Originally published 27 May 2010; corrected 3 June 2010

www.sciencemag.org/cgi/content/full/science.1190414/DC1

Supporting Online Material for

Structure of the Human BK Channel Ca2+-Activation Apparatus at 3.0 Å Resolution

Peng Yuan, Manuel D. Leonetti, Alexander R. Pico, Yichun Hsiung, Roderick MacKinnon*

*To whom correspondence should be addressed. E-mail: mackinn@rockefeller.edu

Published 27 May 2010 on Science Express

DOI: 10.1126/science.1190414

This PDF file includes:

Materials and Methods

Figs. S1 to S5

Tables S1 to S3

References

Correction: Amino acid sequences for hSlo2.2 and gSlo2.2 were corrected in Fig. S4.

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Supplementary Materials

Materials and Methods

Cloning, expression, and purification

HsloM3 (GI: 507922, here referred to as human BK) was generously provided by

Ligia Toro in a pcDNA3 vector and served as the template for subcloning. On the basis

of secondary structure prediction, the CTD construct including residues 341-1056 was

fused to a C-terminal GFP with a deca-histidine affinity tag (GFP-His10) and expressed in

baculovirus-infected sf9 cells using the Bac-to-Bac system (Invitrogen). Infected cells

were cultured at 27°C in supplemented Grace’s insect cell medium (Invitrogen) and

collected 60 hours after infection. Cells were disrupted by sonication in buffer containing

50 mM Hepes pH 8.0, 300 mM NaCl, 10 mM imidazole, and 5 mM 2-mecaptoethanol

supplemented with protease inhibitors including 3 μg/ml aprotinin, 2 mM benzamidine, 5

μg/ml leupeptin, 2 μg/ml pepstatin A, 200 μg/ml 4-(2-Aminoethyl) benzenesulfonyl

fluoride hydrochloride, and 250 μM phenylmethane sulphonylfluoride (Sigma-Aldrich).

Following binding on TALON metal ion affinity resin (Clontech), the CTD fusion

protein was eluted with 300 mM imidazole and incubated overnight with PreScission

protease to remove the C-terminal GFP-His10 tag. The PreScission recognition sequence

and a cloning site (EcoRI) resulted in a stretch of amino acids SNSLEVLFQ attached to

the C-terminus of the CTD protein. The CTD was then isolated by size-exclusion

chromatography in 20 mM Tris pH 8.0, 150 mM NaCl, 50 mM CaCl2, 20 mM

dithiothreitol (DTT), and 1.5 mM tris (2-carboxyethyl) phosphine (TCEP). Peak fractions

corresponding to the CTD were collected and concentrated to 5 mg/ml for crystallization

experiments. All purification procedures were carried out either on ice or at 4°C. For

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selenomethionine-substituted CTD, infected sf9 cells were cultured in standard medium

for 8 hours, collected by centrifugation, cultured again in serum-free medium without

methionine for 8 hours, and then supplemented with 100 mg/L selenomethionine. Cells

were collected after 48 hours and the labeled protein was purified following the same

procedure as for wild type.

A synthetic gene encoding the chicken Slo2.2 channel (GI: 20338417) was

purchased from Biobasic. Based on secondary structure prediction, the chicken Slo2.2

CTD (amino acid sequence 86% identical to human Slo2.2) including residues 347-1201

(yielding diffracting crystals) was expressed and purified following a similar procedure

as described above. However, the size-exclusion chromatography was carried out in 20

mM Tris pH 8.0, 500 mM NaCl, 20 mM DTT, and 1.5 mM TCEP. Purified protein was

concentrated to 5 mg/ml for crystallization.

Crystallization and structure determination

Crystals of human BK CTD were grown at 20°C using hanging-drop vapor

diffusion by mixing equal volume of protein and a reservoir solution containing 50 mM

sodium acetate, 2% (w:v) PEG 4000, 100 mM sodium sulfate, and 100 mM lithium

sulfate (pH 4.8). Hexagonal crystals belonging to space group P6322 grew to a maximum

size of ~0.1 x 0.2 x 0.2 mm3 within a week. Selenomethionine-substituted crystals were

obtained using the same crystallization condition. Crystals were briefly transferred to a

cryoprotectant solution containing 50 mM sodium acetate, 3% (w:v) PEG 4000, 100 mM

sodium sulfate, and 100 mM lithium sulfate, 150 mM NaCl, 50 mM CaCl2, and 30%

ethylene glycol (pH 4.8), flash-frozen and stored in liquid nitrogen. The chicken Slo2.2

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CTD was crystallized at 20°C by hanging-drop vapor diffusion against reservoir solution

containing 50 mM Tris, 9-12% (v:v) PEG 400, and 100 mM potassium phosphate (pH

8.0). Tetragonal crystals belonging to space group I422 grew to full size within a week

and were cryoprotected by increasing the concentration of PEG 400 to 30% in the

reservoir solution supplemented with 500 mM NaCl.

Diffraction data for native crystals of human BK CTD were measured at the

Advanced Photon Source beamline 24 ID-C. Diffraction data for selenomethionine-

substituted crystals of human BK CTD and the chicken Slo2.2 CTD crystals were

collected at beamline X29 from the National Synchrotron Light Source. All diffraction

images were processed with the HKL2000 program suite (1). Initial MAD phasing for

human BK CTD was calculated using SOLVE/RESOLVE (2). Thirteen selenium sites

were found and the solvent-flattened electron density map at 3.3 Å resolution was of high

quality. The protein chain was readily traceable and iterative model building was carried

out in COOT (3). Rounds of refinement were performed with REFMAC (4). The final

model was refined to 3.0 Å resolution with Rwork = 0.246 and Rfree = 0.278. A few

disordered loop regions were not built due to poor electron density and the final refined

model includes residues 343-570, 577-613, 677-806, 817-833, 869-945, 949-1020, and

1025-1056. All side chains but two (L353 and W477) were modeled. The majority

(94.6%) of the residues lie in the most favored region in a Ramachandran plot (5), with

the remaining 5.4% in the additionally allowed regions. One Ca2+ ion and one SO42- were

modeled in the structure. For molecular replacement, a poly-alanine model for the refined

BK CTD structure was used as the search model against the 6.0 Å diffraction data from

the chicken Slo2.2 CTD. A unique solution with a standout Z-score was identified using

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the program PHASER (Table S2) (6). Data collection and structure refinement statistics

are shown in Table S1. All structural illustrations and electron density maps were

prepared with PYMOL (www.pymol.org).

Sequence alignment and analysis

Multiple sequence alignments were built from sequences retrieved directly from

NCBI and the output of Blast (7). The manual alignment of sequences included

information from ClustalW results (8), structure-based similarity groups, and structural

information from the crystal structures of human BK CTD, MthK (9), and Kv paddle

chimera (10). Sequence similarity groups used in the determination of conservation

patterns are based on chemical and structural considerations: 1. DE (negative), 2. KR

(positive), 3. GP (structural), 4. ACFILMTVWY (residues composing the core of RCK

and other α-β-α layered proteins, i.e., primarily hydrophobic).

Electrophysiology

Full length and loop exclusion constructs of the BK gene were transcribed, in

vitro, into RNA for injection into Xenopus leavis oocytes. T7 RNA transcription was

initiated by an hCMV promoter within the original pcDNA3 vector, which also contained

a ribosome binding site, 5’ UTR from Shaker, 3’ UTR from hSlo1(M3), poly-A tail, and

NotI restriction enzyme recognition site for linearization. RNAs were purified by the

Trizol method (Gibco-BRL) or the RNeasy kit (Qiagen) and stored at -80°C. Oocytes

were dissected from anesthetized Xenopus leavis (African clawed frogs) by a survival

surgery. Oocytes were immediately treated with collagenase (2 mg/mL) for 1.5 hours,

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rinsed with a Ca2+-free OR-2 solution (82.5 mM NaCl, 2.5 mM KCl, 1 mM MgCl2, 5

mM Hepes-NaOH pH 7.6) and stored in ND-96 solution (96 mM NaCl, 2 mM KCl, 1.8

mM CaCl2, 1 mM MgCl2, 5 mM Hepes-NaOH pH 7.6) containing fresh gentamycin (50

mg/L). Within 2 days of dissection, oocytes were injected with 50-70 nL of purified

RNAs at full strength or diluted 2-5 fold. Typically, 1-3 days of incubation provided

significant macrocurrents measured in patch-clamp recording.

Patch pipettes were pulled from glass capillary tubing (Warner G85150T-4) on a

programmable Flaming/Brown type puller (Sutter P-97) and fire polished on a

microforge using the resistive heat from a platinum wire. Polished pipettes were

approximately 2 μm in diameter and produced resistances ranging 0.9-2.5 MΩ in series

with recording equipment. Axopatch amplifiers (Axon Instruments 200A and 200B), a

low-pass Bessel filter (Frequency devices 902), a digitizer (Axon Instruments DigiData

1200A) and pClamp8.0 software (Axon Instruments) were used in the recording of data.

Patch pipettes were applied to the surface of freshly devitellinized oocytes to form tight

electrical seals (>10 GΩ), allowing a voltage-clamp on patches of plasma membrane.

Patches were excised in an inside-out configuration, exposing the intracellular face of

membrane and embedded channels to a bath solution controlled by a gravity-flow

perfusion system. Two stock solutions were made to control the preparation of precise,

μM-range, Ca2+-buffered bath solutions: a solution without Ca2+ (0-Ca), containing 130

mM Kgluconate, 20 mM KCl, 20 mM Hepes-KOH pH 7.5, and 5 mM EGTA; and a 1000

μM free-Ca2+ solution (1000-Ca) containing an additional 6 mM CaCl2 buffered by the

EGTA. A series of Ca2+-buffered solutions ranging from 1 μM to 300μM free Ca2+ were

then made by mixing together quantities of 0-Ca and 1000-Ca based on an EGTA-Ca2+

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Kd of 3.86 x 10-7 M. The pipette solution was prepared by the addition of 2 mM MgCl2 to

a 1 - 3 μM Ca2+-buffered solution.

Patches were voltage-clamped at 0 mV between recordings for stability. A

typical episodic voltage protocol consisting of 2 - 3 averaged runs of 16 sweeps stepping

from a holding potential of 0 mV to a range of test voltages in 20 mV steps for 30 ms,

activating channel currents, and then stepping to tail voltage of -60 mV for 10 ms to

produce tail currents. Current signal was filtered at 4 kHz (-3 dB), digitized at 20 kHz,

and recorded onto hard disk. Records were processed and parameterized using Clampfit

(Axon Instruments), Origin (Microcal Software), and Excel (Microsoft) programs. Data

points were extracted from the tail currents 200 μs following the tail voltage step in order

to determine the voltage dependence, and fit with the following equation:

G = Gmax/(1 + exp(-(V-V50)zF/RT)) + AV + B

where G is the conductance measured as tail current divided by tail voltage and V is the

applied test voltage. The linear term accounts for leak current. The Boltzmann term

includes z as the valence, V50 as the voltage at which 50% of the channels are open and F,

R and T, which have their usual meaning. A thorough study of macroscopic BK currents

and current analysis serves as the basis for methodology used here (11, 12).

S8

Fig. S1. (A) Representative experimental electron density at 3.3 Å. (B) Weighted 2fo-fc

electron density at 3.0 Å following refinement. Both (A) and (B) are contoured at 1.0 σ

above mean density.

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Fig. S2. (A) Ribbon representation of the BK CTD with RCK1 in blue and RCK2 in red.

(B) Ribbon representation of the MthK dimer in an open conformation with one subunit

in blue and one in red (PDB ID 1LNQ). Ca2+ ions are shown as yellow spheres.

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Fig. S3. Superposition of the Ca2+ bowl from the human BK CTD (green) and the Ca2+

binding site from the calpain-1 catalytic subunit (blue) (PDB ID 2R9F). The Ca2+ ion is

shown as a yellow sphere.

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Fig. S4. Sequence alignment of Slo family RCK domains. Human sequences

representing the three main families of Slo channels (hSlo1, hSlo2.2 and hSlo3) are

aligned together with a chicken Slo2 family member (gSlo2.2) and MthK. The alignment

of hSlo1 and MthK is based on their known RCK domain structures. The MthK sequence

is repeated to align to the second RCK domain present in Slo family sequences. In

regions with low sequence identity and significant insertions and deletions, manual

alignment was aided by the Slo1 structure. Two levels of shading indicate sequence

identity across all Slo family members (dark blue) versus identity across only 3 of 4

representative members (light blue). The same shading is extended to the MthK sequence

when identical to the Slo family sets. The definition of sequence identity was relaxed to

include D/E and K/R substitutions.

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Fig. S5. Alignment of channel sequences. The Kv paddle chimera is aligned with hSlo1

BK from S1 through S6. The known transmembrane structure of the Kv paddle chimera

is indicated in green. MthK is aligned with BK from S5 through βA of RCK1. The

known transmembrane structure of MthK is indicated in green and the known RCK

structure of both BK and MthK are indicated in blue. Note that all three sequences align

across S5, the pore helix and S6.

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Table S1. Summary of crystallographic data

Datasets Native Selenium gSlo2.2 CTD

Resolution (Å) 3.0 3.3 6.0 Space Group P6322 P6322 I422 Source APS 24ID-C BNL X29 BNL X29 Cell Constants a, b, c (Å) α, β, γ (°)

a = b = 144.5 c = 182.2 90, 90, 120

a = b = 145.1 c = 182.4 90, 90, 120

a = b = 126.3 c = 239.0 90, 90, 90

Peak Inflection Remote

Wavelength (Å) 0.9792 0.9792 0.9793 0.9640 1.0750 Completeness (%) 99.9 (99.9) 99.8 (100) 99.8 (100) 99.8 (100) 95.3 (96.7) Rmerge 0.089 (0.738) 0.105 (0.493) 0.100 (0.533) 0.130 (0.745) 0.108 (0.746) Reflections 23149 17611 17641 17649 2520

/ 19.6 (2.4) 18.8 (3.3) 18.2 (3.1) 13.9 (2.2) 21.4 (3.1) Redundancy 5.8 (5.9) 7.0 (7.2) 7.0 (7.2) 7.0 (7.2) 9.1 (9.6)

MAD Phasing (SOLVE/RESOLVE) Number of sites 13 Resolution (Å) 3.3 Overall Figure of Merit 0.70

Refinement Resolution (Å) 50 - 3.0 Rwork 0.246 (0.328) Rfree 0.278 (0.333) Number of atoms

Protein 4680 Ligands (Ca2+, SO42-) 6

Average B factors (Å2) 67.2 Ramachandron plot

Favored (%) 94.6 Allowed (%) 5.4 Disallowed (%) 0

RMSD Bond lengths (Å) 0.010

RMSD Bond angles (°) 1.215 Rfree was calculated with 5% of the data. Numbers in parentheses represent values in the highest-resolution shell.

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Table S2. Molecular replacement solutions (PHASER)

Fast rotation function table Solution # LLG (Log Likelihood Gain) Z-score 1 11.71 5.46 2 8.33 4.08 3 7.89 3.90 4 7.81 3.87 5 7.50 3.74 6 7.14 3.60 7 7.06 3.56 Fast translation function table Solution # LLG Z-score 1 49.94 9.35 2 21.26 5.53 3 20.40 5.35 4 20.85 5.19 5 18.31 4.61 6 19.53 4.50 7 19.11 4.45

S15

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S. P. Brazier, V. Telezhkin, R. Mears, C. T. Müller, D. Riccardi, P. J. Kemp, Adv Exp

Med Biol 648, 49 (2009).

W. Du et al., Nat Genet 37, 733 (Jul, 2005).

M. Fukao et al., J Biol Chem 274, 10927 (Apr 16, 1999).

S. Hou, R. Xu, S. H. Heinemann, T. Hoshi, Nat Struct Mol Biol 15, 403 (Apr, 2008).

S. Hou et al., J Biol Chem 285, 6434 (Feb 26, 2010).

H. J. Kim et al., Biophys J 94, 446 (Jan 15, 2008).

S. Ling, G. Woronuk, L. Sy, S. Lev, A. P. Braun, J Biol Chem 275, 30683 (Sep 29,

2000).

A. R. Pico, PhD thesis, The Rockefeller University (2003).

L. C. Santarelli, R. Wassef, S. H. Heinemann, T. Hoshi, J Physiol 571, 329 (Mar 1,

2006).

M. Schreiber, L. Salkoff, Biophys J 73, 1355 (Sep, 1997).

J. Z. Sheng et al., Biophys J 89, 3079 (Nov, 2005).

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X. D. Tang et al., Nature 425, 531 (Oct 2, 2003).

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2004).

L. Tian et al., Proc Natl Acad Sci U S A 101, 11897 (Aug 10, 2004).

L. Tian et al., FASEB J 20, 2588 (Dec, 2006).9.

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Y. Yang et al., J Biol Chem 285, 131 (Jan 1, 2010).

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