Huang et al. (2003) GlpT structure (SOM)

1

Supporting Online Materials for

Structure and mechanism of the glycerol-3-phosphate transporter from Escherichia coli

Yafei Huang¶, M. Joanne Lemieux¶&, Jinmei Song, Manfred Auer and Da-Neng Wang*

Skirball Institute of Biomolecular Medicine and Department of Cell Biology,

New York University School of Medicine, 540 First Avenue, New York, NY 10016, USA

¶These authors contributed equally to this work.

& Present address: Dept. of Biochemistry, University of Alberta, Edmonton, Canada T6G 2H6

*To whom correspondence should be addressed. E-mail: wang@saturn.med.nyu.edu

Huang et al. (2003) GlpT structure (SOM)

2

Materials and Methods

Preparation of heavy metal derivatives

GlpT overexpression, purification and crystallization were carried out as described (1, 2). Wild-

type GlpT has 452 amino acids (3), and the crystallized protein construct contains 451 amino

acids (2-452), with Leu2 changed to Gly2 and sequence Arg449-Gly452 to Leu-Val-Pro-Arg.

This protein form binds to substrates in detergent solution, and, upon reconstitution into

proteoliposomes, mediates G3P to Pi exchange (1). GlpT crystals were grown in the presence of

0.15% dodecyl-matoside and 0.06% C12E9 (2). Heavy metal derivative was prepared by soaking

crystals in Na9{P2Nb3W15O62}.nH2O. For seleno-methionine labeled protein (SeMet-GlpT),

transformed E. coli B834 strain was grown in minimal medium with methionine being replaced

by seleno-L-methionine, and purified in the same manner as the wide-type protein (1). Following

confirmation of substitution of methionine with SeMet using matrix-assisted laser

desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (4), SeMet-GlpT crystals

were grown as for the wild-type using a protein concentration of 2 mg/ml in the presence of

5mM of DTT.

Crystallography

Native and tungsten derivative data sets were collected on beamline X25 at BNL-NSLS, and

SeMet data sets from 19-ID at ANL-APS, all from frozen crystals. Diffraction data were indexed

and processed with HKL2000 (5). Both phasing and refinement were carried out with CNS (6).

The location of the tungsten cluster was determined from Patterson maps, which showed a

unique speak at 7σ. The experimental phases to 3.9 Å resolution were calculated using single

isomorphous replacement with anomalous scattering (SIRAS). Following solvent-flattening, the

Huang et al. (2003) GlpT structure (SOM)

3

phases were extended to 3.3 Å. The positions of methionine residues were identified by peaks

(>4σ) in an anomalous (Fse+-Fse

-) Fourier difference map from SeMet data using the tungsten

phases. Model was built using program O (7) and refinement was carried out in iterative cycles

of simulated annealing with torsion-angle dynamics and model rebuilding. The final model

contains sequence Phe5 to Tyr231, and Thr240 to Glu448, in addition to the C-terminal LVP sequence

of the thrombin cleavage site. In addition, a dodecyl-maltoside detergent molecule was found in

the central pore.

Trypsin digestion and mass spectrometry

Inside-out vesicles were prepared from E. coli (LMG194) transformed with GlpT448-His

construct (Gly2-Glu448-His6) by one cycle of French Press (8). The membrane was incubated

with trypsin at an enzyme-to-protein ratio 1:2000 (w/w) (in 50 mM Tris pH 7.5, 100mM NaCl)

at 25 °C for 2 hours, in the presence of G3P (0-50 mM). PMSF was added immediately to the

samples at the end of the incubation, followed by SDS-PAGE and Western blot analysis using an

anti-His-Probe. In a separate experiment, purified GlpT448 at 1.1 mg/ml concentration was

incubated with trypsin, at a ratio of 1:50 (w/w) at 25 °C for 2 hours and in the presence of G3P

(0-50 mM) or Pi (0–200 mM). Samples were analyzed by Coomassie Blue-stained SDS-PAGE.

The molecular masses of proteolytic fragments were measured using MALDI-TOF mass

spectrometry. The Stokes radii of GlpT in the absence and presence of G3P were determined

using analytical size-exclusion chromatography column on HPLC (1).

Huang et al. (2003) GlpT structure (SOM)

4

Fig. S1

Huang et al. (2003) GlpT structure (SOM)

5

Fig. S2

Huang et al. (2003) GlpT structure (SOM)

6

Fig. S3

Huang et al. (2003) GlpT structure (SOM)

7

Fig. S4

Huang et al. (2003) GlpT structure (SOM)

8

Supporting figure legends and movie captions

Fig. S1. Electron density map and crystal packing. (A) Anomalous (Fse+-Fse

-) Fourier difference

map at 4.0 Å resolution from SeMet crystals superimposed with an a carbon trace of GlpT

viewed from within the membrane. All 21 methionine sites in our protein construct were

identified by selenium peaks at 4σ (red), with 18 visible at 5σ (green). (B) Crystal packing of

GlpT in the P3221 space group viewed approximately along the c-axis. Molecules in the unit cell

are divided into three layers perpendicular to the c-axis, which are colored differently. One GlpT

monomer is colored blue. Parallel ribbons composed of GlpT molecules in each layer are rotated

120° relative to each other between layers. In the ribbons the GlpT molecules are arranged in an

alternating antiparallel manner. The packing of GlpT molecules in the crystals illustrates the

delicacy of crystallizing a membrane protein that does not possess a large extramembrane

domain. The membrane spanning portions of the neighboring molecules are in close contact,

indicating fusion of detergent micelles. The contact surface areas between protein molecules

within the ribbon are 2,508 Å2 per molecule. Between the layers, the most important contact is

made by the C-terminus of the last transmembrane a-helix with a cavity formed by four helices

of a molecule from the next layer. This explains our observation that the quality of GlpT crystals

was critically dependent upon the C-terminus of the protein construct (2). The total contact area

between adjacent layers is only 593 Å2 per molecule. Such a small contact area accounts for the

tendency for some GlpT crystals to have partial disorder in the c-direction (2). These and most

other figures in the article were prepared using program Pymol (9).

Fig. S2. Substrate-binding site. (A) Stereo view of electron density at the substrate-binding site.

2Fo-Fc map at 3.3 Å resolution is shown at 1σ contour level. The helixes are colored in the same

Huang et al. (2003) GlpT structure (SOM)

9

scheme as in Fig. 2. (B) A multiple sequence alignment of the H1 and H7 helices of MFS

proteins. Agr45 and Arg269 in the GlpT sequence are indicated by arrowheads. Their equivalent

residues in UhpT are shown to be essential for substrate transport (10).

Fig. S3. Substrate-induced conformational change in L6-7. (A) Western blot analysis of GlpT in

inside-out-vesicle treated with trypsin. Trypsin treatment of inside-out vesicles prepared from E.

coli cells that expressed GlpT construct containing a C-terminal His-tag (GlpT448-His6)

produced a C-terminal fragment of about 20 kDa. The GlpT construct used had a C-terminal

His6-tag for detection by Anti-His-Probe. The molecular weight standards shown in the first lane

were also His6-tagged. (B) Coomassie Blue-stained SDS-PAGE of detergent-purified GlpT

treated with trypsin. This GlpT construct was the same one used for crystallization and did not

contain the His6-tag. Trypsin cleaved GlpT into two fragments, with apparent molecular weights

of 24 and 20 kDa. Mass spectrometry measurements determined their molecular masses to be

26103.4 and 24110.4 Da, respectively. They agree with the calculated masses from the

sequences for Gly2-Lys234 and Ala235-Arg452 of 26108.7 and 24113.6 Da, thus placing the trypsin

sensitive site at Lys234 in L6-7. Interestingly, trypsin cleavage of GlpT, either membrane-bound

or in detergent, was inhibited by the presence of G3P or Pi. Consistent with its higher Kd value

(1), the required concentration of Pi for GlpT protection was higher. It is noted the protein

segment that contains the tryptic site is disordered in the absence of substrate, as shown in our

crystal structure.

Fig. S4. Proposed conformational changes of GlpT upon substrate binding. Simultaneous

binding of a substrate molecule to Arg45 on H1 and Arg269 on H7 pulls the two helixes closer. The

Huang et al. (2003) GlpT structure (SOM)

10

movement of the two helices, transmitted via loops L4-5 and L10-11, brings the N- and C-

terminal domains closer and narrows the cytosolic pore.

Movie S1. Structure of GlpT viewed from within the membrane. The helixes are colored in the

same scheme as in Fig. 2.

Movie S2. Proposed rocker-switch type conformational changes accompany substrate

translocation by GlpT. The crystal structure determined in this work represents the Ci

conformation of the protein. The Co-S conformation was generated by fitting the GlpT model

into the 6.5 map of substrate-bound form of OxlT (11). By separately rotating the two halves

of our GlpT model in opposite directions along an axis at the interface and parallel to the

membrane, we found that a ~6¡ rotation by each domain can generate a structure that fits the

OxlT map reasonably well. The Co conformation was produced by a ~10¡ rotation by each

domain that is sufficient to close the pore on the cytosolic side of the molecule and to open a

pore on the periplasmic side. Finally, the Ci-S conformation was generated by a ~4¡ rotation.

Huang et al. (2003) GlpT structure (SOM)

11

Supporting references

1. M. Auer et al., Biochemistry 40, 6628 (2001).

2. M. J. Lemieux et al., Prot. Sci., Submitted (2003).

3. K. Eiglmeier, W. Boos, S. T. Cole, Mol Microbiol 1, 251 (1987).

4. M. Cadene, B. Chait, Anal Chem 72, 5655 (2000).

5. Z. Otowinowski, W. Miror, Meth. Enzym. 276, Part A, 307 (1997).

6. A. T. Brunger et al., Acta Crystallogr D 54, 905 (1998).

7. T. A. Jones, J. Y. Zou, S. W. Cowan, Kjeldgaard, Acta Crystallogr A 47, 110 (1991).

8. W. W. Reenstra, L. Patel, H. Rottenberg, H. R. Kaback, Biochemistry 19, 1 (1980).

9. http://pymol.sourceforge.net/.

10. M. C. Fann et al., J. Membr. Biol. 164, 187 (1998).

11. T. Hirai et al., Nat. Struct. Biol. 9, 597 (2002).