Calsequestrin, the major calcium storage protein of both cardiac and skeletal muscle, binds and releases large numbers of Ca 2؉ ions for each contraction and relaxation cycle. Here we show that two crystal structures for skeletal and cardiac calsequestrin are nearly superimposable not only for their subunits but also their front-to-front-type dimers. Ca 2؉ binding curves were measured using atomic absorption spectroscopy. This method enables highly accurate measurements even for Ca 2؉ bound to polymerized protein. The binding curves for both skeletal and cardiac calsequestrin were complex, with binding increases that correlated with protein dimerization, tetramerization, and oligomerization. The Ca 2؉ binding capacities of skeletal and cardiac calsequestrin are directly compared for the first time, with ϳ80 Ca 2؉ ions bound per skeletal calsequestrin and ϳ60 Ca 2؉ ions per cardiac calsequestrin, as compared with net charges for these molecules of ؊80 and ؊69, respectively. Deleting the negatively charged and disordered C-terminal 27 amino acids of cardiac calsequestrin results in a 50% reduction of its calcium binding capacity and a loss of Ca 2؉ -dependent tetramer formation. Based on the crystal structures of rabbit skeletal muscle calsequestrin and canine cardiac calsequestrin, Ca 2؉ binding capacity data, and previous lightscattering data, a mechanism of Ca 2؉ binding coupled with polymerization is proposed.Calsequestrin (CSQ) 1 binds and releases large quantities of Ca 2ϩ through its high capacity (40 -50 mol of Ca 2ϩ ion per molecule) and relatively low affinity interactions with Ca 2ϩ (K d ϭ 1 mM) (1). Because of this Ca 2ϩ -buffering capacity of CSQ in the lumenal space, the concentration of free Ca 2ϩ in the sarcoplasmic reticulum (SR) can be maintained below the inhibitory level of the Ca 2ϩ pump (1 mM), and simultaneously, the SR can maintain the ability to rapidly deliver a high capacity Ca 2ϩ signal to the cytoplasm. Even though the lumenal space is minuscule compared with the extracellular space, the high concentrations (ϳ100 mg/ml) of CSQ make the SR an efficient storage compartment for Ca 2ϩ (2).CSQ is associated physically with the RyR protein by a nucleation event that involves CSQ binding to the basic lumenal domains of triadin (3) or junctin (4). These two proteins interact with RyR in the junctional face region of the SR, and this network of interacting proteins assures that high concentrations of Ca 2ϩ are stored very near to the site of Ca 2ϩ release. Ca 2ϩ release from CSQ through the Ca 2ϩ release channel is regulatory but not limiting.The Ca 2ϩ binding and dissociation mechanisms of CSQ are not yet clearly understood. Ca 2ϩ binding sites in CSQ are supposed to be very different from those in the Ca 2ϩ pump (sarco(endo)plasmic reticulum calcium ATPase (SERCA)), calmodulin, and troponin C. CSQ sites need to be made and broken but not over the low cytosolic Ca 2ϩ concentration range or with the same stoichiometry and precision as those formed and subsequently disrupted in the Ca 2ϩ pump or...

Ca2ϩ regulation is coupled to critical signals in eucaryotic cells, and calsequestrin is one of the crucial components for this calcium regulation. Our previous observations of calsequestrins revealed the existence of three thioredoxin-like folds, a basic motif that often provides the platform for small molecule binding. Therefore, we have examined the previously reported trifluoperazine and other pharmaceuticals that have similar heartrelated side effects (such as tachycardia; bradycardia; palpitation; changing PR, QRS, QTc intervals in electrocardiogram; heart failure) for their binding affinity to cardiac calsequestrin (cCSQ) using isothermal titration calorimetry. Our results showed that several antipsychotic phenothiazine derivatives, tricyclic antidepressants, and anthracycline derivatives bind cCSQ with K d in the micromolar range. For these compounds that have a significantly low K d , their effect on Ca 2ϩ binding capacity of cCSQ was checked using equilibrium dialysis and atomic absorption spectroscopy, which clearly showed a significant reduction in Ca 2ϩ binding capacity of cCSQ as a result of this interaction. Furthermore, 8-anilino-1-naphthalene sulfonate (ANS) binding to cCSQ closely resembles ANS binding to flavine or nucleotide binding sites. The combination of this information with the high abundance of CSQ in SR and the high membrane permeability of those drugs led us to the specific hypothesis that there are undesirable and damaging interactions between cCSQ and tricyclic antidepressants, phenothiazine derivatives, anthracyclines, and many other pharmaceutical compounds and to the corollary hypothesis that better understanding of the molecular details of cCSQ-drug interactions could lead to modified drug molecules with reduced heart-related side effects.

The cellular decision regarding whether to undergo proliferation or death is made at the restriction (R)-point, which is disrupted in nearly all tumors. The identity of the molecular mechanisms that govern the R-point decision is one of the fundamental issues in cell biology. We found that early after mitogenic stimulation, RUNX3 binds to its target loci, where it opens chromatin structure by sequential recruitment of Trithorax group proteins and cell-cycle regulators to drive cells to the R-point. Soon after, RUNX3 closes these loci by recruiting Polycomb repressor complexes, causing the cell to pass through the R-point toward S phase. If the RAS signal is constitutively activated, RUNX3 inhibits cell cycle progression by maintaining R-point-associated genes in an open structure. Our results identify RUNX3 as a pioneer factor for the R-point and reveal the molecular mechanisms by which appropriate chromatin modifiers are selectively recruited to target loci for appropriate R-point decisions.

Understanding the structural origins of differences in reduction potentials is crucial to understanding how various electron transfer proteins modulate their reduction potentials and how they evolve for diverse functional roles. Here, the high-resolution structures of several Clostridium pasteurianum rubredoxin (Cp Rd) variants with changes in the vicinity of the redox site are reported in order to increase this understanding. Our crystal structures of [V44L] (at 1.8 A resolution), [V44A] (1.6 A), [V44G] (2.0 A) and [V44A, G45P] (1.5 A) Rd (all in their oxidized states) show that there is a gradual decrease in the distance between Fe and the amide nitrogen of residue 44 upon reduction in the size of the side chain of residue 44; the decrease occurs from leucine to valine, alanine or glycine and is accompanied by a gradual increase in their reduction potentials. Mutation of Cp Rd at position 44 also changes the hydrogen-bond distance between the amide nitrogen of residue 44 and the sulfur of cysteine 42 in a size-dependent manner. Our results suggest that residue 44 is an important determinant of Rd reduction potential in a manner dictated by side-chain size. Along with the electric dipole moment of the 43-44 peptide bond and the 44-42 NH--S type hydrogen bond, a modulation mechanism for solvent accessibility through residue 41 might regulate the redox reaction of the Rds.

Snail contributes to the epithelial-mesenchymal transition by suppressing E-cadherin in transcription processes. The Snail C2H2-type zinc-finger (ZF) domain functions both as a nuclear localization signal which binds to importin β directly and as a DNA-binding domain. Here, a 2.5 Å resolution structure of four ZF domains of Snail1 complexed with importin β is presented. The X-ray structure reveals that the four ZFs of Snail1 are required for tight binding to importin β in the nuclear import of Snail1. The shape of the ZFs in the X-ray structure is reminiscent of a round snail, where ZF1 represents the head, ZF2-ZF4 the shell, showing a novel interaction mode, and the five C-terminal residues the tail. Although there are many kinds of C2H2-type ZFs which have the same fold as Snail, nuclear import by direct recognition of importin β is observed in a limited number of C2H2-type ZF proteins such as Snail, Wt1, KLF1 and KLF8, which have the common feature of terminating in ZF domains with a short tail of amino acids.

Rubredoxin is a small iron-sulfur (FeS4) protein involved in oxidation-reduction reactions. The side chain of Leu41 near the iron-sulfur center has two conformations, which we suggested previously serve as a gate for a water molecule during the electron transfer process. To establish the role of residue 41 in electron transfer, an [L41A] mutant of Clostridium pasteurianum rubredoxin was constructed and crystallized in both oxidation states. Despite the lack of the gating side chain in this protein, the structure of the reduced [L41A] rubredoxin reveals a specific water molecule in the same position as observed in the reduced wild-type rubredoxin. In contrast, both the wild-type and [L41A] rubredoxins in the oxidized state do not have water molecules in this location. The reduction potential of the [L41A] variant was approximately 50 mV more positive than wild-type. Based on these observations, it is proposed that the site around the Sgamma of Cys9 serves as a port for an electron acceptor. Lastly, the Fe-S distances of the reduced rubredoxin are expanded, while the hydrogen bonds between Sgamma of the cysteines and the backbone amide nitrogens are shortened compared to its oxidized counterpart. This small structural perturbation in the Fe(II)/Fe(III) transition is closely related to the small energy difference which is important in an effective electron transfer agent.

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