Mediating molecular recognition by methionine oxidation

Conformational switching by oxidation of methionine in the carboxyl-terminal domain of calmodulin

Asokan Anbanandam, Ramona J. Bieber Urbauer, Ryan K. Bartlett, Heather Smallwood, Thomas C. Squier, Jeffrey L. Urbauer

Research output: Contribution to journalArticle

37 Citations (Scopus)

Abstract

The C-terminus of calmodulin (CaM) functions as a sensor of oxidative stress, with oxidation of methionine 144 and 145 inducing a nonproductive association of the oxidized CaM with the plasma membrane Ca2+-ATPase (PMCA) and other target proteins to downregulate cellular metabolism. To better understand the structural underpinnings and mechanism of this switch, we have engineered a CaM mutant (CaM-L7) that permits the site-specific oxidation of M144 and M145, and we have used NMR spectroscopy to identify structural changes in CaM and CaM-L7 and changes in the interactions between CaM-L7 and the CaM-binding sequence of the PMCA (C28W) due to methionine oxidation. In CaM and CaM-L7, methionine oxidation results in nominal secondary structural changes, but chemical shift changes and line broadening in NMR spectra indicate significant tertiary structural changes. For CaM-L7 bound to C28W, main chain and side chain chemical shift perturbations indicate that oxidation of M144 and M145 leads to large tertiary structural changes in the C-terminal hydrophobic pocket involving residues that comprise the interface with C28W. Smaller changes in the N-terminal domain also involving residues that interact with C28W are observed, as are changes in the central linker region. At the C-terminal helix, 1Hα, 13Cα, and 13CO chemical shift changes indicate decreased helical character, with a complete loss of helicity for M144 and M145. Using 13C- filtered, 13C-edited NMR experiments, dramatic changes in intermolecular contacts between residues in the C-terminal domain of CaM-L7 and C28W accompany oxidation of M144 and M145, with an essentially complete loss of contacts between C28W and M144 and M145. We propose that the inability of CaM to fully activate the PMCA after methionine oxidation originates in a reduced helical propensity for M144 and M145, and results primarily from a global rearrangement of the tertiary structure of the C-terminal globular domain that substantially alters the interaction of this domain with the PMCA.

Original languageEnglish (US)
Pages (from-to)9486-9496
Number of pages11
JournalBiochemistry
Volume44
Issue number27
DOIs
StatePublished - Jun 12 2005
Externally publishedYes

Fingerprint

Molecular recognition
Calmodulin
Methionine
Oxidation
Calcium-Transporting ATPases
Cell membranes
Chemical shift
Cell Membrane
Nuclear magnetic resonance
Oxidative stress
Metabolism
Nuclear magnetic resonance spectroscopy

All Science Journal Classification (ASJC) codes

  • Biochemistry

Cite this

Mediating molecular recognition by methionine oxidation : Conformational switching by oxidation of methionine in the carboxyl-terminal domain of calmodulin. / Anbanandam, Asokan; Bieber Urbauer, Ramona J.; Bartlett, Ryan K.; Smallwood, Heather; Squier, Thomas C.; Urbauer, Jeffrey L.

In: Biochemistry, Vol. 44, No. 27, 12.06.2005, p. 9486-9496.

Research output: Contribution to journalArticle

Anbanandam, Asokan ; Bieber Urbauer, Ramona J. ; Bartlett, Ryan K. ; Smallwood, Heather ; Squier, Thomas C. ; Urbauer, Jeffrey L. / Mediating molecular recognition by methionine oxidation : Conformational switching by oxidation of methionine in the carboxyl-terminal domain of calmodulin. In: Biochemistry. 2005 ; Vol. 44, No. 27. pp. 9486-9496.
@article{cee73bae6ef140efa3315486db9e0cff,
title = "Mediating molecular recognition by methionine oxidation: Conformational switching by oxidation of methionine in the carboxyl-terminal domain of calmodulin",
abstract = "The C-terminus of calmodulin (CaM) functions as a sensor of oxidative stress, with oxidation of methionine 144 and 145 inducing a nonproductive association of the oxidized CaM with the plasma membrane Ca2+-ATPase (PMCA) and other target proteins to downregulate cellular metabolism. To better understand the structural underpinnings and mechanism of this switch, we have engineered a CaM mutant (CaM-L7) that permits the site-specific oxidation of M144 and M145, and we have used NMR spectroscopy to identify structural changes in CaM and CaM-L7 and changes in the interactions between CaM-L7 and the CaM-binding sequence of the PMCA (C28W) due to methionine oxidation. In CaM and CaM-L7, methionine oxidation results in nominal secondary structural changes, but chemical shift changes and line broadening in NMR spectra indicate significant tertiary structural changes. For CaM-L7 bound to C28W, main chain and side chain chemical shift perturbations indicate that oxidation of M144 and M145 leads to large tertiary structural changes in the C-terminal hydrophobic pocket involving residues that comprise the interface with C28W. Smaller changes in the N-terminal domain also involving residues that interact with C28W are observed, as are changes in the central linker region. At the C-terminal helix, 1Hα, 13Cα, and 13CO chemical shift changes indicate decreased helical character, with a complete loss of helicity for M144 and M145. Using 13C- filtered, 13C-edited NMR experiments, dramatic changes in intermolecular contacts between residues in the C-terminal domain of CaM-L7 and C28W accompany oxidation of M144 and M145, with an essentially complete loss of contacts between C28W and M144 and M145. We propose that the inability of CaM to fully activate the PMCA after methionine oxidation originates in a reduced helical propensity for M144 and M145, and results primarily from a global rearrangement of the tertiary structure of the C-terminal globular domain that substantially alters the interaction of this domain with the PMCA.",
author = "Asokan Anbanandam and {Bieber Urbauer}, {Ramona J.} and Bartlett, {Ryan K.} and Heather Smallwood and Squier, {Thomas C.} and Urbauer, {Jeffrey L.}",
year = "2005",
month = "6",
day = "12",
doi = "10.1021/bi0504963",
language = "English (US)",
volume = "44",
pages = "9486--9496",
journal = "Biochemistry",
issn = "0006-2960",
publisher = "American Chemical Society",
number = "27",

}

TY - JOUR

T1 - Mediating molecular recognition by methionine oxidation

T2 - Conformational switching by oxidation of methionine in the carboxyl-terminal domain of calmodulin

AU - Anbanandam, Asokan

AU - Bieber Urbauer, Ramona J.

AU - Bartlett, Ryan K.

AU - Smallwood, Heather

AU - Squier, Thomas C.

AU - Urbauer, Jeffrey L.

PY - 2005/6/12

Y1 - 2005/6/12

N2 - The C-terminus of calmodulin (CaM) functions as a sensor of oxidative stress, with oxidation of methionine 144 and 145 inducing a nonproductive association of the oxidized CaM with the plasma membrane Ca2+-ATPase (PMCA) and other target proteins to downregulate cellular metabolism. To better understand the structural underpinnings and mechanism of this switch, we have engineered a CaM mutant (CaM-L7) that permits the site-specific oxidation of M144 and M145, and we have used NMR spectroscopy to identify structural changes in CaM and CaM-L7 and changes in the interactions between CaM-L7 and the CaM-binding sequence of the PMCA (C28W) due to methionine oxidation. In CaM and CaM-L7, methionine oxidation results in nominal secondary structural changes, but chemical shift changes and line broadening in NMR spectra indicate significant tertiary structural changes. For CaM-L7 bound to C28W, main chain and side chain chemical shift perturbations indicate that oxidation of M144 and M145 leads to large tertiary structural changes in the C-terminal hydrophobic pocket involving residues that comprise the interface with C28W. Smaller changes in the N-terminal domain also involving residues that interact with C28W are observed, as are changes in the central linker region. At the C-terminal helix, 1Hα, 13Cα, and 13CO chemical shift changes indicate decreased helical character, with a complete loss of helicity for M144 and M145. Using 13C- filtered, 13C-edited NMR experiments, dramatic changes in intermolecular contacts between residues in the C-terminal domain of CaM-L7 and C28W accompany oxidation of M144 and M145, with an essentially complete loss of contacts between C28W and M144 and M145. We propose that the inability of CaM to fully activate the PMCA after methionine oxidation originates in a reduced helical propensity for M144 and M145, and results primarily from a global rearrangement of the tertiary structure of the C-terminal globular domain that substantially alters the interaction of this domain with the PMCA.

AB - The C-terminus of calmodulin (CaM) functions as a sensor of oxidative stress, with oxidation of methionine 144 and 145 inducing a nonproductive association of the oxidized CaM with the plasma membrane Ca2+-ATPase (PMCA) and other target proteins to downregulate cellular metabolism. To better understand the structural underpinnings and mechanism of this switch, we have engineered a CaM mutant (CaM-L7) that permits the site-specific oxidation of M144 and M145, and we have used NMR spectroscopy to identify structural changes in CaM and CaM-L7 and changes in the interactions between CaM-L7 and the CaM-binding sequence of the PMCA (C28W) due to methionine oxidation. In CaM and CaM-L7, methionine oxidation results in nominal secondary structural changes, but chemical shift changes and line broadening in NMR spectra indicate significant tertiary structural changes. For CaM-L7 bound to C28W, main chain and side chain chemical shift perturbations indicate that oxidation of M144 and M145 leads to large tertiary structural changes in the C-terminal hydrophobic pocket involving residues that comprise the interface with C28W. Smaller changes in the N-terminal domain also involving residues that interact with C28W are observed, as are changes in the central linker region. At the C-terminal helix, 1Hα, 13Cα, and 13CO chemical shift changes indicate decreased helical character, with a complete loss of helicity for M144 and M145. Using 13C- filtered, 13C-edited NMR experiments, dramatic changes in intermolecular contacts between residues in the C-terminal domain of CaM-L7 and C28W accompany oxidation of M144 and M145, with an essentially complete loss of contacts between C28W and M144 and M145. We propose that the inability of CaM to fully activate the PMCA after methionine oxidation originates in a reduced helical propensity for M144 and M145, and results primarily from a global rearrangement of the tertiary structure of the C-terminal globular domain that substantially alters the interaction of this domain with the PMCA.

UR - http://www.scopus.com/inward/record.url?scp=21844479556&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=21844479556&partnerID=8YFLogxK

U2 - 10.1021/bi0504963

DO - 10.1021/bi0504963

M3 - Article

VL - 44

SP - 9486

EP - 9496

JO - Biochemistry

JF - Biochemistry

SN - 0006-2960

IS - 27

ER -