Crystal structure of the trimeric N-terminal domain of ciliate Euplotes octocarinatus centrin binding with calcium ions

Abstract

Centrin is a member of the EF-hand superfamily of calcium-binding proteins, a highly conserved eukaryotic protein that binds to Ca²⁺ . Its self-assembly plays a causative role in the fiber contraction that is associated with the cell division cycle and ciliogenesis. In this study, the crystal structure of N-terminal domain of ciliate Euplotes octocarinatus centrin (N-EoCen) was determined by using the selenomethionine single-wavelength anomalous dispersion method. The protein molecules formed homotrimers. Every protomer had two putative Ca²⁺ ion-binding sites I and II, protomer A, and C bound one Ca²⁺ ion, while protomer B bound two Ca²⁺ ions. A novel binding site III was observed and the Ca²⁺ ion was located at the center of the homotrimer. Several hydrogen bonds, electrostatic, and hydrophobic interactions between the protomers contributed to the formation of the oligomer. Structural studies provided insight into the foundation for centrin aggregation and the roles of calcium ions.

Keywords: aggregation; calcium coordination; centrin; crystal structure; oligomer.

Figure 1

Overall structure of N‐EoCen. (A) Topology diagram of N‐EoCen. Helices are shown in blue and loops are green lines. (B) Schematic representation of N‐EoCen from the front (left) and the back (right). N‐ter, N terminal end; C‐ter, C‐terminal end. Two Ca²⁺ ion binding sites are indicated. (C) The triangle trimer of N‐EoCen is shown as a ribbon and electron potential surface representation from front view, side view, and back view. Positive regions are blue, negative regions are red, and neutral regions are shown in white.

Figure 2

Sequence and structure comparison. (A) Sequence alignment21 of N‐EoCen (PDB code: http://firstglance.jmol.org/fg.htm?mol=5Z1Q, http://firstglance.jmol.org/fg.htm?mol=2JOJ), N‐HuCen (PDB code: 1ZMZ), N‐CrCen (PDB code: 3QRX), C‐PtCam (PDB code: 1CLM), N‐MuCen (PDB code: 5D43). The strictly conserved residues are shown in white text with a red background, while the conserved residues are represented as red text with blue frames. The residues involved in calcium binding are indicated by blue arrows and red arrows. The residues that were disordered in the structures are indicated by black dashed lines. The sequence number and the secondary structure elements corresponding to N‐EoCen are on the top of the figure. (B) Structural comparison. Every structure is colored different color. http://firstglance.jmol.org/fg.htm?mol=5Z1Q: Blue, http://firstglance.jmol.org/fg.htm?mol=2JOJ: Red, http://firstglance.jmol.org/fg.htm?mol=1ZMZ: cyan, http://firstglance.jmol.org/fg.htm?mol=3QRX: Black, http://firstglance.jmol.org/fg.htm?mol=1CLM: claybank, 5D43: magenta. (C) Structural analyses of intermolecular interactions. Protomer A and protomer B are colored with yellow and claybank in the cartoon, respectively. The key residues, responsible for intermolecular interactions, are shown as colored sticks. (D) Close‐up view of three calcium binding sites. Site I (a), Site II (b), and Site III (c). The magenta molecule represents a Ca²⁺ ion and red molecules represent water molecules. The key residues, responsible for Ca²⁺ ion binding, are shown as colored sticks. (d) Fo‐Fc electron density omit map of Ca²⁺ ion at binding site III at 5 σ is shown.