Download Voltage dependent K+ channel

X-ray structure of a voltage –dependent
K+ channel
NATURE | VOL 423 | 1 MAY 2003
Youxing Jiang*, Alice Lee, Jiayun Chen, Vanessa Ruta,
Martine Chait & Roderick MacKinnon
HTSD-NMR
석사 1학기 전 병 영
<Introduction>
•Voltage dependent K+ channel are members of the family of voltagedependent cation (K+, Na+ and Ca2+) channels
• allow ion conduction in response to changes in cell membrane
voltage.
• the structure of KvAP, a voltage-dependent K+ channel from
thermophilic archaebacteria Aeropyrum pernix.
• voltage dependent cation channel contain six hydrophobic segments
per subunit, S1–S6 (S5~S6 : pore & ion selectivity ,S1~S4 : voltage
sensor)
Certain charged amino acids within the voltage sensors, particularly
the first four arginines in S4, account for most of the gating charge.
< X-ray crystallography overview >
Bragg’s law : “2 x d sin θ =λ”
X-ray 결정크기 : 0.1mm(minimum)
1012 molecule(50x50x50 Å)
<Procedure of Structure Analysis>
cDNA Cloning and Construction of Expression Plasmids
147aa(isolated voltage sensor), pQE60 expression vector, E.coli XL-1
Blue cell
Expression of Proteins
Induction : O.D 600 ≃ 1.0 0.4mM IPTG, 37 ℃ incubation
Harvest하여 50mM tris Ph8.0, 100mM KCl, Leupeptin, Pepstatin,
Aprotinin, phenylmethylsulphonyl fluoride에서 lysis
Protein Purification
Protein lysate를 3h동안 room temperature에서 40mM decylmaltoside
(DM)를 첨가 , TALON Co2+ affinity column으로 purify함
1unit of thrombin per 2mg of protein 21 ℃
Further purification ~ superdex 200(10/30)column
MALDI-TOF mass spec으로 확인.
Protein Crystallization
Crystal growth: Sitting Drop vapor diffusion
protein과 동일 volume의 reservoir solution ㅡmix
,20 ℃
Fab 6E1-KvAP complex crystal
PEGMME350, 150~200mM CdCl2,
100mM sodium acetate, pH 5.0
cell dimension a=b=189.4Å , c=150.5Å
a=β=γ=90 ℃
Fab 33H1-isolated voltage-sensor complex crystal
Cell dimension a=264.7Å, b=61.6Å, c=46.1Å
a=γ=90 ℃, β=90.35 ℃
Diffraction images from crystals and diffraction data collection
Determination of protein structures
CHESS A1, F1 & NSLS X25 beam line / 모든 data는 CCP4 program
( DENZO , SCALEPACK)
Accumulation of 3-D Structure Information
< Crystal growth technique >
Sitting Drop vapor diffusion
Figure 1
Structure of the KvAP channel.
a, Sequence alignment of KvAP and
the Shaker K+ channel.
b, Structure of the KvAP channel
bound to four Fab fragments
,viewed down the four-fold axis
from the intracellular side.
Figure 2
Stereo view of the KvAP pore and
comparison with the KcsA K+ channel.
a, An electron density map at 1.0 σ contour
(blue mesh) was calculated from the Fab
model used in molecular replacement after
rigid-body refinement.
b, The a-carbon traces of the KvAP pore
(blue) and the KcsA K+ channel (green)
are shown from the side
c, And from the intracellular solution.
Table1
Data listed are those used
for refinement.
Numerous data sets were also
collected at CHESS F1 and
NSLS X25.
Figure 3
Architecture of
the KvAP channel.
a, Stereo image of the
KvAP channel tetramer
viewed from the intracellular
side of the membrane.
b, Stereo image of the
KvAP channel tetramer viewed
from the side with the
intracellular solution below.
c, a single KvAP subunit vie
wed from the same perspective
as in b.
d, A schematic diagram of
the KvAP subunit topology is
shown with an orange selectivity
filter and an arrow to indicate
the ion pathway.
Figure 4
Functional and structural
analysis of the isolated
voltage-sensor domain.
a, The isolated voltage sensor
retains its ability to bind tarantula
toxins that specifically inhibit
voltage sensors.
b, VSTX1 inhibits KvAP channel
Currents elicited by a á100 mV
depolarization.
c, Structure of the isolated voltage
sensor (cyan) bound to Fab 33H1
green).
Figure 5
Structure of the isolated
voltage sensor.
a, Stereo view with helical elements
labelled in cyan and selected amino
acids labelled in black using the
single-letter code.
b–d, The voltage sensor from the
beginning of S2 to the end of the
voltage-sensor paddle is shown
for the full-length KvAP channel
Figure 6
The voltage-sensor paddle
is conserved.
a, Stereo view of the
voltage-sensor paddle.
b, A multiple sequence alignmen
of a portion of the voltage
sensor from a variety of
voltage-dependent K+ channels
Figure 7
Effect of Fabs on
voltage-sensor
conformation.
a, Stereo view of two
subunits (blue and red)
from the full-length
channel
b, Identical view of the
pore (S5–S6) with the
isolated voltage sensor
docked according to the
position of S2 in the fulllength channel.
Figure 8
Hypothesis for gating
charge movements.
a, The conventional model of
voltage dependent gating
b, In the new model
<CONCLUSION>
1. The channel contains a central ion conduction pore surrounded by voltage
sensors, which form what we call ‘voltage-sensor paddles’—hydrophobic,
cationic, helix–turn–helix structures on the channel’s outer perimeter.
2. Flexible hinges suggest that the voltage-sensor paddles move In response
to membrane voltage changes, carrying their positive charge across the
membrane.