced the SD in full-length InsP3R, and an InsP3R in which its C-terminal transmembrane region was replaced by that from RyR1 was gated by InsP3 and blocked by ryanodine. Activation mechanisms are conserved between InsP3R and RyR. Allosteric modulation of two similar domain interfaces within an N-terminal subunit reorients the first domain (SD or A domain), allowing it, through interactions of the second domain of an adjacent subunit (IBC-β or B domain), to gate the pore.

Structural insights into Ca2+-dependent regulation of inositol 1,4,5-trisphosphate receptors by CaBP1

Calcium-binding protein 1 (CaBP1), a neuron-specific member of the calmodulin (CaM) superfamily, modulates Ca2+-dependent activity of inositol 1,4,5-trisphosphate receptors (InsP3Rs). Here we present NMR structures of CaBP1 in both Mg2+-bound and Ca2+-bound states and their structural interaction with InsP3Rs. CaBP1 contains four EF-hands in two separate domains. The N-domain consists of EF1 and EF2 in a closed conformation with Mg2+ bound at EF1. The C-domain binds Ca2+ at EF3 and EF4, and exhibits a Ca2+-induced closed to open transition like that of CaM. The Ca2+-bound C-domain contains exposed hydrophobic residues (Leu132, His134, Ile141, Ile144, and Val148) that may account for selective binding to InsP3Rs. Isothermal titration calorimetry analysis reveals a Ca2+-induced binding of the CaBP1 C-domain to the N-terminal region of InsP3R (residues 1-587), whereas CaM and the CaBP1 N-domain did not show appreciable binding. CaBP1 binding to InsP3Rs requires both the suppressor and ligand-binding core domains, but has no effect on InsP3 binding to the receptor. We propose that CaBP1 may regulate Ca2+-dependent activity of InsP3Rs by promoting structural contacts between the suppressor and core domains.

NMR structure of DREAM: Implications for Ca(2+)-dependent DNA binding and protein dimerization.

DREAM (calsenilin/KChIP3) is an EF-hand calcium-binding protein that binds to specific DNA sequences and regulates Ca2+-induced transcription of prodynorphin and c-fos genes. Here, we present the atomic-resolution structure of Ca2+-bound DREAM in solution determined by nuclear magnetic resonance (NMR) spectroscopy. Pulsed-field gradient NMR diffusion experiments and 15N NMR relaxation analysis indicate that Ca2+-bound DREAM forms a stable dimer in solution. The structure of the first 77 residues from the N-terminus could not be determined by our NMR analysis. The C-terminal DREAM structure (residues 78-256) contains four EF-hand motifs arranged in a tandem linear array, similar to that seen in KChIP1, recoverin, and other structures of the neuronal calcium sensor (NCS) branch of the calmodulin superfamily. Mg2+ is bound at the second EF-hand, whereas Ca2+ is bound functionally at the third and fourth sites. The first and second EF-hands form an exposed hydrophobic groove on the protein surface lined by side-chain atoms of L96, F100, F114, I117, Y118, F121, F122, Y151, L155, L158, and L159 that are highly conserved in all NCS proteins. An exposed leucine near the C-terminus (L251) is suggested to form intermolecular contacts with leucine residues in the hydrophobic groove (L155, L158, and L159). Positively charged side chains of Arg and Lys (Lys87, Lys90, Lys91, Arg98, Lys101, Arg160, and Lys166) are clustered on one side of the protein surface and may mediate electrostatic contacts with DNA targets. We propose that Ca2+-induced dimerization of DREAM may partially block the putative DNA-binding site, which may suggest as to how Ca2+ abolishes DREAM binding to DNA to activate the transcription of prodynorphin and other downstream genes in pain control.

Structural analysis of Mg2+ and Ca2+ binding, myristoylation, and dimerization of the neuronal calcium sensor and visinin-like protein 1, VILIP-1

Visinin-like protein 1 (VILIP-1) belongs to the neuronal calcium sensor family of Ca2+-myristoyl switch proteins that regulate signal transduction in the brain and retina. Here we analyze Ca2+ and Mg2+ binding, characterize metal-induced conformational changes, and determine structural effects of myristoylation and dimerization. Mg2+ binds functionally to VILIP-1 at EF3 (ΔH = +1.8 kcal/mol and KD = 20 μm). Unmyristoylated VILIP-1 binds two Ca2+ sequentially at EF2 and EF3 (KEF3 = 0.1 μm and KEF2 = 1–4 μm), whereas myristoylated VILIP-1 binds two Ca2+ with lower affinity (KD = 1.2 μm) and positive cooperativity (Hill slope = 1.5). NMR assignments and structural analysis indicate that Ca2+-free VILIP-1 contains a sequestered myristoyl group like that of recoverin. NMR resonances of the attached myristate exhibit Ca2+-dependent chemical shifts and NOE patterns consistent with Ca2+-induced extrusion of the myristate. VILIP-1 forms a dimer in solution independent of Ca2+ and myristoylation. The dimerization site is composed of residues in EF4 and the loop region between EF3 and EF4, confirmed by mutagenesis. We present the structure of the VILIP-1 dimer and a Ca2+-myristoyl switch to provide structural insights into Ca2+-induced trafficking of nicotinic acetylcholine receptors.

CaBP1, a neuronal Ca2+ sensor protein, inhibits inositol trisphosphate receptors by clamping intersubunit interactions

Calcium-binding protein 1 (CaBP1) is a neuron-specific member of the calmodulin superfamily that regulates several Ca2+ channels, including inositol 1,4,5-trisphosphate receptors (InsP3Rs). CaBP1 alone does not affect InsP3R activity, but it inhibits InsP3-evoked Ca2+ release by slowing the rate of InsP3R opening. The inhibition is enhanced by Ca2+ binding to both the InsP3R and CaBP1. CaBP1 binds via its C lobe to the cytosolic N-terminal region (NT; residues 1–604) of InsP3R1. NMR paramagnetic relaxation enhancement analysis demonstrates that a cluster of hydrophobic residues (V101, L104, and V162) within the C lobe of CaBP1 that are exposed after Ca2+ binding interact with a complementary cluster of hydrophobic residues (L302, I364, and L393) in the β-domain of the InsP3-binding core. These residues are essential for CaBP1 binding to the NT and for inhibition of InsP3R activity by CaBP1. Docking analyses and paramagnetic relaxation enhancement structural restraints suggest that CaBP1 forms an extended tetrameric turret attached by the tetrameric NT to the cytosolic vestibule of the InsP3R pore. InsP3 activates InsP3Rs by initiating conformational changes that lead to disruption of an intersubunit interaction between a “hot-spot” loop in the suppressor domain (residues 1–223) and the InsP3-binding core β-domain. Targeted cross-linking of residues that contribute to this interface show that InsP3 attenuates cross-linking, whereas CaBP1 promotes it. We conclude that CaBP1 inhibits InsP3R activity by restricting the intersubunit movements that initiate gating.

Nuclear magnetic resonance structure of calcium-binding protein 1 in a Ca2+-bound closed state: Implications for target recognition

Calcium‐binding protein 1 (CaBP1), a neuron‐specific member of the calmodulin (CaM) superfamily, regulates the Ca2+‐dependent activity of inositol 1,4,5‐triphosphate receptors (InsP3Rs) and various voltage‐gated Ca2+ channels. Here, we present the NMR structure of full‐length CaBP1 with Ca2+ bound at the first, third, and fourth EF‐hands. A total of 1250 nuclear Overhauser effect distance measurements and 70 residual dipolar coupling restraints define the overall main chain structure with a root‐mean‐squared deviation of 0.54 Å (N‐domain) and 0.48 Å (C‐domain). The first 18 residues from the N‐terminus in CaBP1 (located upstream of the first EF‐hand) are structurally disordered and solvent exposed. The Ca2+‐saturated CaBP1 structure contains two independent domains separated by a flexible central linker similar to that in calmodulin and troponin C. The N‐domain structure of CaBP1 contains two EF‐hands (EF1 and EF2), both in a closed conformation [interhelical angles = 129° (EF1) and 142° (EF2)]. The C‐domain contains EF3 and EF4 in the familiar Ca2+‐bound open conformation [interhelical angles = 105° (EF3) and 91° (EF4)]. Surprisingly, the N‐domain adopts the same closed conformation in the presence or absence of Ca2+ bound at EF1. The Ca2+‐bound closed conformation of EF1 is reminiscent of Ca2+‐bound EF‐hands in a closed conformation found in cardiac troponin C and calpain. We propose that the Ca2+‐bound closed conformation of EF1 in CaBP1 might undergo an induced‐fit opening only in the presence of a specific target protein, and thus may help explain the highly specialized target binding by CaBP1.

Double electron-electron resonance probes Ca2+-induced conformational changes and dimerization of recoverin

Recoverin, a member of the neuronal calcium sensor (NCS) branch of the calmodulin superfamily, is expressed in retinal photoreceptor cells and serves as a calcium sensor in vision. Ca²⁺-induced conformational changes in recoverin cause extrusion of its covalently attached myristate (termed Ca²⁺-myristoyl switch) that promotes translocation of recoverin to disk membranes during phototransduction in retinal rod cells. Here we report double electron-electron resonance (DEER) experiments on recoverin that probe Ca²⁺-induced changes in distance as measured by the dipolar coupling between spin-labels strategically positioned at engineered cysteine residues on the protein surface. The DEER distance between nitroxide spin-labels attached at C39 and N120C is 2.5 ± 0.1 nm for Ca²⁺-free recoverin and 3.7 ± 0.1 nm for Ca²⁺-bound recoverin. An additional DEER distance (5-6 nm) observed for Ca²⁺-bound recoverin may represent an intermolecular distance between C39 and N120. ¹⁵N NMR relaxation analysis and CW-EPR experiments both confirm that Ca²⁺-bound recoverin forms a dimer at protein concentrations above 100 μM, whereas Ca²⁺-free recoverin is monomeric. We propose that Ca²⁺-induced dimerization of recoverin at the disk membrane surface may play a role in regulating Ca²⁺-dependent phosphorylation of dimeric rhodopsin. The DEER approach will be useful for elucidating dimeric structures of NCS proteins in general for which Ca²⁺-induced dimerization is functionally important but not well understood.

Structural Insights into Activation of the Retinal L-type Ca2+ Channel (Cav1.4) by Ca2+-binding Protein 4 (CaBP4)

Background: Cav1.4 is regulated by CaBP4, which is required for continuous release of neurotransmitter in retinal photoreceptor cells. Results: CaBP4 contains two separate EF-hand lobes that bind Ca2+ and form a collapsed structure around the IQ motif in Cav1.4. Conclusion: CaBP4 is suggested to activate Cav1.4 by disrupting an interaction between IQ and ICDI. Significance: CaBP4 mutations associated with congenital stationary night blindness impair its binding to IQ. CaBP4 modulates Ca2+-dependent activity of L-type voltage-gated Ca2+ channels (Cav1.4) in retinal photoreceptor cells. Mg2+ binds to the first and third EF-hands (EF1 and EF3), and Ca2+ binds to EF1, EF3, and EF4 of CaBP4. Here we present NMR structures of CaBP4 in both Mg2+-bound and Ca2+-bound states and model the CaBP4 structural interaction with Cav1.4. CaBP4 contains an unstructured N-terminal region (residues 1–99) and four EF-hands in two separate lobes. The N-lobe consists of EF1 and EF2 in a closed conformation with either Mg2+ or Ca2+ bound at EF1. The C-lobe binds Ca2+ at EF3 and EF4 and exhibits a Ca2+-induced closed-to-open transition like that of calmodulin. Exposed residues in Ca2+-bound CaBP4 (Phe137, Glu168, Leu207, Phe214, Met251, Phe264, and Leu268) make contacts with the IQ motif in Cav1.4, and the Cav1.4 mutant Y1595E strongly impairs binding to CaBP4. We conclude that CaBP4 forms a collapsed structure around the IQ motif in Cav1.4 that we suggest may promote channel activation by disrupting an interaction between IQ and the inhibitor of Ca2+-dependent inactivation domain.

1H, 15N, and 13C chemical shift assignments of calcium-binding protein 1 with Ca2+ bound at EF1, EF3 and EF4

Calcium-binding protein 1 (CaBP1) regulates inositol 1,4,5-trisphosphate receptors (InsP3Rs) and a variety of voltage-gated Ca2+ channels in the brain. We report complete NMR chemical shift assignments of the Ca2+-saturated form of CaBP1 with Ca2+ bound at EF1, EF3 and EF4 (residues 1–167, BMRB no. 16862).