Colbran, Roger

Hoffman, Laurel

Kaufmann, Kristian

Mchaourab, Hassane

Meiler, Jens

Elsevier - Publisher Connector 

Wednesday, February 24, 2010 675aCalcium Signaling Proteins
3518-Pos
Characterization of Conformational Transitions in Dream Protein using
Fluorescence Spectroscopy
Gangadhar Dhulipala, Jaroslava Miksovska.
Florida International University, Miami, FL, USA.
DREAM (Down Stream Regulatory Antagonist Modulator) belongs to an im-
portant class of calcium binding proteins that are involved in transducing the
calcium signal into a biological response in neuronal tissue. DREAM is
a unique calcium binding protein that directly binds to DNA and regulates
the transcription of prodynorphin and c-fos genes in a calcium dependent fash-
ion. Here we report the conformational transitions in DREAM upon calcium
binding by monitoring fluorescence properties of tryptophane residue which
is located in proximity to the high affinity calcium binding EF-hand 3. Ca2þ
association to the protein leads to the hypsochromic shift in the Trp emission
spectrum (max ¼ 338nm) compared to the apo form (max ¼ 343 nm) indicating
that the calcium induced conformational transition strongly alters fluorescent
probe environment. The changes in the Trp emission spectra are strongly influ-
enced by DREAM concentration confirming that alteration of oligomerization
state of DREAM can be readily monitored using fluorescence data. The ligand
induced changes in the tryptophan environment were further confirmed by the
fluorescence quenching studies using iodide and acrydine quenchers. Trp prop-
erties were also analyzed using fluorescence lifetime measurements in fre-
quency domain. In both forms, apo- and Ca2þ bond, the tryptophan lifetime
can be described using two exponential decay model , with 1 ¼ 1.5 ns and
1=3.5 ns. The Ca
2þ association to protein does not significantly alter tryptophan
lifetime but increase the fraction of protein with the lifetime of 1.5 ns.3519-Pos
New Aspects of Lipid-Protein Interactions Revealed by Calmodulin Bind-
ing to the Lipid Mediator Sphingosylphosphorylcholine
Erika Kovacs1, Veronika Harmat2, Judit Toth1, Beata G. Vertessy1,
Karoly Modos3, Jozsef Kardos2, Karoly Liliom1.
1Institute of Enzymology, Budapest, Hungary, 2Eotvos Lorand University,
Budapest, Hungary, 3Semmelweis University, Budapest, Hungary.
Lipid-protein interactions drive core cellular signaling events and regulate the
function of most membrane proteins. Despite their significance, molecular de-
tails of these interactions are scarcely known. Here we provide mechanistic in-
sight into the binding of the lipid mediator sphingosylphosphorylcholine to cal-
modulin, the most central and ubiquitous regulator protein in calcium signaling,
and present a crystal structure of their complex. Thermodynamic and kinetic
analysis of their interaction revealed a peculiar stoichiometry-dependent bind-
ing process. At low protein-to-lipid ratios, calmodulin binds with high affinity
to lipid micelles. At high protein-to-lipid ratios, however, the resulting complex
adopts a compact globular conformation with calmodulin embracing a few lipid
molecules, as can be seen in our crys-
tal structure. Intriguingly, the sphin-
golipid occupies the binding pocket
for the amphipatic alpha-helices of
calmodulin’s target proteins. This
finding explains the competitive inhi-
bition of calmodulin function by the
signaling lipid, and proposes an ut-
terly novel type of endogenous regu-
lation for the calcium sensor protein.
The binding model presented here
might be widely applicable to other
lipid-protein interactions as well.3520-Pos
Substitutions for a Semi-Conserved IQ Motif Glycine Differentially Affect
the Stabilities of Different Ca2þ-Bound States of a Calmodulin-IQ Domain
Complex
Anthony Persechini, DJ Black.
University of Missouri-Kansas City, Kansas City, MO, USA.
We have determined the effects of substituting Ala, Arg or Met for a semi-con-
served Gly residue on the stabilities of different Ca2þ-bound states of a refer-
ence calmodulin-IQ domain complex. The substitutions are all destabilizing in
the absence of Ca2þ, with free energy increases of ~2 kJ/mol for the Ala and
Arg substituted complexes, and ~6 kJ/mol for the Met substituted complex.
When Ca2þ is bound only to the C-ter pair of Ca2þ-binding sites in calmodulinthe free energy of the Ala substituted complex is essentially unaffected, and the
free energies of the Arg and Met substituted complexes decrease by ~4 and ~6
kJ/mol. In contrast, the free energy of the reference complex increases by ~4
kJ/mol. When the N-ter Ca2þ-binding sites in the intermediate C-ter Ca2þ-
bound reference and substituted complexes are occupied their free energies
all decrease by ~4 kJ/mol. This suggests that the N-ter calmodulin lobe un-
dergoes a similar conformational change in all the complexes, which therefore
does not involve direct interactions with the semi-conserved position. When
Ca2þ is bound only to the N-ter pair of Ca2þ-binding sites the free energy
of the Arg substituted complex is unaffected, but the free energies of the
Ala and Met substituted complexes increase by ~13 and ~9 kJ/mol, and the
free energy of reference complex increases by ~7 kJ/mol. This is consistent
with a destabilizing interaction between the Ca2þ-bound N-ter calmodulin
lobe and the calmodulin-binding sequence that requires a non-polar residue
at the semi-conserved position. Our results are overall consistent with a model
in which the N-ter calmodulin lobe has two different binding sites on the ref-
erence CaM-binding sequence, one of which contains the semi-conserved Gly
position.3521-Pos
Conformational Changes of CaMKII: A Model of Activation
Laurel Hoffman, Kristian Kaufmann, Roger Colbran, Jens Meiler,
Hassane Mchaourab.
Vanderbilt University, Nashville, TN, USA.
The ubiquitously expressed calcium/calmodulin dependent protein kinase II
(CaMKII) functions as a transducer of calcium (Ca2þ) signaling by responding
to the amplitude, duration, and frequency of Ca2þ transients. Autophosphory-
lation at Thr286 following binding of calcium-calmodulin (Ca2þ/CaM) leads
to a Ca2þ-independent activity referred to as a conformational memory of prior
activation. While CaM binding, autophosphorylation, and catalytic-regulatory
domain autoinhibition have been linked to CaMKII function, the underlying
structural and dynamic framework of activation and conformational memory
is poorly understood. Here we utilize site-directed spin labeling and electron
paramagnetic resonance (SDSL-EPR) to explore the conformational changes
associated with CaMKII activation and conformational memory. The structure
of the regulatory domain was investigated via spin label mobility under several
conditions representing various intermediates of activation. Inter-domain
movements were also examined through distance measurements between reg-
ulatory and catalytic domains. We found that CaMKII activation is associated
with regulatory-catalytic domain disengagement, causing a disruption of auto-
inhibition and producing significant conformational changes which are propa-
gated throughout the regulatory domain. Here we detail a mechanistic descrip-
tion of activation and are currently using EPR data to computationally model
conformational changes associated with CaMKII activation.3522-Pos
Anomalous Calcium Dependent Binding of Calmodulin to KCNQ2 Potas-
sium Channels
Alessandro Alaimo1, Pilar Areso2, Alvaro Villarroel1.
1Unidad de Biofı´sica CSIC-UPV/EHU, Leioa, Spain, 2Universidad del Paı´s
Vasco, Leioa, Spain.
Often, the free concentration of the ubiquitous Ca2þ sensor calmodulin (CaM)
is limiting, probably below 50 nM in excitable cells, because most of it is
bound to targets inside the cell. However, it is generally assumed that the bind-
ing characteristics do not depend on CaM concentration, and it has been
generally studied at much larger concentrations. We show here that this as-
sumption does not hold true. We have found that Ca2þ favors or reduces the
association between KCNQ2 potassium channels and CaM depending on the
free CaM concentration, shifting abruptly between negative and positive coop-
erativity when the concentration of CaM is below or above ~26 nM, respec-
tively. With CaM concentrations below 26 nM there was a ~four-fold decrease
in the binding affinity in the presence of Ca2þ, shifting the Kd from ~4.7 to ~22
nM. In contrast, with CaM concentrations above 26 nM there was a ~two-fold
increase in the binding affinity in the presence of Ca2þ, shifting the Kd from
~54 nM to ~22 nM. In addition, when 12.5 nM CaM was used in the assay,
the EC50 for Ca
2þ binding changed from ~302 nM to ~608 nM or ~645 nM
in the presence of molar excess of the KCNQ2 or KCNQ3 CaM binding sites,
respectively. In contrast, using 100 nM CaM in the assay, the affinity for Ca2þ
binding increased slightly in the presence of molar excess of the KCNQ2 bind-
ing protein, lowering the EC50 to ~287 nM. The anomalous CaM-dependent
behavior of CaM adds complexity to the integration of calcium signaling inside
the cell.


Conformational Changes of CaMKII: A Model of Activation 

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Conformational Changes of CaMKII: A Model of Activation

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