The Structural Basis For Calcium Signal Transduction

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The Structural Basis For Calcium Signal
Transduction
March 25, 2003

• Walter J. Chazin
• Center for Structural Biology
• Vanderbilt University, Nashville TN
• E-mail Walter.Chazin_at_vanderbilt.edu
• http//structbio.vanderbilt.edu/chazin

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By what mechanisms are calcium signals read and
translated into biochemical response?What is
the structural basis for the function of the
proteins involved?How is the specificity of
different signalling pathways generated?
Calcium Signal Transduction
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Direct, rational and efficient targeting of
biological activity with high selectivityAb
initio design of biologicalfunction/
activityApplications in Therapeuticsand
Biotechnology
Why Go To All The Trouble?
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Calcium Signal Transduction
Ca2
mM
0.1?10 mM
mM
Target
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Function by converting the ionic signal into a
biochemical responseCalcium signal transduction
involvesa Ca2 binding induced switch froman
off state to an on statethat can interact
with target(s)Calmodulin is the paradigm system
EF-hand Calcium Binding Proteins
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Step 1 Calcium-induced Activation
Structure Response to Ca2 binding
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Binding of Ca2 in the EF-hand

• Calcium Coordination
• 7 oxygen atoms from
• 3 mono-dentate side chains
• 1 backbone carbonyl
• 1 water H-bonded to side chain
• 1 bi-dentate side chain
• Specific geometry pentagonal bi-pyrimid

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5
9
3
1
12
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The Functional Unit EF-hand DomainNo Isolated
EF-hands
2 EF-hands
Calmodulin (2 domains)
Domain
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The change in calcium concentration in the cell
has to be small 50-100 foldThis poses special
challenges for the proteins that must have a
clean separation between on and off
statesCooperative binding of Ca2 ions is an
essential property for signalling
Generating Clean Signal Readout
What is cooperativity? How is it generated?
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The Structural Effect Induced by the Binding of
Calcium
Ca2
CaM-N

• The structure of the EF-hand domain is changed

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Activation of Typical Ca2 Sensors
Calmodulin N-terminal Domain
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Step 2 Interaction with Targets
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Example Calmodulin-Mediated Activation of
Protein Kinases
Myosin light chain kinase
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Now we understand how the calcium signal is read
and transduced into biochemical response.The
next step is to determine how the diversity in
the functions of EF-hand proteins is
achieved.EF-hand proteins function as both Ca2
sensors and signal modulators
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More Than Just Ca2 Sensors!!! EF-hand Proteins
as Signal Modulators

• Shape signal
• Buffer
• Transport

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EF-hand proteins have homologous sequences and
very similar structures, yet diversity in
functionHow does nature fine-tune the protein
sequence to achieve diversity and specificity of
biological action?1. Differences in the
response to Ca2
Structure-Function Relationships
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Ca2-Induced Conformational Changes
Calmodulin (N domain)
Calbindin D9K
(Signal Modulator)
(Sensor)
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EF-hand proteins have homologous sequences and
very similar structures, yet diversity in
functionHow does nature fine-tune the protein
sequence to achieve diversity and specificity of
biological action?2. Differences in structural
organization
Functional Diversity and Specificity
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S100 Proteins Unique Architecture

• S100 proteins have a unique dimeric structure

Potts. et al., 1995
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Basic Structural Unit is 4 EF-Hands!

• Interdigitated side chains
• A single contiguous hydrophobic core

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Mode of Action Must Be Unique

• S100 proteins have a unique dimeric structure
• The mode of signal transduction must be distinct
  from calmodulin
• Smaller changes in conformation

Potts. et al., 1995
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Ca2-induced Conformational ChangeS100s Are
Different From Calmodulin Ca2 Sensor
S100B
CaM-N

• S100 protein response is much smaller than
  typical Ca2 sensors

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Mode of Action Must Be Unique

• S100 proteins have a unique dimeric structure
• The mode of signal transduction must be distinct
  from calmodulin
• Smaller changes in conformation
• Activation must be different from CaM

Potts. et al., 1995
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The CaM Wrap-around Paradigm
Ca2
Target
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S100s Bind Targets Differently
p53
Calmodulin/MLC Kinase
S100B/p53

• No wrap-around possible for S100 proteins!
• Dimeric structure has 2 symmetric binding sites

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S100 Protein Quaternary Structure
Dimer
Hexamer
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Many S100 Proteins Oligomerize
S100A9
S100A12
Dimer
Tetramer
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Functional Diversity and Specificity Diversity
from Differences in Quaternary Structure?
Tetramer
Dimer
Hexamer
Octamer
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EF-hand proteins have homologous sequences and
very similar structures, yet diversity in
functionHow does nature fine-tune the protein
sequence to achieve diversity and specificity of
biological action?3. Differences in target
binding
Structure-Function Relationships
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Specificity Calmodulin vs CentrinDifferences in
the Binding Sites for Different Proteins
calmodulin centrin

• Extremely similar structures, but subtle details
  different

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Structural Basis of Functional Diversity
opposite charge
extra cleft
extra pocket
Centrin
Calmodulin

• Also a critical role for target to match the
  binding site!

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Differences in Hydrophobic Surface
Apo S100A6
Apo S100B
Ca2 loaded S100A6
Ca2 loaded S100B

• Differences in D hydrophobic surface induced by
  Ca2 binding

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Differences in Electrostatic Surface
S100B-P53
S100A11-Annexin-I

• Complemented by the properties of the target

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Functional Diversity and SpecificityDifferent
Binding Modes for Different Proteins
S100A10/annexin II
S100A11/annexin I
S100B/p53
S100A9/Chaps
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Functional Diversity and SpecificityA New
Concept!!
S100B-Ndr
S100B-p53

• Different binding modes for the same
• S100 protein with different targets!!

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Summary of Factors Providing Functional Diversity
and Specificity

• Differences in the architecture and responses to
  the binding of calcium ions
• Sequence variability of residues at the surface
  alters the character of binding sites
• The complementarity of the binding surface and
  target leads to different binding modes
• Different modes for different proteins
• Multiple modes for each protein?