PDBsum entry 2jm3

Metal binding protein

PDB id

2jm3

Contents

			Protein chain

					91 a.a.

			Metals

					_ZN

References listed in PDB file

Key reference

Title

Solution structure of the thap domain from caenorhabditis elegans c-Terminal binding protein (ctbp).

Authors

C.K.Liew, M.Crossley, J.P.Mackay, H.R.Nicholas.

Ref.

J Mol Biol, 2007, 366, 382-390. [DOI no: 10.1016/j.jmb.2006.11.058]

PubMed id

17174978

Abstract

The THAP (Thanatos-associated protein) domain is a recently discovered zinc-binding domain found in proteins involved in transcriptional regulation, cell-cycle control, apoptosis and chromatin modification. It contains a single zinc atom ligated by cysteine and histidine residues within a Cys-X(2-4)-Cys-X(35-53)-Cys-X(2)-His consensus. We have determined the NMR solution structure of the THAP domain from Caenorhabditis elegans C-terminal binding protein (CtBP) and show that it adopts a fold containing a treble clef motif, bearing similarity to the zinc finger-associated domain (ZAD) from Drosophila Grauzone. The CtBP THAP domain contains a large, positively charged surface patch and we demonstrate that this domain can bind to double-stranded DNA in an electrophoretic mobility-shift assay. These data, together with existing reports, indicate that THAP domains might exhibit a functional diversity similar to that observed for classical and GATA-type zinc fingers.



	Figure 2. Figure 2. Solution structure of C. elegans CtBP-TD. Zinc atoms are shown as yellow spheres. (a) Stereo view of the 20 lowest energy structures. Poorly defined regions of the structure are shown in magenta. (b) A ribbon diagram of a representative structure in the same orientation as (a). Side-chains of the zinc ligating residues are shown in red. (c) The family of structures showing residues with well-ordered side-chains. The side-chains of highly conserved residues are shown in purple. Methods: DNA encoding the THAP domain (residues 1–89) of C. elegans CtBP was amplified from C. elegans cDNA using PCR and this fragment was subcloned into the pGEX-2T expression vector. The resulting plasmid was transformed into E. coli BL21(DE3) cells, which were grown in minimal medium supplemented with ^15NH[4]Cl and [^13C]glucose. Cell growth and protein expression were carried out in a fermentor.^32 CtBP-TD was expressed as a GST fusion protein and was purified from the cell lysate using glutathione Sepharose beads. The THAP domain was released from the GST using thrombin, leaving Gly-Ser at the N terminus of the protein. This protein was then further purified using gel-filtration chromatography. The protein fractions were concentrated up to a concentration of 1 mM in buffer containing 20 mM NaH[2]PO[4] (pH 6.5), 100 mM NaCl, 1 mM DTT and 0.01% (w/v) NaN[3], with 0.1 mM DSS added as an internal reference. All NMR spectra were acquired at 298 K on a Bruker DRX 600 spectrometer, equipped with a triple resonance cryoprobe and z-axis pulsed field gradients. HNCA, HN(CO)CA, HNCO, HN(CA)CO, HNCACB and CBCA(CO)NH experiments were recorded for backbone assignment. C(CO)NH-TOCSY, H(CCO)NH-TOCSY, HCCH-TOCSY and HCCH- correlated spectroscopy (COSY) experiments enabled the assignment of side-chains. Interproton distances were derived from a ^15N-HSQC-NOESY (τ[m] = 100 ms), a ^13C-HSQC-NOESY (τ[m] = 80 ms) and a homonuclear ^1H-NOESY (τ[m] = 100 ms) spectrum. All spectra were processed with TopSpin (Bruker, Karlsruhe) and analyzed using Sparky [http://www.cgl.ucsf.edu/home/sparky/]. Backbone and ψ dihedral angle restraints were determined from an HNHA spectrum as well as from backbone chemical shifts using TALOS.^33 Pro33 was found to be in the cis conformation, based on the chemical shift difference between the C^β and C^γ atoms of the proline.^34 The zinc coordination geometry was defined as tetrahedral using angle (Zn-C-N at 125°, S-Zn-S at 109°, S-Zn-N^ε2and C-S-Zn at 107°) and bond (2.3 Å for Zn-S and 2 Å for Zn-N) constraints. Structures were calculated using the experimentally derived restraints with ARIA 1.2/CNS.^[35.]^ and ^[36.] Eight iterations of structure calculations were performed using the standard protocols provided with the software. Figure 2. Solution structure of C. elegans CtBP-TD. Zinc atoms are shown as yellow spheres. (a) Stereo view of the 20 lowest energy structures. Poorly defined regions of the structure are shown in magenta. (b) A ribbon diagram of a representative structure in the same orientation as (a). Side-chains of the zinc ligating residues are shown in red. (c) The family of structures showing residues with well-ordered side-chains. The side-chains of highly conserved residues are shown in purple. Methods: DNA encoding the THAP domain (residues 1–89) of C. elegans CtBP was amplified from C. elegans cDNA using PCR and this fragment was subcloned into the pGEX-2T expression vector. The resulting plasmid was transformed into E. coli BL21(DE3) cells, which were grown in minimal medium supplemented with ^15NH[4]Cl and [^13C]glucose. Cell growth and protein expression were carried out in a fermentor.[3]^32 CtBP-TD was expressed as a GST fusion protein and was purified from the cell lysate using glutathione Sepharose beads. The THAP domain was released from the GST using thrombin, leaving Gly-Ser at the N terminus of the protein. This protein was then further purified using gel-filtration chromatography. The protein fractions were concentrated up to a concentration of 1 mM in buffer containing 20 mM NaH[2]PO[4] (pH 6.5), 100 mM NaCl, 1 mM DTT and 0.01% (w/v) NaN[3], with 0.1 mM DSS added as an internal reference. All NMR spectra were acquired at 298 K on a Bruker DRX 600 spectrometer, equipped with a triple resonance cryoprobe and z-axis pulsed field gradients. HNCA, HN(CO)CA, HNCO, HN(CA)CO, HNCACB and CBCA(CO)NH experiments were recorded for backbone assignment. C(CO)NH-TOCSY, H(CCO)NH-TOCSY, HCCH-TOCSY and HCCH- correlated spectroscopy (COSY) experiments enabled the assignment of side-chains. Interproton distances were derived from a ^15N-HSQC-NOESY (τ[m] = 100 ms), a ^13C-HSQC-NOESY (τ[m] = 80 ms) and a homonuclear ^1H-NOESY (τ[m] = 100 ms) spectrum. All spectra were processed with TopSpin (Bruker, Karlsruhe) and analyzed using Sparky [http://www.cgl.ucsf.edu/home/sparky/]. Backbone [4]phi and ψ dihedral angle restraints were determined from an HNHA spectrum as well as from backbone chemical shifts using TALOS.[5]^33 Pro33 was found to be in the cis conformation, based on the chemical shift difference between the C^β and C^γ atoms of the proline.[6]^34 The zinc coordination geometry was defined as tetrahedral using angle (Zn-C-N at 125°, S-Zn-S at 109°, S-Zn-N^ε2and C-S-Zn at 107°) and bond (2.3 Å for Zn-S and 2 Å for Zn-N) constraints. Structures were calculated using the experimentally derived restraints with ARIA 1.2/CNS.[7]^[35.]^ and [8]^[36.] Eight iterations of structure calculations were performed using the standard protocols provided with the software.		Figure 4. Figure 4. Electrostatic surface potentials of CtBP-TD, hTHAP1-TD model and hTHAP2-TD in two orientations. The molecules in (a) and (b) are related to each other by a 120° rotation about the indicated axis. The ribbon diagrams reflect the orientation of the molecules represented as surface plots directly below. Figure 4. Electrostatic surface potentials of CtBP-TD, hTHAP1-TD model and hTHAP2-TD in two orientations. The molecules in (a) and (b) are related to each other by a 120° rotation about the indicated axis. The ribbon diagrams reflect the orientation of the molecules represented as surface plots directly below.

PROCHECK

	Headers