An intra-molecular disulfide cross-link stabilizes an inward-oriented transport intermediate conformation of the TonB-dependent transporters

Shimei Gong, Katarzyna Niedzielska and Robert NakamotoDepartment of Molecular Physiology and Biophysics, University of Virginia, Charlottesville, VA USA

Abstract

BtuB

BtuF

BtuCD

ExbB

ExbD

TonB

A395

Ton box

S120

Crystallographic structure of BtuB (Chimento et al. 2003). The amino terminal hatch domain is in dark blue; the carboxyl terminal 22 stranded ß barrel, in blue; B12 (Cyanocobalamin, CBL), in green; the amino terminal Ton box is indicated by stick figures. Residue S120 in the hatch is in yellow space filling and A395 in the barrel is in red. For the clarity, the front of the barrel was removed in the right panel.

Reaction of Biotin-maleimide with E. coli cells expressing BtuB with Cys substitutions

Lack of reactivity indicates that Cys is buried or in a disulfide

Strains btuB carrying BtuB variants and TonB ExbBD grown in minimal medium Collected in log phase and reacted with 15 μg/ml BMCC (1-biotinamido-4-(4-[maleimidoethylcyclohexane]carboxamido)butane) for 15min The reactions were stopped by addition of 10 mM DTT Cell extracts were resolved in SDS-PAGE, transferred, blotted with neutravidinvisualized by chemiluminescence

WT

B12

S120

C

D6C D6C/S

120C

V10C

V10C/

S120

C

− + − + − + − + − + − +BMCC labeling

Anti- BtuBimmunoblot

Model of vitamin B12 (CBL) transport system. CBL is transported into the periplasm through the outer membrane receptor/transporter BtuB. Periplasmic protein BtuF binds CBL and transfers it to the inner membrane ABC transporter BtuCD. Current models state that the proton motive force provides energy for BtuB transport. The energy is believed to be transduced to the outer membrane via direct interactions between the inner membrane TonB-ExbB/D complex and the TBTD amino terminal Ton Box motif.

ReferencesCadieux, N, Phan PG, Cafiso DS and Kadner, R.J. (2003) Proc. Natl. Acad. Sci. U.S.A. 100, 10688-10693Cadieux, N and Kadner, R.J. (1990) Proc. Natl. Acad. Sci. U.S.A. 96, 10673-10678Chimento, D.P., Mohanty, A., Kadner R.J. and Wiener, M.C.(2003a) Nature Struct. Biol, 10, 391-401Chimento. D.P. , Kadner, R.J. and Wiener, M.C.,(2003b) J. Mol. Biol. 332, 999-1014Eisenhower, H.A., Shames, S., Pawelek, P. D. and Coulton, J. W., (2005) J. Biol, Chem. 280, 30574-30580Nikaido, H (2003) Microbiol. Mol. Biol. Rev. 67, 593-656Kulp, A and Kuehn M.J., . (2010) Annu. Rev. Microbiol. 64, 163-184Ellis T. N. and Kuehn M. J., (2010) Microbiol and Mol. Biol Reviews, 74, 81-94Schwechheimer C. and Kuehn M. J., (2013) 195, 4161-4173

A disulfide cross-link between S120C with an amino terminal Ton box Cys blocks accessibility of the Ton box into the periplasm and greatly reduces the rate of B12 uptake. Transport is recovered upon reduction of the crosslink and occurs in the absence of the pmf.

These results suggest an activate transport conformation that is not predicted by any of the TBDT transporters. Our data indicate that the Ton box play critical roles in establishing the proper conformation for transport.

Cbl binds to the cross-linked BtuB in isolated outer membrane fragments but not to the whole cells indicating the Cbl binding site of the cross-linked form is oriented towards the periplasmic side.

The TBDT family members FecA and FhuA with cysteines in equivalent positions also form stoichiometric disulfide bonds.

Conclusions

Transport of [57Co]CBL is activated upon reduction of the double Cys mutant disulfide.

Suggests the cross-linked conformation is the activated state.

[57Co]CBL Uptake Method

btuB strains carrying BtuB derivatives grown in minimal medium

Cultures were harvested in log phase and washed with phosphate buffer

Uptake is started by addition of 10 nM [57Co]CBL

1 ml aliquots of the reaction mixture were taken every 10 min

Cells are collected on Millipore filters, washed twice with 10 mM LiCl

Different amounts of DTT were added at 30 min and uptake continued for 40 min

Filters are dried and counted in scintillation cocktail.

DTTpmol

CBL

/109 c

ells

The Gram negative bacteria TonB-dependent transporter(TBDT) BtuB translocatesvitamin B12 across the outer membrane. The amino-terminal lumenal domain fits within the 22-stranded β-barrel and contains several determinants for Cbl binding. Current models suggest that the TBDT carries out active transport driven by energy from the proton motive force(pmf), which is transmitted via direct interactions between the inner membrane protein TonB and the BtuB amino-terminal Ton Box motif. The lumenal domain must undergo large conformationalchanges to accommodate passage of the substrate through the barrel, but the molecular features of the mechanism are unknown. We show that disulfide bonds can be induced to form in whole cells between three pairs of cysteines introduced at barrel-lumenal domain contact points, consistent with the x-ray crystallographic structures. Howerer, we also find spontaneously formed, stoichometric cross-links between cysteines in the Ton Box motif and in place of Ser-120 that indicate a conformation different from the x-ray structures. The TBDT family members FecA and FhuA with cysteines in equivalent positions also form stoichiometric disulfide bonds. Cbl uptake through BtuB is blocked by the Ton Box Cys-S120C cross-link and activity is recovered upon reduction. Significantly, Cbl binds to the cross-linked BtuB in isolated outer membrane fragments but not whole cells, indicating that the Cbl binding site of the cross-linked form is oriented towards the periplasmic side. In contrast, the transporter is oriented outwards in all other condition, included in the absence of a pmf. These results suggest an alternating transport mechanism.

C

CN

-Cbl

bou

nd, p

mol

s/m

g pr

otei

n

CN

-Cbl

bou

nd, p

mol

s/10

9 cel

ls

A B

CN

-Cbl

bou

nd, %

of m

axim

um

Time in min

DTT − −+ +WT V10C/S120C

Effect of btuB variants on cobalamin binding (A) and release (B) in vitro. After cell envelopes had been incubated with 15nM labeled Cbl for 15min, 1ml samples were taken, filtered, washed, dried and measured the binding activity. Binding results were expressed as pmol per mg protein(A). After the maximum binding, 2.5μM unlabeled CNCbl were added to the reaction. Samples were taken at time points and activity were measured (B). The release results were expressed as a percentage of maximum bound of CNCbl. wt ; , S120C; , V10C ; , S120C/V10C.

Effect of btuB variants on cobalamin binding in vivo(C). Cells were grown in minimal medium to log phase, washed with phosphate buffer, incubation with 2mM DNP for 5 min before adding 5nM labeled CNCbl. After incubation for 10min, 50mM DTT were added and 1ml samples were taken at 20min, filtered, washed, dried and measured. Activity were expressed as pmols per 109 cells.

The Ton Box intra-molecular disulfide spontaneously forms in FecA and FhuA similar to BtuB and cause a gel shift

BtuB-TonBox Cys-S120 disulfide was confirmed by mass spectrometry analysis (data not shown). Cysteines were introduced in the equivalent positions in FecA and FhuA(2×Cys).

Coomassie stained gels of isolated outer membrane preparations from strains RK5173 (TonB+) and RK5043(ΔtonB) with plasmids carrying (A) WT BtuB or BtuB(10C-120C), (B) WT FecA or FecA(84C-207C), (C) WT FhuA or FhuA(7C-143C).

While BtuB does not show a gel shift, both FecA and FhuA are shifted with the double cysteine mutation(2×Cys; (-)) in non-reducing conditions. With DTT, the mobilities are similar to the non-cysteine containing WT( ).

FecA is not well processed in the ΔtonB strain, whereas FhuA is apparent in the outer membrane preparation from strain RK5043. The mobility of the FhuA-7C-143C mutant band shifts (Panel C, right) indicating that the intra-molecular cross-link forms spontaneously in the absence of TonB.

Detected BtuB conformation states

The substrate (vitamin B12, red octagon) binding-induced extension and disordering of the TonBox (red bar) (Conformation#1 to #2) has been detected in vitro(×× ) and in vivo(×× ) in the presence or absence of TonB or pmf (proton motive force, ΔμΗ+ ) in BtuB and FecA. The substrate binding site is oriented outwards (large black arrows).

The substrate binding site of the S120C mutant (yellow side chain; #3) is oriented outward and bound substrate is occluded.

The cross-linked double mutant TonBox Cys-120C (yellow bar and side chain; #4 and #5) is a conformation with the substrate binding site oriented to the periplasm (large blue arrows) and bound substrate is occluded.

The binding sites of the occluded states (#3 and #4) are still accessible and bind substrate directly. These conformations may be the intermediates that interconvert from #3 to #4 in a pmf-and TonB-dependent manner during the transport cycle.

The TonBox Cys-120C cross-link can be reduced and reformed in the presence of an oxidizing agent such as Cu-phenanthroline, but it is not certain if this process is pmf or TonB dependent. This step may represent the interconversion from periplasmic (#5) to outward (#1) oriented conformations.