Rn [34]. The complex structure of CaM and IQ peptide from Lc-

Rn [34]. The complex structure of CaM and IQ peptide from Lc-, P/Q-, and R-type voltage-dependent Ca2+ channels is similar, wherein the central helix of CaM unwinds and peptides are wrapped by two lobes of CaM ( [35,36]; 3BXK and 3BXL). Similarly, the structure of apo CaM bound to the first two IQ motifs of the murine myosin V heavy chain adopts a unique conformation, in which central helix unwinds and the N and C lobes wrap around the peptides [37].All the above CaM complex structures adopt a cis conformation. However, in the current study, the conformation of CaM was in an extended form, and the two globular lobes were widely separated. As noted above, residues Ala74-Asp79 were unwound by one turn and formed a sharp bend at Arg75. However, incubating Nm/Ng or their IQ peptides with Ca2+/CaM did not show any spectral changes to indicate any conformational change in CaM [38]. Nonetheless, the observed conformation of CaM in this study 58-49-1 web represents a novel trans structure of CaM. The implication of this conformation is yet to be studied. The bending of the central helix is a key feature of the conformational dynamics of CaM in recognizing the target [9].Figure 3. Comparison of cis and trans conformations of Calmodulin. A: Side-by-side comparison of the novel trans (current structure, blue) and cis (pdb code 1PRW, magenta) conformations of CaM, which show the unwinding region of central helix in both structures in cyan and yellow, respectively. B: Side-by-side comparison of the novel trans (current structure, blue) and extended trans (pdb code 3CLN, dark salmon) 1527786 conformations of CaM. In the extended trans conformation of CaM, no unwinding of the central helix was observed. The positions of the metal ions in the current novel trans (blue) are labeled as Ca2+ (Green) and Zn2+ (grey). In wrapped cis (1PRW, magenta) and extended trans (3CLN, dark salmon), all sites (EF1EF4) were occupied by Ca2+ (Green). doi:10.1371/journal.pone.0054834.gA Novel Conformation of CalmodulinFigure 4. Coordination of Zn2+ ion: A Zn2+ ion (grey) bound to His108 and Lys95 of chain A (magenta), and Asp81 and Glu85 from chain B (blue). A similar Zn2+ ion is also present in chain A. doi:10.1371/journal.pone.0054834.gIt has to be noted that the Ca2+/CaM (buffer supplemented with 10 mM CaCl2) crystals were grown in conditions containing 5?0 mM ZnCl2. A previous flow dialysis study showed that CaM has two BI-78D3 web higher (80?00 mM) affinity Zn2+ ion sites and four or five lower affinity Zn2+-binding sites [22,39]. Nevertheless, the previously reported Zn2+-bound, N-lobe CaM structure resembles the apo CaM structures 1662274 (i.e. the closed form of CaM) [39]; by comparison, CaM adopts an open form in the current structure. Further, no similar bend at Arg75 was observed in the previous structure despite Zn2+ binding to both EF-hand motifs in the Nlobe [39]. Based on the heavy atom peak heights in the anomalous map and the refinement statistics comparison (R-values and Bfactors) we assigned the observed electron density as Ca2+ ions in the EF-hand motifs and a Zn2+ ion near His108 (Figure 4). However the possibility of having less occupancy Zn2+ that might mimic a fully occupied Ca2+ cannot be ruled out. We have observed that lowering the ZnCl2 concentration in crystallization conditions reduces the nucleation and results in good quality crystals. The observed Zn2+ ion at chain A is coordinating with His108, Lys95 (chain A), Asp81 and Glu81 (chain B) and vice versa for chain B. A similar coo.Rn [34]. The complex structure of CaM and IQ peptide from Lc-, P/Q-, and R-type voltage-dependent Ca2+ channels is similar, wherein the central helix of CaM unwinds and peptides are wrapped by two lobes of CaM ( [35,36]; 3BXK and 3BXL). Similarly, the structure of apo CaM bound to the first two IQ motifs of the murine myosin V heavy chain adopts a unique conformation, in which central helix unwinds and the N and C lobes wrap around the peptides [37].All the above CaM complex structures adopt a cis conformation. However, in the current study, the conformation of CaM was in an extended form, and the two globular lobes were widely separated. As noted above, residues Ala74-Asp79 were unwound by one turn and formed a sharp bend at Arg75. However, incubating Nm/Ng or their IQ peptides with Ca2+/CaM did not show any spectral changes to indicate any conformational change in CaM [38]. Nonetheless, the observed conformation of CaM in this study represents a novel trans structure of CaM. The implication of this conformation is yet to be studied. The bending of the central helix is a key feature of the conformational dynamics of CaM in recognizing the target [9].Figure 3. Comparison of cis and trans conformations of Calmodulin. A: Side-by-side comparison of the novel trans (current structure, blue) and cis (pdb code 1PRW, magenta) conformations of CaM, which show the unwinding region of central helix in both structures in cyan and yellow, respectively. B: Side-by-side comparison of the novel trans (current structure, blue) and extended trans (pdb code 3CLN, dark salmon) 1527786 conformations of CaM. In the extended trans conformation of CaM, no unwinding of the central helix was observed. The positions of the metal ions in the current novel trans (blue) are labeled as Ca2+ (Green) and Zn2+ (grey). In wrapped cis (1PRW, magenta) and extended trans (3CLN, dark salmon), all sites (EF1EF4) were occupied by Ca2+ (Green). doi:10.1371/journal.pone.0054834.gA Novel Conformation of CalmodulinFigure 4. Coordination of Zn2+ ion: A Zn2+ ion (grey) bound to His108 and Lys95 of chain A (magenta), and Asp81 and Glu85 from chain B (blue). A similar Zn2+ ion is also present in chain A. doi:10.1371/journal.pone.0054834.gIt has to be noted that the Ca2+/CaM (buffer supplemented with 10 mM CaCl2) crystals were grown in conditions containing 5?0 mM ZnCl2. A previous flow dialysis study showed that CaM has two higher (80?00 mM) affinity Zn2+ ion sites and four or five lower affinity Zn2+-binding sites [22,39]. Nevertheless, the previously reported Zn2+-bound, N-lobe CaM structure resembles the apo CaM structures 1662274 (i.e. the closed form of CaM) [39]; by comparison, CaM adopts an open form in the current structure. Further, no similar bend at Arg75 was observed in the previous structure despite Zn2+ binding to both EF-hand motifs in the Nlobe [39]. Based on the heavy atom peak heights in the anomalous map and the refinement statistics comparison (R-values and Bfactors) we assigned the observed electron density as Ca2+ ions in the EF-hand motifs and a Zn2+ ion near His108 (Figure 4). However the possibility of having less occupancy Zn2+ that might mimic a fully occupied Ca2+ cannot be ruled out. We have observed that lowering the ZnCl2 concentration in crystallization conditions reduces the nucleation and results in good quality crystals. The observed Zn2+ ion at chain A is coordinating with His108, Lys95 (chain A), Asp81 and Glu81 (chain B) and vice versa for chain B. A similar coo.