Dataset Information

A Dynamic Protein-Protein Coupling between the TonB-Dependent Transporter FhuA and TonB.

ABSTRACT: Bacterial outer membrane TonB-dependent transporters function by executing cycles of binding and unbinding to the inner membrane protein TonB. In the vitamin B12 transporter BtuB and the ferric citrate transporter FecA, substrate binding increases the periplasmic exposure of the Ton box, an energy-coupling segment. This increased exposure appears to enhance the affinity of the transporter for TonB. Here, continuous wave and pulse EPR spectroscopy were used to examine the state of the Ton box in the Escherichia coli ferrichrome transporter FhuA. In its apo state, the Ton box of FhuA samples a broad range of positions and multiple conformational substates. When bound to ferrichrome, the Ton box does not extend further into the periplasm, although the structural states sampled by the FhuA Ton box are altered. When bound to a soluble fragment of TonB, the TonB-FhuA complex remains heterogeneous and dynamic, indicating that TonB does not make strong, specific contacts with either the FhuA barrel or the core region of the transporter. This result differs from that seen in the crystal structure of the TonB-FhuA complex. These data indicate that unlike BtuB and FecA, the periplasmic exposure of the Ton box in FhuA does not change significantly in the presence of substrate and that allosteric control of transporter-TonB interactions functions by a different mechanism than that seen in either BtuB or FecA. Moreover, the data indicate that models involving a rotation of TonB relative to the transporter are unlikely to underlie the mechanism that drives TonB-dependent transport.

SUBMITTER: Sarver JL

PROVIDER: S-EPMC5811355 | biostudies-literature | 2018 Feb

REPOSITORIES: biostudies-literature

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A Dynamic Protein-Protein Coupling between the TonB-Dependent Transporter FhuA and TonB.

Sarver Jessica L JL Zhang Michael M Liu Lishan L Nyenhuis David D Cafiso David S DS

Biochemistry 20180126 6

Bacterial outer membrane TonB-dependent transporters function by executing cycles of binding and unbinding to the inner membrane protein TonB. In the vitamin B<sub>12</sub> transporter BtuB and the ferric citrate transporter FecA, substrate binding increases the periplasmic exposure of the Ton box, an energy-coupling segment. This increased exposure appears to enhance the affinity of the transporter for TonB. Here, continuous wave and pulse EPR spectroscopy were used to examine the state of the ...[more]

PMID: 29338257

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Project description:Abstract Hydrogen‐deuterium exchange mass spectrometry (HDX‐MS) is a powerful tool that monitors protein dynamics in solution. However, the reversible nature of HDX labels has largely limited the application to in vitro systems. Here, we describe a protocol for measuring HDX‐MS in living Escherichia coli cells applied to BtuB, a TonB‐dependent transporter found in outer membranes (OMs). BtuB is a convenient and biologically interesting system for testing in vivo HDX‐MS due to its controllable HDX behavior and large structural rearrangements that occur during the B12 transport cycle. Our previous HDX‐MS study in native OMs provided evidence for B12 binding and breaking of a salt bridge termed the Ionic Lock, an event that leads to the unfolding of the amino terminus. Although purified OMs provide a more native‐like environment than reconstituted systems, disruption of the cell envelope during lysis perturbs the linkage between BtuB and the TonB complex that drives B12 transport. The in vivo HDX response of BtuB's plug domain (BtuBp) to B12 binding corroborates our previous in vitro findings that B12 alone is sufficient to break the Ionic Lock. In addition, we still find no evidence of B12 binding‐induced unfolding in other regions of BtuBp that could enable B12 passage. Our protocol was successful in reporting on the HDX of several endogenous E. coli proteins measured in the same measurement. Our success in performing HDX in live cells opens the possibility for future HDX‐MS studies in a native cellular environment. IMPORTANCE We present a protocol for performing in vivo HDX‐MS, focusing on BtuB, a protein whose native membrane environment is believed to be mechanistically important for B12 transport. The in vivo HDX‐MS data corroborate the conclusions from our previous in vitro HDX‐MS study of the allostery initiated by B12 binding. Our success with BtuB and other proteins opens the possibility for performing additional HDX‐MS studies in a native cellular environment.

Dataset Information

A Dynamic Protein-Protein Coupling between the TonB-Dependent Transporter FhuA and TonB.

Publications

A Dynamic Protein-Protein Coupling between the TonB-Dependent Transporter FhuA and TonB.

Similar Datasets

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