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.
The above figure is reprinted
by permission from Elsevier:
J Mol Biol
(2007,
366,
382-390)
copyright 2007.
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.