Biochemistry 305Biochemistry 305

Calcium Signal TransductionCalcium Signal Transduction

Walter ChazinWalter Chazin5140 BIOSCI/MRBIII5140 BIOSCI/MRBIII

E-mail: Walter.Chazin@ vanderbilt.eduE-mail: Walter.Chazin@ vanderbilt.eduhttp://structbio.vanderbilt.edu/chazinhttp://structbio.vanderbilt.edu/chazin

Jan. 28-30, 2003Jan. 28-30, 2003

By what mechanisms are calcium By what mechanisms are calcium signals read and translated into signals read and translated into biochemical response?biochemical response?

What is the structural basis for the What is the structural basis for the function of the proteins involved?function of the proteins involved?

How is the specificity of different How is the specificity of different signalling pathways generated?signalling pathways generated?

Calcium Signal TransductionCalcium Signal Transduction

Ca2+

Target

Calcium Signal TransductionCalcium Signal Transduction

mM

M

0.110 M

1 slide

Function by converting the ionic signal Function by converting the ionic signal into a biochemical responseinto a biochemical response

Calcium signal transduction involves Calcium signal transduction involves binding induced switch from an off binding induced switch from an off state to an on state that interacts with state to an on state that interacts with target(s)target(s)

Calmodulin is the paradigm systemCalmodulin is the paradigm system

EF-hand Calcium Binding ProteinsEF-hand Calcium Binding Proteins

Step 1: Calcium-induced ActivationStep 1: Calcium-induced Activation

Ca2+

CaM-N

Step 2: Interaction with TargetsStep 2: Interaction with Targets

Target

Calmodulin-Mediated Activation Calmodulin-Mediated Activation of Protein Kinasesof Protein Kinases

Myosin lightchain kinase

The change is calcium concentration in The change is calcium concentration in the cell has to be small: 50-100 foldthe cell has to be small: 50-100 fold

This poses special challenges for the This poses special challenges for the proteins that must have a clean proteins that must have a clean separation between on and off statesseparation between on and off states

Cooperative binding of CaCooperative binding of Ca2+2+ ions is an ions is an essential property for signallingessential property for signalling

Generating Clean Signal ReadoutGenerating Clean Signal Readout

10 slides

What is cooperativity? How is it generated? What is cooperativity? How is it generated?

The binding of the 1st ion causes the The binding of the 1st ion causes the structure to switch to a conformation structure to switch to a conformation that is close to the on statethat is close to the on state

The binding of the first ion causes the The binding of the first ion causes the protein to organize and become less protein to organize and become less flexible, like the on stateflexible, like the on state

Detailed Analysis of CaDetailed Analysis of Ca2+2+ Binding Binding

The enthalpic (structure) and entropic The enthalpic (structure) and entropic (dynamics) penalties associated with (dynamics) penalties associated with binding calcium ions are largely paid binding calcium ions are largely paid by the first ion, thereby facilitating by the first ion, thereby facilitating binding of the second ionbinding of the second ion

Mechanism for CooperativityMechanism for Cooperativity

Now we understand how the calcium Now we understand how the calcium signal is read and transduced into signal is read and transduced into biochemical responsebiochemical response

The next step is to examine how the The next step is to examine how the diversity in the functions of EF-hand diversity in the functions of EF-hand proteins is achieved.proteins is achieved.

And we have only looked at part of the And we have only looked at part of the picture so far!!!!!picture so far!!!!!

More Than Just CaMore Than Just Ca2+2+ Sensors!!! Sensors!!!EF-hand Protein Signal ModulatorsEF-hand Protein Signal Modulators

• Shape signal• Buffer• Transport

EF-hand proteins have homologous EF-hand proteins have homologous sequences and very similar structures, yet sequences and very similar structures, yet diversity in functiondiversity in function

How does nature fine-tune the properties How does nature fine-tune the properties of the protein sequence to achieve this of the protein sequence to achieve this diverse set of activities?diverse set of activities?

- Differences in structural organization- Differences in structural organization- Differences in the response to Ca- Differences in the response to Ca2+2+

Structure-Function RelationshipsStructure-Function Relationships

4 slides

Much structural information is available Much structural information is available that shows the structural basis for the that shows the structural basis for the diverse range of activitiesdiverse range of activities

Analyze calcium-induced opening of Analyze calcium-induced opening of different EF-hand proteinsdifferent EF-hand proteins

- Residue interactions - Residue interactions - Changes in packing interactions- Changes in packing interactions

What Controls Response to CaWhat Controls Response to Ca2+2+??

1 slide

Interactions at the helical interfaces in the Interactions at the helical interfaces in the hydrophobic core dictate response to Cahydrophobic core dictate response to Ca2+2+ binding binding

If understanding is complete, it should be If understanding is complete, it should be possible to control the response to Capossible to control the response to Ca2+2+ binding by adjusting the sequencebinding by adjusting the sequence

Re-engineer the calcium signal modulator Re-engineer the calcium signal modulator calbindin Dcalbindin D9k9k to respond to the binding of to respond to the binding of

calcium in the manner of the calcium calcium in the manner of the calcium sensor calmodulinsensor calmodulin

Can function be altered through directed Can function be altered through directed design?design?

Testing the HypothesisTesting the Hypothesis

Direct, rational and efficient targeting of Direct, rational and efficient targeting of biological activity with high selectivitybiological activity with high selectivity

Ab initioAb initio design of biological function/ design of biological function/ activityactivity

Applications in Therapeutics and Applications in Therapeutics and BiotechnologyBiotechnology

Why Go To All The Trouble?Why Go To All The Trouble?