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Title: Crystallization and preliminary X-ray analysis of the Ca{sup 2+}-bound C-terminal lobe of troponin C in complex with a troponin I-derived peptide fragment from Akazara scallop

Abstract

Recombinant TnC was expressed in Escherichia coli, purified, complexed with a 24-residue synthetic peptide derived from scallop troponin I (TnI) and crystallized. Troponin C (TnC) is the Ca{sup 2+}-binding component of troponin and triggers muscle contraction. TnC of the invertebrate Akazara scallop can bind only one Ca{sup 2+} at the C-terminal EF-hand motif. Recombinant TnC was expressed in Escherichia coli, purified, complexed with a 24-residue synthetic peptide derived from scallop troponin I (TnI) and crystallized. The crystals diffracted X-rays to 1.80 Å resolution and belonged to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 32.1, b = 42.2, c = 60.0 Å. The asymmetric unit was assumed to contain one molecular complex of the Akazara scallop TnC C-lobe and TnI fragment, with a Matthews coefficient of 1.83 Å{sup 3} Da{sup −1} and a solvent content of 33.0%.

Authors:
 [1];  [2]; ;  [1]; ; ;  [3];  [4];  [1]
  1. Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, Bunkyo-ku, Tokyo 113-8657 (Japan)
  2. (Japan)
  3. Laboratory of Biochemistry and Biotechnology, Graduate School of Fisheries Sciences, Hokkaido University, Hakodate, Hokkaido 041-8611 (Japan)
  4. Department of Physiology II, The Jikei University School of Medicine, 3-19-18 Nishi-shinbashi, Minato-ku, Tokyo 105-8461 (Japan)
Publication Date:
OSTI Identifier:
22360337
Resource Type:
Journal Article
Resource Relation:
Journal Name: Acta Crystallographica. Section F; Journal Volume: 63; Journal Issue: Pt 6; Other Information: PMCID: PMC2335068; PMID: 17554181; PUBLISHER-ID: bo5017; OAI: oai:pubmedcentral.nih.gov:2335068; Copyright (c) International Union of Crystallography 2007; Country of input: International Atomic Energy Agency (IAEA)
Country of Publication:
United Kingdom
Language:
English
Subject:
75 CONDENSED MATTER PHYSICS, SUPERCONDUCTIVITY AND SUPERFLUIDITY; CONTRACTION; CRYSTALLIZATION; CRYSTALS; ESCHERICHIA COLI; RESOLUTION; SOLVENTS; SPACE GROUPS

Citation Formats

Yumoto, Fumiaki, Department of Physiology II, The Jikei University School of Medicine, 3-19-18 Nishi-shinbashi, Minato-ku, Tokyo 105-8461, Nagata, Koji, Miyauchi, Yumiko, Ojima, Takao, Tanaka, Hiroyuki, Nishita, Kiyoyoshi, Ohtsuki, Iwao, and Tanokura, Masaru, E-mail: amtanok@mail.ecc.u-tokyo.ac.jp. Crystallization and preliminary X-ray analysis of the Ca{sup 2+}-bound C-terminal lobe of troponin C in complex with a troponin I-derived peptide fragment from Akazara scallop. United Kingdom: N. p., 2007. Web. doi:10.1107/S1744309107024712.
Yumoto, Fumiaki, Department of Physiology II, The Jikei University School of Medicine, 3-19-18 Nishi-shinbashi, Minato-ku, Tokyo 105-8461, Nagata, Koji, Miyauchi, Yumiko, Ojima, Takao, Tanaka, Hiroyuki, Nishita, Kiyoyoshi, Ohtsuki, Iwao, & Tanokura, Masaru, E-mail: amtanok@mail.ecc.u-tokyo.ac.jp. Crystallization and preliminary X-ray analysis of the Ca{sup 2+}-bound C-terminal lobe of troponin C in complex with a troponin I-derived peptide fragment from Akazara scallop. United Kingdom. doi:10.1107/S1744309107024712.
Yumoto, Fumiaki, Department of Physiology II, The Jikei University School of Medicine, 3-19-18 Nishi-shinbashi, Minato-ku, Tokyo 105-8461, Nagata, Koji, Miyauchi, Yumiko, Ojima, Takao, Tanaka, Hiroyuki, Nishita, Kiyoyoshi, Ohtsuki, Iwao, and Tanokura, Masaru, E-mail: amtanok@mail.ecc.u-tokyo.ac.jp. Fri . "Crystallization and preliminary X-ray analysis of the Ca{sup 2+}-bound C-terminal lobe of troponin C in complex with a troponin I-derived peptide fragment from Akazara scallop". United Kingdom. doi:10.1107/S1744309107024712.
@article{osti_22360337,
title = {Crystallization and preliminary X-ray analysis of the Ca{sup 2+}-bound C-terminal lobe of troponin C in complex with a troponin I-derived peptide fragment from Akazara scallop},
author = {Yumoto, Fumiaki and Department of Physiology II, The Jikei University School of Medicine, 3-19-18 Nishi-shinbashi, Minato-ku, Tokyo 105-8461 and Nagata, Koji and Miyauchi, Yumiko and Ojima, Takao and Tanaka, Hiroyuki and Nishita, Kiyoyoshi and Ohtsuki, Iwao and Tanokura, Masaru, E-mail: amtanok@mail.ecc.u-tokyo.ac.jp},
abstractNote = {Recombinant TnC was expressed in Escherichia coli, purified, complexed with a 24-residue synthetic peptide derived from scallop troponin I (TnI) and crystallized. Troponin C (TnC) is the Ca{sup 2+}-binding component of troponin and triggers muscle contraction. TnC of the invertebrate Akazara scallop can bind only one Ca{sup 2+} at the C-terminal EF-hand motif. Recombinant TnC was expressed in Escherichia coli, purified, complexed with a 24-residue synthetic peptide derived from scallop troponin I (TnI) and crystallized. The crystals diffracted X-rays to 1.80 Å resolution and belonged to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 32.1, b = 42.2, c = 60.0 Å. The asymmetric unit was assumed to contain one molecular complex of the Akazara scallop TnC C-lobe and TnI fragment, with a Matthews coefficient of 1.83 Å{sup 3} Da{sup −1} and a solvent content of 33.0%.},
doi = {10.1107/S1744309107024712},
journal = {Acta Crystallographica. Section F},
number = Pt 6,
volume = 63,
place = {United Kingdom},
year = {Fri Jun 01 00:00:00 EDT 2007},
month = {Fri Jun 01 00:00:00 EDT 2007}
}
  • Akazara scallop (Chlamys nipponensis akazara) troponin C (TnC) of striated adductor muscle binds only one Ca{sup 2+} ion at the C-terminal EF-hand motif (Site IV), but it works as the Ca{sup 2+}-dependent regulator in adductor muscle contraction. In addition, the scallop troponin (Tn) has been thought to regulate muscle contraction via activating mechanisms that involve the region spanning from the TnC C-lobe (C-lobe) binding site to the inhibitory region of the TnI, and no alternative binding of the TnI C-terminal region to TnC because of no similarity between second TnC-binding regions of vertebrate and the scallop TnIs. To clarify themore » Ca{sup 2+}-regulatory mechanism of muscle contraction by scallop Tn, we have analyzed the Ca{sup 2+}-binding properties of the complex of TnC C-lobe and TnI peptide, and their interaction using isothermal titration microcalorimetry, nuclear magnetic resonance, circular dichroism, and gel filtration chromatography. The results showed that single Ca{sup 2+}-binding to the Site IV leads to a structural transition not only in Site IV but also Site III through the structural network in the C-lobe of scallop TnC. We therefore assumed that the effect of Ca{sup 2+}-binding must lead to a change in the interaction mode between the C-lobe of TnC and the TnI peptide. The change should be the first event of the transmission of Ca{sup 2+} signal to TnI in Tn ternary complex.« less
  • Crystals of the complex of the first von Willebrand type C domain (VWC1) of crossveinless 2 (CV2) bound to bone morphogenetic protein 2 (BMP2) exist in two tetragonal crystal forms belonging to either space group P4{sub 1}2{sub 1}2 or I4{sub 1}, with one complete BMP2 dimer and two CV2 VWC1 domains per asymmetric unit, and diffract to 2.6 Å resolution. Crossveinless 2 (CV2) is a member of the chordin family, a protein superfamily that modulates the activity of bone morphogenetic proteins such as BMP2. The BMPs represent a large group of secreted proteins that control many steps during embryonal developmentmore » and in tissue and organ homeostasis in the adult organism. The gene encoding the first von Willebrand type C domain (VWC1) of CV2 was cloned, expressed in Escherichia coli and purified to homogeneity. The binary complex of CV2 VWC1 and BMP2 was purified and subjected to crystallization. Crystals of SeMet-labelled proteins were obtained in two different forms belonging to the tetragonal space groups P4{sub 1}2{sub 1}2 and I4{sub 1}, with unit-cell parameters a = b = 86.7, c = 139.2 Å and a = b = 83.7, c = 139.6 Å, respectively. Initial analysis suggests that a complete binary complex consisting of one BMP2 dimer bound to two CV2 VWC1 domains is present in the asymmetric unit.« less
  • A C-terminal fragment of the Epstein–Barr virus lytic switch protein ZEBRA has been crystallized in complex with DNA. A C-terminal fragment of the Epstein–Barr virus immediate-early transcription factor ZEBRA has been expressed as a recombinant protein in Escherichia coli and purified to homogeneity. The fragment behaves as a dimer in solution, consistent with the presence of a basic region leucine-zipper (bZIP) domain. Crystals of the fragment in complex with a DNA duplex were grown by the hanging-drop vapour-diffusion technique using polyethylene glycol 4000 and magnesium acetate as crystallization agents. Crystals diffract to better than 2.5 Å resolution using synchrotron radiationmore » (λ = 0.976 Å). Crystals belong to space group C2, with unit-cell parameters a = 94.2, b = 26.5, c = 98.1 Å, β = 103.9°.« less
  • Peptide deformylase (PDF) from B. cereus has been overexpressed, purified and crystallized in ligand-free and actinonin-bound forms. Diffraction data have been collected from these crystals to 1.7 and 2.0 Å resolution, respectively. In bacteria, protein expression initiates with an N-formyl group and this needs to be removed in order to ensure proper bacterial growth. These formylation and deformylation processes are unique to eubacteria; therefore, inhibition of these would provide a novel antibacterial therapy. Deformylation is carried out by peptide deformylase (PDF). PDF from Bacillus cereus, one of the major pathogenic bacteria, was cloned into expression plasmid pET-28a (Novagen), overexpressed inmore » Escherichia coli BL21 (DE3) and purified to high quality. Crystals have been obtained of both ligand-free PDF and PDF to which actinonin, a highly potent naturally occurring inhibitor, is bound. Both crystals belong to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 42.72, b = 44.04, c = 85.19 Å and a = 41.31, b = 44.56, c = 84.47 Å, respectively. Diffraction data were collected to 1.7 Å resolution for the inhibitor-free crystals and to 2.0 Å resolution for the actinonin-bound crystals.« less
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