Supporting Information to
Substrate specificity and ion coupling in the Na+/betaine symporter BetP
Camilo Perez1, Caroline Koshy1, Susanne Ressl1,2, Sascha Nicklisch3, 4, Reinhard
Krämer4 and Christine Ziegler1
1
Max-Planck Institute of Biophysics, Department of Structural Biology, 60438
Frankfurt am Main
2
The Howard Hughes Medical Institute and Departments of Molecular and Cellular
Physiology, Neurology and Neurological Sciences, Structural Biology, and Photon
Science, Stanford University, Stanford, CA94305, USA
3
Marine Science Institute, University of California, Santa Barbara, USA
4
Institut of Biochemistry, University of Cologne, Zülpicher Str. 47, 50674 Köln
Supplementary Methods
Cell culturing and protein purification
Cell culture and protein preparation methods have been described previously
(Rübenhagen et al, 2000). Uptake of [14C] betaine was measured in E. coli MKH13
cells (Haardt et al, 1995). E. coli DH5αmcr (Grant et al, 1990) was used for the
heterologous expression of strep-betP. Cells were grown at 37°C in LB medium
supplemented with carbenicillin (50 g/ml) and induction was initiated with
anhydrotetracycline (200 g/l). Cells were harvested at 4 C by centrifugation and
resuspended in buffer containing 100 mM Tris-HCl (pH 8) and protease inhibitor
1
Pefabloc 0.24mg/ml. Membranes were isolated from disrupted cells and solubilized
with 2,5% β-dodecyl-maltoside (DDM) when purified protein was subsequently
crystallized, or 1,3% of the same detergent when protein was reconstituted. The
protein was then loaded on a StrepII-Tactin macroprep® column, washed with 50
mM Tris-HCl (pH 7.5), 500 mM NaCl, 8.6 % Glycerol, 0.05-0.1 % DDM, and eluted
with 5 mM desthiobiotin, 50 mM Tris-HCl (pH 7.5), 200 mM NaCl, 8.7% glycerol
and 0.6% Cymal-5, if used for crystallization, or 0.05 % DDM if used for
reconstitution. Prior to crystallization the protein was loaded onto a Superose 6 (GE
Healthcare) size-exclusion column equilibrated with 20 mM Tris-HCl (pH 7.5), 200
mM NaCl and 0.6% Cymal-5. This purified protein was concentrated at 4 °C to
approx. 10 mg/ml at 3000 g in a Vivaspin tube (Vivascience) with a 100k -molecularweight cut-off.
Site-directed mutagenesis
The QuickChangeTM kit (Stratagene) and Pfu Turbo DNA polymerase were applied
for nucleotide mutagenesis (primer sequences are listed in Table SII) in pASKIBA5betP (Schiller et al, 2004) and pASK IBA7betPN29EEE44/45/46AAA (Ressl
etal, 2009) plasmids. All the plasmids were fully sequenced and the specific
mutations confirmed.
Protein reconstitution into liposomes
Functional reconstitution of the proteins was performed as described (Rübenhagen et
al, 2000). Briefly, liposomes (20 mg phospholipid/ml) from E. coli polar lipid extract
phospholipids (Avanti Polar Lipids, Alabaster, U.S.A.) were prepared by extrusion
2
through polycarbonate filters (400 nm pore-size) and diluted 1:4 in buffer (100 mM
KPi, pH 7.5). After saturation with Triton X-100, the liposomes were mixed with
purified protein at a lipid/protein ratio of 30:1 (w/w). BioBeads at ratios (w/w) of 5
(BioBeads/Triton X-100) and 10 (BioBeads/DDM) were added to remove the
detergent. Finally, the proteoliposomes were centrifuged and washed before being
frozen in liquid nitrogen and stored at -80°C.
Transport assays
Uptake of [14C] betaine in E. coli cells was performed as described (Ott et al, 2008).
E. coli MKH13 cells expressing a particular strep-betP mutant were cultivated at 37
C in LB medium containing carbenicillin (50 g/ml), and induced at a OD600 of 0.5
by adding anhydrotetracycline (200g/l). After 2 h the cells were harvested and
washed in buffer containing 25 mM KPi buffer (pH 7.5), 100 mM NaCl, then
resuspended in the same buffer containing 20 mM glucose. For uptake measurements
the external osmolality was adjusted using different concentrations of KCl. Cells were
incubated for 3 minutes at 37C before the addition of 250 µM [14C] betaine. Betaine
uptake was measured at various intervals after cell samples were passed through glass
fiber filters (APFF02500, Millipore, Schwalbach, Germany), and washed twice with
2.5 ml of 0.6 M KPi buffer. The radioactivity retained on the filters was quantified by
liquid scintillation counting. Immuno-blotting against the N-terminal StrepII-tag of
the different BetP variants in membranes of E. coli MKH13 using StrepII-TAG
specific antibody confirmed that mutant forms of BetP were synthesized to
approximately the same level in cells.
3
Uptake of [14C] betaine into proteoliposomes was measured as described (Ott et al,
2008). Extruded proteoliposomes were diluted in potassium-free buffer (20 mM NaPi
pH 7.5, 25 mM NaCl, 1 µM valinomycin) or sodium-free buffer (100 mM KPi, 50
mM 2-(N-morpholino) ethanesulfonic acid (MES) pH 7.5-5.5, 10 nM valinomycin)
containing
1-400
µM
[14C]-glycine
betaine
(Moravek
Biochemicals
and
Radiochemicals, U.S.A) or 25-800 µM [14C]-Choline (Amersham radiochemicals,
England). High osmolalities of the external buffer were adjusted with proline.
To measure uptake rates, samples were filtered at various times through nitrocellulose
filters, and the scintillation was counted. Betaine and choline uptake rates were
calculated as described (Schiller et al, 2006). The kinetic constants were derived by
Michaelis-Menten curve fitting of the uptakes rates versus the substrate concentration
with GraphPad Prism version 5.0c for Mac OS X, GraphPad Software (Motulsky,
1999).
Crystallization and structure determination
BetPN29/G153D was crystallized reproducibly in the presence of 5 mM choline
with a reservoir solution of 100 mM Na-tri-citrate (pH 5.3–5.6), 100 mM NaCl, 17–
24% PEG 400 (Ressl et al, 2009). Crystals grew at 18 °C upon mixing the protein
solution in 1:1 or 1:2 ratio with the reservoir solution. A dataset to 3.35 Å was
collected at ESRF-ID29 and processed using the XDS package (Kabsch, 1993). The
crystal structure was determined by molecular replacement against the structure of
chain C of BetP (PDB entry 2WIT) using the Phaser program (Read, 2007). The
structure was refined using the Phenix refinement program (Terwilliger et al, 2008)
and by manual building in the COOT program (Emsley & Cowtan, 2004). The
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substrate position was located under observance of a clear positive peak in the Fo-Fc
difference electron-density map of the binding site after several refinement rounds.
The regions in the model that showed no densities were deleted to avoid incorrect
building. In chain A this regions correspond to the residues 30-56, 270-275, 499-515
and 584-595; in chain B to 30-56, 272-277 and 559-595; and in chain C to 30-56,
272-275 and 569-595.
Modeling studies
Homology models of BetT, with and without choline as substrate, were generated.
BetP (PDB entry: 2WIT), which shares a sequence identity of 35% with BetT (from
E. coli), was used as template. Sequences were aligned with T-Coffee (Notredame et
al, 2000) and edited manually using Jalview (Waterhouse et al, 2009). The terminal
ends of BetT were not included in the alignment due to differences in their length
with the template BetP. Eleven residues from the N-terminus and 146 residues from
the C-terminus were deleted while ensuring that helices matched with those of the
template and that important features, such as the Gly-x-Gly-x-Gly motif in the
binding region, were maintained. These steps led to improved alignment scores.
Modeling was performed using MODELLER 9v6 (Bino & Andrej, 2003), and the
model with the lowest score was validated using PROCHECK (Laskowski et al,
1993). Eight residues (1.8%) were in the generously allowed regions of the
Ramachandran plot, and 1 residue (0.2%) was in the disallowed region. An additional
validation step was performed using MolProbity (Chen et al, 2010 and Davis et al,
2007). Over 92% residues were in the Ramachandran favored regions while 1.15%
were outliers and 2.1% with poor rotamers. These deviations were examined and were
5
found to mainly be in loop regions or in low electron density regions in the template.
They were thus left unaltered.
The coordinates for choline were extracted by a subsequent alignment of BetT with
ChoX (PDB entry: 2REG) and were used in the ligand-modeling module of
MODELLER. Verification of the correct coordinating rotamer for Asp 97 in COOT
(Emsley & Cowtan, 2004) optimized this model. Validation using PROCHECK
resulted in 4 residues (0.9%) in the generously allowed regions and 2 residues (0.4%)
in the disallowed region of the Ramachandran plot. Additional MolProbity validation
of the optimized model reported 94.81% residues in Ramachandran favoured regions
with a similar 1.15% outlier and 3.5% poor rotamer residue distribution. These loop
region residues were also left unaltered due to insufficient information from the
template.
References
Bino J, Andrej S (2003) Comparative protein structure modeling by iterative
alignment, model building and model assessment. Nucl Acids Res 31(14): 3982–3992
Emsley P, Cowtan K (2004) Coot: model-building tools for molecular graphics. Acta
Crystallogr D 60: 2126–2132
Chen VB, Arendall WB 3rd, Headd JJ, Keedy DA, Immormino RM, Kapral GJ,
Murray LW, Richardson JS, Richardson DC (2009) MolProbity: all-atom structure
validation for macromolecular crystallography. Acta Crystallogr D Biol Crystallogr
66(Pt 1):12-21
Davis IW, Leaver-Fay A, Chen VB, Block JN, Kapral GJ, Wang X, Murray LW,
Arendall WB 3rd, Snoeyink J, Richardson JS, Richardson DC (2007) MolProbity: all-
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atom contacts and structure validation for proteins and nucleic acids. Nucleic Acids
Res 35:W375-83
Grant S, Jessee J, Bloom F, Hanahan D (1990) Differential plasmid rescue from
transgenic mouse DNAs into Escherichia coli K12. J Bacteriol 166: 6605–6612
Haardt M, Kempf B, Faatz E, Bremer E (1995) The osmoprotectant proline betaine is
a major substrate for the binding-protein-dependent transport system ProU of
Escherichia coli K-12. Mol Gen Genet 246: 783–786
Kabsch W (1993) Automatic processing of rotation diffraction data from crystals of
initially unknown symmetry and cell constants. J Appl Cryst 26: 795–800
Laskowski R, Macarthur M, Moss D, Thornton J (1993) PROCHECK: a program to
check the stereochemical quality of protein structures. J Appl Cryst 26 (2): 283-291
Notredame C, Higgins D, Heringa J (2000) T-coffee: A novel method for fast and
accurate multiple sequence alignment. J Mol Biol 302 (1): 205-217
Read R (2007) Phaser crystallographic software. J Appl Cryst 40: 658-674
Schiller D, Ott V, Krämer R, Morbach S (2006). Influence of membrane composition
on osmosensing process of the betaine carrier BetP from Corynebacterium
glutamicum. J Biol Chem 281: 7737–7746
Terwilliger T, Grosse-Kunstleve R, Afonine P, Moriarty N, Zwart P, Hung L, Read R,
Adams P (2008) Iterative model building, structure refinement and density
modification with the PHENIX AutoBuild wizard. Acta Crystallogr D 64: 61–69
Waterhouse A, Procter J, Martin D, Clamp M, Barton G (2009) Jalview Version 2—a
multiple sequence alignment editor and analysis workbench. Bioinformatics 25:
1189–1191
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Supplementary Figure Legends
Figure S1 (A) A homology model of BetT was determined in an inward facing
substrate occluded conformation with the crystal structure of BetP (PDB entry 2WIT)
as a template. The superposed aromatic box of the homology model of BetT (salmon)
and BetP (PDB entry 2WIT) (blue) is shown. In BetT, a Tyr135 is located at the same
position as the Trp189 in BetP. (B) Binding site in the choline binding protein ChoX
(PDB entry 2REG) from E.coli. The trimethylammonium group of choline (black) is
coordinated by Trp43, Trp90 and Trp205 (orange), while its hydroxyl group is
coordinated by Asp157 and Asn156 (blue).
Figure S2 (A) Comparison of the Na2 site and choline position in BetP-G153D.
Choline is shown in black and the Na2 site as blue dotted sphere. (B) Na2 site in
BetP, LeuT, Mhp1 and vSGLT is located between the first helix of the first repeat
(salmon) and the third helix of the second repeat (orange).
Figure S3 (A) Homology model of BetT with the quaternary ammonium group of
choline docked to an aromatic box formed by Trp140 and Tyr143 in TM4 (green),
and by Trp316 and Trp319 in TM8 (blue). Asp97 coordinates the hydroxyl group of
choline (corresponds to Asp153 in BetP-G153D) in TM3 (salmon). (B)
Superimposition of TM3, TM5 and TM10 of BetPG153D (yellow) to the BetT
homology model (salmon) shown in (A). Asp97 is oriented towards the aromatic box,
while in the BetP-G153D structure Asp153 forms a hydrogen bond to Ser253 in TM5.
In BetT, a threonine residue (Thr198) is found at this position, which might be also
suitable to form a similar hydrogen bond.
8
Supplementary Tables
Table SI. Independent superposition of TM helices of BetP-G153D and BetP
TM’s
r.m.s.d. (Å)
1
0.9
2
0.7
Bundle (4-3,8-9)
0.6
Hash (5-6,10-11)
0.8
7
1.0
12
0.5
Each TM was extracted from the inward-open (PDB entry 3PO3) and inward-occluded (PDB entry 2WIT)
structures and “LSQ” superimposed in COOT (Emsley & Cowtan, 2004). The RMSD is for the entire length of the
TM helices.
Table SII. Primers sequences with exchanged codons underlined. Only sense primers
are listed
Oligonucleotide
Sequence 5’ - 3’
G149A
ATG ATG TTT GCT GCA GCT ATG GGT ATT GGT TTG
G151A
TTT GCT GCA GGT ATG GCT ATT GGT TTG ATG TTC
G153A
GCA GGT ATG GGT ATT GCT TTG ATG TTC TAC GGA
G153D
GCA GGT ATG GGT ATT GAT TTG ATG TTC TAC GGA
W189Y
ACG ACA ATG TTC CAC TAC ACC TTG CAT CCA TGG
9
Figure S1
10
Figure S2
11
FigureS3
12