1. Biochemistry 305 Calcium Signal Transduction Walter Chazin 5140 BIOSCI/MRBIII E-mail: Walter.Chazin@ vanderbilt.edu http://structbio.vanderbilt.edu/chazin Jan. 28-30, 2003

2. 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

3. Ca 2+ Target Calcium Signal Transduction mM MM 0.1  10  M 1 slide

4. Function by converting the ionic signal into a biochemical response Calcium signal transduction involves binding induced switch from an off state to an on state that interacts with target(s) Calmodulin is the paradigm system EF-hand Calcium Binding Proteins

5. Step 1: Calcium-induced Activation Ca 2+ CaM-N

6. Step 2: Interaction with Targets Target

7. Calmodulin-Mediated Activation of Protein Kinases Myosin light chain kinase

8. The change is calcium concentration in the cell has to be small: 50-100 fold This poses special challenges for the proteins that must have a clean separation between on and off states Cooperative binding of Ca 2+ ions is an essential property for signalling Generating Clean Signal Readout 10 slides What is cooperativity? How is it generated?

9. The binding of the 1st ion causes the structure to switch to a conformation that is close to the on state The binding of the first ion causes the protein to organize and become less flexible, like the on state Detailed Analysis of Ca 2+ Binding

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

11. Now we understand how the calcium signal is read and transduced into biochemical response The next step is to examine how the diversity in the functions of EF-hand proteins is achieved. And we have only looked at part of the picture so far!!!!!

12. More Than Just Ca 2+ Sensors!!! EF-hand Protein Signal Modulators Shape signal Buffer Transport

13. EF-hand proteins have homologous sequences and very similar structures, yet diversity in function How does nature fine-tune the properties of the protein sequence to achieve this diverse set of activities? - Differences in structural organization - Differences in the response to Ca 2+ Structure-Function Relationships 4 slides

14. Much structural information is available that shows the structural basis for the diverse range of activities Analyze calcium-induced opening of different EF-hand proteins - Residue interactions - Changes in packing interactions What Controls Response to Ca 2+ ? 1 slide Interactions at the helical interfaces in the hydrophobic core dictate response to Ca 2+ binding

15. If understanding is complete, it should be possible to control the response to Ca 2+ binding by adjusting the sequence Re-engineer the calcium signal modulator calbindin D 9k to respond to the binding of calcium in the manner of the calcium sensor calmodulin Can function be altered through directed design? Testing the Hypothesis

16. Direct, rational and efficient targeting of biological activity with high selectivity Ab initio design of biological function/ activity Applications in Therapeutics and Biotechnology Why Go To All The Trouble?