Modulation of effector affinity by hinge region mutations also modulates switching activity in an engineered allosteric TEM1 β-lactamase switch

Jin Kim, Marc Ostermeier

Research output: Contribution to journalArticle

Abstract

RG13 is an engineered allosteric β-lactamase (BLA) for which maltose is a positive effector. RG13 is a hybrid protein between TEM1 BLA and maltose-binding protein (MBP). Maltose binding to MBP is known to convert the open form of the protein to the closed form through conformational changes about the hinge region. We have constructed and genetically selected several variants of RG13 modified in the hinge region of the MBP domain and explored their effect on β-lactam hydrolysis, maltose affinity and maltose-induced switching. Hinge mutations that increased maltose affinity the most (and thus presumably close the apo-MBP domain the most) also abrogated switching the most. We provide evidence for a model of RG13 switching in which there exists a threshold conformation between the open to closed form of the MBP domain that divides states that catalyze β-lactam hydrolysis with different relative rates of acylation and deacylation.

Original languageEnglish (US)
Pages (from-to)44-51
Number of pages8
JournalArchives of Biochemistry and Biophysics
Volume446
Issue number1
DOIs
StatePublished - Feb 1 2006

Fingerprint

Maltose-Binding Proteins
Maltose
Hinges
Switches
Modulation
Mutation
Lactams
Hydrolysis
Acylation
Conformations
Proteins
Protein Domains

Keywords

  • β-lactamase
  • Allostery
  • Maltose-binding protein
  • Switch

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology

Cite this

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abstract = "RG13 is an engineered allosteric β-lactamase (BLA) for which maltose is a positive effector. RG13 is a hybrid protein between TEM1 BLA and maltose-binding protein (MBP). Maltose binding to MBP is known to convert the open form of the protein to the closed form through conformational changes about the hinge region. We have constructed and genetically selected several variants of RG13 modified in the hinge region of the MBP domain and explored their effect on β-lactam hydrolysis, maltose affinity and maltose-induced switching. Hinge mutations that increased maltose affinity the most (and thus presumably close the apo-MBP domain the most) also abrogated switching the most. We provide evidence for a model of RG13 switching in which there exists a threshold conformation between the open to closed form of the MBP domain that divides states that catalyze β-lactam hydrolysis with different relative rates of acylation and deacylation.",
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AU - Ostermeier, Marc

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N2 - RG13 is an engineered allosteric β-lactamase (BLA) for which maltose is a positive effector. RG13 is a hybrid protein between TEM1 BLA and maltose-binding protein (MBP). Maltose binding to MBP is known to convert the open form of the protein to the closed form through conformational changes about the hinge region. We have constructed and genetically selected several variants of RG13 modified in the hinge region of the MBP domain and explored their effect on β-lactam hydrolysis, maltose affinity and maltose-induced switching. Hinge mutations that increased maltose affinity the most (and thus presumably close the apo-MBP domain the most) also abrogated switching the most. We provide evidence for a model of RG13 switching in which there exists a threshold conformation between the open to closed form of the MBP domain that divides states that catalyze β-lactam hydrolysis with different relative rates of acylation and deacylation.

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