Abstract
The molybdate importer (ModBC-A of Archaeoglobus fulgidus) and the vitamin B12 importer (BtuCD-F of Escherichia coli) are members of the type I and type II ABC importer families. Here we study the influence of substrate and nucleotide binding on complex formation and stability. Using native mass spectrometry we show that the interaction between the periplasmic substrate-binding protein (SBP) ModA and the transporter ModBC is dependent upon binding of molybdate. By contrast, vitamin B12 disrupts interactions between the transporter BtuCD and the SBP BtuF. Moreover, while ATP binds cooperatively to BtuCD-F, and acts synergistically with vitamin B12 to destabilize the BtuCD-F complex, no effect is observed for ATP binding on the stability of ModBC-A. These observations not only highlight the ability of mass spectrometry to capture these importer-SBP complexes but allow us to add molecular detail to proposed transport mechanisms.
Keywords:
ABC importers; ATP hydrolysis; BtuCD-F; ModBC-A; cooperativity; molybdate; native mass spectrometry; vitamin B(12).
Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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ATP-Binding Cassette Transporters / chemistry*
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ATP-Binding Cassette Transporters / genetics
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ATP-Binding Cassette Transporters / metabolism
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Adenosine Triphosphate / chemistry*
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Adenosine Triphosphate / metabolism
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Amino Acid Sequence
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Archaeoglobus fulgidus / genetics
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Archaeoglobus fulgidus / metabolism*
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Binding Sites
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Cloning, Molecular
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Crystallography, X-Ray
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Escherichia coli / genetics
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Escherichia coli / metabolism*
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Escherichia coli Proteins / chemistry*
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Escherichia coli Proteins / genetics
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Escherichia coli Proteins / metabolism
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Gene Expression
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Genetic Vectors / chemistry
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Genetic Vectors / metabolism
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Ion Transport
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Models, Molecular
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Molybdenum / chemistry*
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Molybdenum / metabolism
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Periplasmic Binding Proteins / chemistry*
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Periplasmic Binding Proteins / genetics
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Periplasmic Binding Proteins / metabolism
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Protein Binding
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Protein Conformation, alpha-Helical
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Protein Conformation, beta-Strand
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Protein Interaction Domains and Motifs
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Protein Multimerization
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Recombinant Proteins / chemistry
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Recombinant Proteins / genetics
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Recombinant Proteins / metabolism
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Sequence Alignment
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Sequence Homology, Amino Acid
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Species Specificity
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Substrate Specificity
Substances
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ATP-Binding Cassette Transporters
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BtuC peptide, E coli
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BtuD peptide, E coli
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Escherichia coli Proteins
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Periplasmic Binding Proteins
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Recombinant Proteins
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btuF protein, E coli
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molybdate
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Molybdenum
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Adenosine Triphosphate