A 1.3-Å Structure of Zinc-bound N-terminal Domain of Calmodulin Elucidates Potential Early Ion-binding Step

Abstract

SummaryCalmodulin (CaM) is a 16.8 kDa calcium binding protein involved in calcium-signal transduction. It is the canonical member of the EF-hand family of proteins, which are characterized by a helixloop-helix calcium-binding motif. CaM is comprised of N-and C-terminal globular domains (NCaM and C-CaM), and within each domain there are two EF-hand motifs. Upon binding calcium, CaM undergoes a significant, global conformational change involving reorientation of the four helix bundles in each of its two domains. This conformational change upon ion binding is a key component of the signal transduction and regulatory roles of CaM, yet the precise nature of this transition is still unclear. Here, we present a 1.3Å structure of zinc-bound N-terminal calmodulin (N-CaM) solved by single wavelength anomalous diffraction (SAD) phasing of a selenomethionyl-N-CaM. Our zincbound N-CaM structure differs from previously reported CaM structures and resembles calcium-free apo-CaM despite the zinc binding to both EF-hand motifs. Structural comparison with calcium-free apo-CaM, calcium-loaded CaM, and a crosslinked calcium-loaded CaM suggests that our zinc-bound N-CaM reveals an intermediate step in the initiation of metal ion binding at the first EF-hand motif. Our data also suggests that metal ion coordination by two key residues in the first metal-binding site represents an initial step in the conformational transition induced by metal binding. This is followed by reordering of the N-terminal region of the helix exiting from this first binding loop. This conformational switch should be incorporated into models of either step-wise conformational transition or flexible, dynamic energetic state-sampling based transition.

X-ray Structures of Magnesium and Manganese Complexes with the N-Terminal Domain of Calmodulin: Insights into the Mechanism and Specificity of Metal Ion Binding to an EF-Hand

“…A search for the crystallization conditions using the Hampton Research Crystal Screens I and II yielded tetragonal crystals (space-group P4 3 2 1 2), which contained also Zn 2+ ions in addition to Mg 2+ . Similar crystals of N-CaM obtained in the presence of Zn 2+ only have been previously reported by Warren et al (39). Upon further search we have obtained triclinic crystals of N-CaM in the presence of Mg 2+ only.…”

“…The structure was solved by molecular replacement using the coordinates of N-CaM structure obtained in the presence of Zn 2+ only (39)(PDB code 2PQ3). Both Ca 2+ -binding loops of N-CaM in the Mg/Zn-N-CaM crystal contain an octahedrally coordinated metal ion.…”

An allosteric model of calmodulin explains differential activation of PP2B and CaMKII

“…Rather, the T state represents a collection of structures that may differ somewhat in the conformation of the calcium-binding sites, but whose overall structure resembles that of apo calmodulin. The existence of an ion-bound form that resembles the apo conformation has recently been established (51). Likewise, the R state is a collection of structures that resemble the reported open structure of active calmodulin (52,53).…”

Calcium-Enhanced Twitching Motility in Xylella fastidiosa Is Linked to a Single PilY1 Homolog

“…Here, examination of elements, such as Ca, Mg, Fe, Zn, and Cu, previously shown to promote or suppress X. fastidiosa biofilm formation (29) indicated that only Ca increased twitching motility in vitro. Ca binding was confirmed for the recombinant PilY1-1611 protein, and while Mg was also detected in the purified protein, the failure of this element to modulate twitching suggests that this could be due to the fact that Ca-binding motifs can also bind other divalent cations (49,50). Chelation of extracellular Ca by EGTA produced larger (on agar plates) and faster (in microfluidic chambers) decreases in twitching than intracellular chelation (BAPTA/AM), a finding consistent with the easy removal of Ca from exposed proteins or other molecules.…”