High-affinity and cooperative binding of oxidized calmodulin by methionine sulfoxide reductase

Yijia Xiong, Baowei Chen, Heather Smallwood, Ramona J. Bieber Urbauer, Lye Meng Markille, Nadezhda Galeva, Todd D. Williams, Thomas C. Squier

Research output: Contribution to journalArticle

19 Citations (Scopus)

Abstract

Methionines can play an important role in modulating protein-protein interactions associated with intracellular signaling, and their reversible oxidation to form methionine sulfoxides [Met(O)] in calmodulin (CaM) and other signaling proteins has been suggested to couple cellular redox changes to protein functional changes through the action of methionine sulfoxide reductases (Msr). Prior measurements indicate the full recovery of target protein activation upon the stereospecific reduction of oxidized CaM by MsrA, where the formation of the S-stereoisomer of Met(O) selectively inhibits the CaM-dependent activation of the Ca-ATPase. However, the physiological substrates of MsrA remain unclear, as neither the binding specificities nor affinities of protein targets have been measured. To assess the specificity of binding and its possible importance in the maintenance of CaM function, we have measured the kinetics of repair and the binding affinity between oxidized CaM and MsrA. Reduction of Met(O) in fully oxidized CaM by MsrA is sensitive to the protein fold, as repair of the intact protein is incomplete, with >6 Met(O) remaining in each CaM following MsrA reduction. In contrast, following proteolytic digestion, MsrA is able to fully reduce one-half of the oxidized methionines, indicating that surface-accessible Met(O) within folded proteins need not be substrates for MsrA repair. Mutation of the active site (i.e., C72S) in MsrA permitted equilibrium-binding measurements using both ensemble and single-molecule fluorescence correlation spectroscopy measurements. We observe cooperative binding of two MsrA to each CaMox with an apparent affinity (K = 70 ± 10 nM) that is 3 orders of magnitude greater than the Michaelis constant (KM = 68 ± 4 μM). The high-affinity and cooperative interaction between MsrA and CaMox suggests an important regulatory role of MsrA in the binding and reduction of Met(O) in functionally sensitive proteins, such that multiple MsrA proteins are recruited to simultaneously bind and reduce Met(O) in highly oxidized proteins. Given the suggested role of Met(O) in modulating reversible binding interactions between proteins associated with cellular signaling, these results indicate an ability of MsrA to selectively reduce Met(O) within highly surface-accessible sequences to maintain cellular function as part of an adaptive response to oxidative stress.

Original languageEnglish (US)
Pages (from-to)14642-14654
Number of pages13
JournalBiochemistry
Volume45
Issue number49
DOIs
StatePublished - Dec 12 2006
Externally publishedYes

Fingerprint

Calmodulin
Proteins
Repair
Methionine
methionine sulfoxide reductase
Methionine Sulfoxide Reductases
Chemical activation
Cell signaling
Stereoisomerism
Oxidative stress
Fluorescence Spectrometry
Substrates
Oxidation-Reduction
Adenosine Triphosphatases
Digestion
Catalytic Domain
Oxidative Stress
Fluorescence
Maintenance
Spectroscopy

All Science Journal Classification (ASJC) codes

  • Biochemistry

Cite this

Xiong, Y., Chen, B., Smallwood, H., Bieber Urbauer, R. J., Markille, L. M., Galeva, N., ... Squier, T. C. (2006). High-affinity and cooperative binding of oxidized calmodulin by methionine sulfoxide reductase. Biochemistry, 45(49), 14642-14654. https://doi.org/10.1021/bi0612465

High-affinity and cooperative binding of oxidized calmodulin by methionine sulfoxide reductase. / Xiong, Yijia; Chen, Baowei; Smallwood, Heather; Bieber Urbauer, Ramona J.; Markille, Lye Meng; Galeva, Nadezhda; Williams, Todd D.; Squier, Thomas C.

In: Biochemistry, Vol. 45, No. 49, 12.12.2006, p. 14642-14654.

Research output: Contribution to journalArticle

Xiong, Y, Chen, B, Smallwood, H, Bieber Urbauer, RJ, Markille, LM, Galeva, N, Williams, TD & Squier, TC 2006, 'High-affinity and cooperative binding of oxidized calmodulin by methionine sulfoxide reductase', Biochemistry, vol. 45, no. 49, pp. 14642-14654. https://doi.org/10.1021/bi0612465
Xiong, Yijia ; Chen, Baowei ; Smallwood, Heather ; Bieber Urbauer, Ramona J. ; Markille, Lye Meng ; Galeva, Nadezhda ; Williams, Todd D. ; Squier, Thomas C. / High-affinity and cooperative binding of oxidized calmodulin by methionine sulfoxide reductase. In: Biochemistry. 2006 ; Vol. 45, No. 49. pp. 14642-14654.
@article{bddb74b2e5bf427e8d9e8a56e6db1563,
title = "High-affinity and cooperative binding of oxidized calmodulin by methionine sulfoxide reductase",
abstract = "Methionines can play an important role in modulating protein-protein interactions associated with intracellular signaling, and their reversible oxidation to form methionine sulfoxides [Met(O)] in calmodulin (CaM) and other signaling proteins has been suggested to couple cellular redox changes to protein functional changes through the action of methionine sulfoxide reductases (Msr). Prior measurements indicate the full recovery of target protein activation upon the stereospecific reduction of oxidized CaM by MsrA, where the formation of the S-stereoisomer of Met(O) selectively inhibits the CaM-dependent activation of the Ca-ATPase. However, the physiological substrates of MsrA remain unclear, as neither the binding specificities nor affinities of protein targets have been measured. To assess the specificity of binding and its possible importance in the maintenance of CaM function, we have measured the kinetics of repair and the binding affinity between oxidized CaM and MsrA. Reduction of Met(O) in fully oxidized CaM by MsrA is sensitive to the protein fold, as repair of the intact protein is incomplete, with >6 Met(O) remaining in each CaM following MsrA reduction. In contrast, following proteolytic digestion, MsrA is able to fully reduce one-half of the oxidized methionines, indicating that surface-accessible Met(O) within folded proteins need not be substrates for MsrA repair. Mutation of the active site (i.e., C72S) in MsrA permitted equilibrium-binding measurements using both ensemble and single-molecule fluorescence correlation spectroscopy measurements. We observe cooperative binding of two MsrA to each CaMox with an apparent affinity (K = 70 ± 10 nM) that is 3 orders of magnitude greater than the Michaelis constant (KM = 68 ± 4 μM). The high-affinity and cooperative interaction between MsrA and CaMox suggests an important regulatory role of MsrA in the binding and reduction of Met(O) in functionally sensitive proteins, such that multiple MsrA proteins are recruited to simultaneously bind and reduce Met(O) in highly oxidized proteins. Given the suggested role of Met(O) in modulating reversible binding interactions between proteins associated with cellular signaling, these results indicate an ability of MsrA to selectively reduce Met(O) within highly surface-accessible sequences to maintain cellular function as part of an adaptive response to oxidative stress.",
author = "Yijia Xiong and Baowei Chen and Heather Smallwood and {Bieber Urbauer}, {Ramona J.} and Markille, {Lye Meng} and Nadezhda Galeva and Williams, {Todd D.} and Squier, {Thomas C.}",
year = "2006",
month = "12",
day = "12",
doi = "10.1021/bi0612465",
language = "English (US)",
volume = "45",
pages = "14642--14654",
journal = "Biochemistry",
issn = "0006-2960",
publisher = "American Chemical Society",
number = "49",

}

TY - JOUR

T1 - High-affinity and cooperative binding of oxidized calmodulin by methionine sulfoxide reductase

AU - Xiong, Yijia

AU - Chen, Baowei

AU - Smallwood, Heather

AU - Bieber Urbauer, Ramona J.

AU - Markille, Lye Meng

AU - Galeva, Nadezhda

AU - Williams, Todd D.

AU - Squier, Thomas C.

PY - 2006/12/12

Y1 - 2006/12/12

N2 - Methionines can play an important role in modulating protein-protein interactions associated with intracellular signaling, and their reversible oxidation to form methionine sulfoxides [Met(O)] in calmodulin (CaM) and other signaling proteins has been suggested to couple cellular redox changes to protein functional changes through the action of methionine sulfoxide reductases (Msr). Prior measurements indicate the full recovery of target protein activation upon the stereospecific reduction of oxidized CaM by MsrA, where the formation of the S-stereoisomer of Met(O) selectively inhibits the CaM-dependent activation of the Ca-ATPase. However, the physiological substrates of MsrA remain unclear, as neither the binding specificities nor affinities of protein targets have been measured. To assess the specificity of binding and its possible importance in the maintenance of CaM function, we have measured the kinetics of repair and the binding affinity between oxidized CaM and MsrA. Reduction of Met(O) in fully oxidized CaM by MsrA is sensitive to the protein fold, as repair of the intact protein is incomplete, with >6 Met(O) remaining in each CaM following MsrA reduction. In contrast, following proteolytic digestion, MsrA is able to fully reduce one-half of the oxidized methionines, indicating that surface-accessible Met(O) within folded proteins need not be substrates for MsrA repair. Mutation of the active site (i.e., C72S) in MsrA permitted equilibrium-binding measurements using both ensemble and single-molecule fluorescence correlation spectroscopy measurements. We observe cooperative binding of two MsrA to each CaMox with an apparent affinity (K = 70 ± 10 nM) that is 3 orders of magnitude greater than the Michaelis constant (KM = 68 ± 4 μM). The high-affinity and cooperative interaction between MsrA and CaMox suggests an important regulatory role of MsrA in the binding and reduction of Met(O) in functionally sensitive proteins, such that multiple MsrA proteins are recruited to simultaneously bind and reduce Met(O) in highly oxidized proteins. Given the suggested role of Met(O) in modulating reversible binding interactions between proteins associated with cellular signaling, these results indicate an ability of MsrA to selectively reduce Met(O) within highly surface-accessible sequences to maintain cellular function as part of an adaptive response to oxidative stress.

AB - Methionines can play an important role in modulating protein-protein interactions associated with intracellular signaling, and their reversible oxidation to form methionine sulfoxides [Met(O)] in calmodulin (CaM) and other signaling proteins has been suggested to couple cellular redox changes to protein functional changes through the action of methionine sulfoxide reductases (Msr). Prior measurements indicate the full recovery of target protein activation upon the stereospecific reduction of oxidized CaM by MsrA, where the formation of the S-stereoisomer of Met(O) selectively inhibits the CaM-dependent activation of the Ca-ATPase. However, the physiological substrates of MsrA remain unclear, as neither the binding specificities nor affinities of protein targets have been measured. To assess the specificity of binding and its possible importance in the maintenance of CaM function, we have measured the kinetics of repair and the binding affinity between oxidized CaM and MsrA. Reduction of Met(O) in fully oxidized CaM by MsrA is sensitive to the protein fold, as repair of the intact protein is incomplete, with >6 Met(O) remaining in each CaM following MsrA reduction. In contrast, following proteolytic digestion, MsrA is able to fully reduce one-half of the oxidized methionines, indicating that surface-accessible Met(O) within folded proteins need not be substrates for MsrA repair. Mutation of the active site (i.e., C72S) in MsrA permitted equilibrium-binding measurements using both ensemble and single-molecule fluorescence correlation spectroscopy measurements. We observe cooperative binding of two MsrA to each CaMox with an apparent affinity (K = 70 ± 10 nM) that is 3 orders of magnitude greater than the Michaelis constant (KM = 68 ± 4 μM). The high-affinity and cooperative interaction between MsrA and CaMox suggests an important regulatory role of MsrA in the binding and reduction of Met(O) in functionally sensitive proteins, such that multiple MsrA proteins are recruited to simultaneously bind and reduce Met(O) in highly oxidized proteins. Given the suggested role of Met(O) in modulating reversible binding interactions between proteins associated with cellular signaling, these results indicate an ability of MsrA to selectively reduce Met(O) within highly surface-accessible sequences to maintain cellular function as part of an adaptive response to oxidative stress.

UR - http://www.scopus.com/inward/record.url?scp=33845411006&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=33845411006&partnerID=8YFLogxK

U2 - 10.1021/bi0612465

DO - 10.1021/bi0612465

M3 - Article

VL - 45

SP - 14642

EP - 14654

JO - Biochemistry

JF - Biochemistry

SN - 0006-2960

IS - 49

ER -