smFRET Probing Reveals Substrate-Dependent Conformational Dynamics of E. coli Multidrug MdfA

AbstractTo enable the processing of chemical gradients, chemotactic bacteria possess large arrays of transmembrane chemoreceptors, the histidine kinase CheA, and the adaptor protein CheW, organized as coupled core-signaling units (CSU). Despite decades of study, important questions surrounding the molecular mechanisms of sensory signal transduction remain unresolved, owing especially to the lack of a high-resolution CSU structure. Here, we use cryo-electron tomography and sub-tomogram averaging to determine a structure of the Escherichia coli CSU at sub-nanometer resolution. Based on our experimental data, we use molecular simulations to construct an atomistic model of the CSU, enabling a detailed characterization of CheA conformational dynamics in its native structural context. We identify multiple, distinct conformations of the critical P4 domain as well as asymmetries in the localization of the P3 bundle, offering several novel insights into the CheA signaling mechanism.

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Conformational dynamics and membrane interactions of the E. coli outer membrane protein FecA: A molecular dynamics simulation study

Biochimica et Biophysica Acta (BBA) - Biomembranes ◽

10.1016/j.bbamem.2012.08.021 ◽

2013 ◽

Vol 1828 (2) ◽

pp. 284-293 ◽

Cited By ~ 41

Author(s):

Thomas J. Piggot ◽

Daniel A. Holdbrook ◽

Syma Khalid

Keyword(s):

Molecular Dynamics ◽

Molecular Dynamics Simulation ◽

Membrane Protein ◽

Outer Membrane ◽

Simulation Study ◽

Outer Membrane Protein ◽

Conformational Dynamics ◽

Dynamics Simulation ◽

Membrane Interactions ◽

E Coli

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Oversized galactosides as a probe for conformational dynamics in LacY

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1800706115 ◽

2018 ◽

Vol 115 (16) ◽

pp. 4146-4151 ◽

Cited By ~ 3

Author(s):

Irina Smirnova ◽

Vladimir Kasho ◽

Xiaoxu Jiang ◽

Hong-Ming Chen ◽

Stephen G. Withers ◽

...

Keyword(s):

Binding Site ◽

Conformational Dynamics ◽

Dissociation Rate ◽

Binding Kinetics ◽

Lactose Permease ◽

E Coli ◽

Open Conformation ◽

Dissociation Rate Constants ◽

Kinetics Of ◽

Micromolar Range

Binding kinetics of α-galactopyranoside homologs with fluorescent aglycones of different sizes and shapes were determined with the lactose permease (LacY) of Escherichia coli by FRET from Trp151 in the binding site of LacY to the fluorophores. Fast binding was observed with LacY stabilized in an outward-open conformation (kon = 4–20 μM−1·s−1), indicating unobstructed access to the binding site even for ligands that are much larger than lactose. Dissociation rate constants (koff) increase with the size of the aglycone so that Kd values also increase but remain in the micromolar range for each homolog. Phe27 (helix I) forms an apparent constriction in the pathway for sugar by protruding into the periplasmic cavity. However, replacement of Phe27 with a bulkier Trp does not create an obstacle in the pathway even for large ligands, since binding kinetics remain unchanged. High accessibility of the binding site is also observed in a LacY/nanobody complex with partially blocked periplasmic opening. Remarkably, E. coli expressing WT LacY catalyzes transport of α- or β-galactopyranosides with oversized aglycones such as bodipy or Aldol518, which may require an extra space within the occluded intermediate. The results confirm that LacY specificity is strictly directed toward the galactopyranoside ring and also clearly indicate that the opening on the periplasmic side is sufficiently wide to accommodate the large galactoside derivatives tested here. We conclude that the actual pathway for the substrate entering from the periplasmic side is wider than the pore diameter calculated in the periplasmic-open X-ray structures.

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