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March 25, 2003. The Structural Basis For Calcium Signal Transduction. Walter J. Chazin Center for Structural Biology Vanderbilt University, Nashville TN E-mail: Walter.Chazin@vanderbilt.edu http://structbio.vanderbilt.edu/chazin. Calcium Signal Transduction.

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the structural basis for calcium signal transduction

March 25, 2003

The Structural Basis For Calcium Signal Transduction

Walter J. Chazin

Center for Structural Biology

Vanderbilt University, Nashville TN

E-mail: Walter.Chazin@vanderbilt.edu

http://structbio.vanderbilt.edu/chazin

slide2

Calcium Signal Transduction

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?

slide3

Why Go To All The Trouble?

Direct, rational and efficient targeting of biological activity with high selectivityAb initio design of biologicalfunction/ activityApplications in Therapeuticsand Biotechnology

slide4

Calcium Signal Transduction

mM

Ca2+

0.110 mM

mM

Target

slide5

EF-hand Calcium Binding Proteins

Function by converting the ionic signal into a biochemical responseCalcium signal transduction involvesa Ca2+ binding induced switch froman “off state” to an “on state”that can interact with target(s)Calmodulin is the paradigm system

step 1 calcium induced activation
Step 1: Calcium-induced Activation

Structure

Response to Ca2+ binding

slide7

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

7

5

9

3

1

12

the functional unit ef hand domain no isolated ef hands
The Functional Unit: EF-hand DomainNo Isolated EF-hands

2 EF-hands

Calmodulin

(2 domains)

Domain

slide9

Generating Clean Signal Readout

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

What is cooperativity? How is it generated?

the structural effect induced by the binding of calcium
The Structural Effect Induced by the Binding of Calcium

Ca2+

CaM-N

  • The structure of the EF-hand domain is changed
activation of typical ca 2 sensors
Activation of Typical Ca2+ Sensors

Accessible Hydrophobic Surface

Calmodulin N-terminal Domain

slide14

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

slide15

More Than Just Ca2+ Sensors!!!

EF-hand Proteins as Signal Modulators

  • Shape signal
  • Buffer
  • Transport
slide16

Structure-Function Relationships

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+

ca 2 induced conformational changes
Ca2+-Induced Conformational Changes

Calmodulin

(N domain)

Calbindin D9K

(Signal Modulator)

(Sensor)

slide18

Functional Diversity and Specificity

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

slide19

S100 Proteins: Unique Architecture

  • S100 proteins have a unique dimeric structure

Potts. et al., 1995

slide20

Basic Structural Unit is 4 EF-Hands!

  • Interdigitated side chains
  • A single contiguous hydrophobic core
slide21

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

ca 2 induced conformational change s100s are different from calmodulin ca 2 sensor
Ca2+-induced Conformational ChangeS100s Are Different From Calmodulin Ca2+ Sensor

S100B

CaM-N

  • S100 protein response is much smaller than typical Ca2+ sensors
slide23

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

s100s bind targets differently

MLCK

S100s Bind Targets Differently

p53

Calmodulin/MLC Kinase

S100B/p53

  • No wrap-around possible for S100 proteins!
  • Dimeric structure has 2 symmetric binding sites
many s100 proteins oligomerize
Many S100 Proteins Oligomerize

S100A9

S100A12

Dimer

Tetramer

functional diversity and specificity diversity from differences in quaternary structure
Functional Diversity and Specificity Diversity from Differences in Quaternary Structure?

Tetramer

Dimer

Hexamer

Octamer

slide29

Structure-Function Relationships

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

specificity calmodulin vs centrin differences in the binding sites for different proteins
Specificity: Calmodulin vs CentrinDifferences in the Binding Sites for Different Proteins

calmodulin

centrin

  • Extremely similar structures, but subtle details different
structural basis of functional diversity
Structural Basis of Functional Diversity

opposite

charge

extra

cleft

extra

pocket

Centrin

Calmodulin

  • Also a critical role for target to match the binding site!
slide32

Differences in Hydrophobic Surface

Apo S100A6

Apo S100B

Ca2+ loaded S100A6

Ca2+ loaded S100B

  • Differences in D hydrophobic surface induced by Ca2+ binding
slide33

Differences in Electrostatic Surface

S100B-P53

S100A11-Annexin-I

  • Complemented by the properties of the target
functional diversity and specificity different binding modes for different proteins
Functional Diversity and SpecificityDifferent Binding Modes for Different Proteins

S100A10/annexin II

S100A11/annexin I

S100B/p53

S100A9/Chaps

functional diversity and specificity a new concept
Functional Diversity and SpecificityA New Concept!!

S100B-Ndr

S100B-p53

  • Different binding modes for the same
  • S100 protein with different targets!!
summary of factors providing functional diversity and specificity
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?