1. Mapping Conformational Transitions in the Cyclic AMP Receptor Protein, Class-3, 23-Nov-09 Mapping Conformational Transitions in the Cyclic AMP Receptor Protein

2. Monod-Wyman-Changeux model The model makes a number of simple assumptions: The protein is an oligomer (>1 polypeptide chain) The protein can exist in 2 states: Tense (T) and Relaxed (R) T-state has low affinity for oxygen (K T large) R-state has high affinity for oxygen (K R small) All the subunits of any one molecule are either in the T-state or the R-state (concerted model)

3. Mapping Conformational Transitions in the Cyclic AMP Receptor Protein, Class-3, 23-Nov-09 The Concerted model for allosteric proteins Monod, Wyman and Changeux, MWC model

4. Mapping Conformational Transitions in the Cyclic AMP Receptor Protein, Class-3, 23-Nov-09 Assumes that subunits can change tertiary conformation one at a time in response to binding of substrate. Cooperativity arises because the presence of some subunits carrying substrate favours the strong binding state in adjacent subunits, whose sites are not yet filled. As substrate binding progresses almost all the sites become strong binding. Characterized by existence of molecules with some subunits in weak binding and some in strong binding state. Koshland, Nemethy, Filmer (sequential) model

5. Mapping Conformational Transitions in the Cyclic AMP Receptor Protein, Class-3, 23-Nov-09 The sequential model for allosteric proteins Daniel Koshland Increasing O 2 binding affinity

6. Mapping Conformational Transitions in the Cyclic AMP Receptor Protein, Class-3, 23-Nov-09 Global regulation by catabolite activation/ repression Diauxic growth High glucose no cAMP CAP does not bind not positive control for dozens of operons Low glucose cAMP is produced CAP binds many operons are activated

7. Mapping Conformational Transitions in the Cyclic AMP Receptor Protein, Class-3, 23-Nov-09 1.cAMP binds CRP, allosterically changing the conformation of the protein 2.The CRP-cAMP complex binds to target 22 bp DNA 3.Binding of CRP-cAMP complex to the DNA changes the conformation of both the protein and the DNA 4.Altered conformation modulates transcription initiation by RNA polymerase Effect of cAMP on CRP and transcription initiation

8. Mapping Conformational Transitions in the Cyclic AMP Receptor Protein, Class-3, 23-Nov-09 Kolb et al., Ann Rev Biochem, 1993 cAMP-binding domain DNA-binding domain C-helix HTH motif Structure of the cAMP Receptor Protein

9. Mapping Conformational Transitions in the Cyclic AMP Receptor Protein, Class-3, 23-Nov-09 34 Å 42 Å Comparison of cAMP+CRP with cAMP+CRP+DNA 1O3T Chen et al., 2001 1G6N Passner et al., 2000

10. Mapping Conformational Transitions in the Cyclic AMP Receptor Protein, Class-3, 23-Nov-09 Evidence for cAMP-mediated structural changes in CRP 1.Free CRP binds DNA with low affinity in absence of cAMP, with high affinity and sequence specificity in the presence of cAMP 2.CRP is resistant to proteolysis, but is readily digested in the presence of micromolar concentrations of cAMP 3.DTNB induces intersubunit disulfide bond formation between Cys178s in the CRP dimer in the presence of micromolar concentrations of cAMP but not in its absence 4.Binding of ANS results in an increase in fluorescence intensity with a shift of emission maximum from 530nm to 480 nm. In the presence of micromolar concentration of cAMP, this fluorescence signal is decreased 5.Three cAMP analogs: 1.Induce structural changes and activate transcription 2.Induce structural changes but do not promote DNA binding or transcription activation 3.Bind CRP but do not induce structural changes

11. Mapping Conformational Transitions in the Cyclic AMP Receptor Protein, Class-3, 23-Nov-09 No. of subunits per asymmetric unit2 No. of protein atoms3328 No. of water molecules per asymmetric unit38 Resolution range (Å)33.3 – 2.9 (3.2- 2.9) R cryst (%)22.3 (27.6) R free (%)29.6 (37.2) rmsd from ideal values Bond distances (Å)0.004 Bond angles (°)0.631 Average B-factor (Å 2 ) Overall chain A53.8 Main chain A52.8 Side chain A55.2 Overall chain B35.6 Main chain B33.0 Side chain B38.3 Water30.7 Sulfate ion48.8 Structure validation PROCHECK-Ramachandran plot (%) Core91.6 Allowed8.4 MolProbity Clash score (all atoms)0.91 (100th percentile) Clash score (B < 40)0 Rotamer outliers (%)2.31 C β deviations No. > 250 Protein Data Bank code3H3U Crystallographic statistics Mtb CRP: Rv3676 ~22 kDa P2 1 2 1 2 1 a=54.1, b=84.6, c= 101.2

12. Mapping Conformational Transitions in the Cyclic AMP Receptor Protein, Class-3, 23-Nov-09 Overall Structure of Apo form of Mtb CRP

13. Mapping Conformational Transitions in the Cyclic AMP Receptor Protein, Class-3, 23-Nov-09 Superposition of monomers of Mtb CRP cAMP binding domain DNA binding domain

14. Mapping Conformational Transitions in the Cyclic AMP Receptor Protein, Class-3, 23-Nov-09 E. coli CRP-Binding site of cAMP

15. Mapping Conformational Transitions in the Cyclic AMP Receptor Protein, Class-3, 23-Nov-09 M. tb CRP-Binding site

16. Mapping Conformational Transitions in the Cyclic AMP Receptor Protein, Class-3, 23-Nov-09 cAMP binding site comparison

17. Mapping Conformational Transitions in the Cyclic AMP Receptor Protein, Class-3, 23-Nov-09 Grey: cAMP-bound form Blue: cAMP-free form

18. Mapping Conformational Transitions in the Cyclic AMP Receptor Protein, Class-3, 23-Nov-09 cAMP Helix C Helix F Helix E Aligned DI

19. Mapping Conformational Transitions in the Cyclic AMP Receptor Protein, Class-3, 23-Nov-09

20. LEU 157 ILU 71 LEU 183 LEU 68 C F mtb 1G6N

21. Mapping Conformational Transitions in the Cyclic AMP Receptor Protein, Class-3, 23-Nov-09 Apo- structure of CRP Mt CRP Ec + cAMP complex (1G6N) CRP Ec + cAMP+ DNA complex (1O3T) CRP Ec + cAMP+ DNA+ RNA polymerase C- Domain complex (1LB2) Angle between C-helices of the two monomers (°) 18 24 25 Buried accessible areas Buried accessible surface area of A and B chains respectively (%) 16+18 11+6 12+12 Area buried by the two chains (Å 2 ) A Chain463307347310 B Chain550166336310 Angle between E and F helices A chain ( o )108.099.0104.0 B chain ( o )91.0102.0104.0 Buried surface areas between cAMP-binding and DNA-binding domains

22. Mapping Conformational Transitions in the Cyclic AMP Receptor Protein, Class-3, 23-Nov-09 Green- DNA bound CRP Grey- cAMP bound CRP Blue- cAMP free CRP Superposition of Monomers of CRP A chain and B chain

23. Mapping Conformational Transitions in the Cyclic AMP Receptor Protein, Class-3, 23-Nov-09 Helix E: Q170 – G177 and Helix F: S179 – E181 1O3T Helix E: Q170 – G177 and Helix F: S179 – E181 1G6N Difference distance map between cAMP+CRP and cAMP+CRP+DNA structures

24. Mapping Conformational Transitions in the Cyclic AMP Receptor Protein, Class-3, 23-Nov-09 Difference distance map between apo CRP and cAMP+CRP structures Helix E: Q177 – V184 and Helix F: R188 – T190 Our Structue Helix E: T168 – G177 and Helix F: S180 – R181 1G6N

25. Mapping Conformational Transitions in the Cyclic AMP Receptor Protein, Class-3, 23-Nov-09 1G6N 3H3U

26. Mapping Conformational Transitions in the Cyclic AMP Receptor Protein, Class-3, 23-Nov-09 Normal Mode Analysis Elastic Network Model as implemented in the ElNemo server Two end- structures: one with cAMP and one without Homology models of cAMP-free E. coli CRP and cAMP-bound Mtb CRP Anlayze low frequency normal modes with maximum overlap and collectivity

27. Mapping Conformational Transitions in the Cyclic AMP Receptor Protein, Class-3, 23-Nov-09 Dynamic Cross Correlation Map for 1G6N and model of E. coli cAMP-free CRP.

28. Mapping Conformational Transitions in the Cyclic AMP Receptor Protein, Class-3, 23-Nov-09 Dynamic Cross Correlation Map for 3H3U and Mtb cAMP-bound CRP model.

29. Mapping Conformational Transitions in the Cyclic AMP Receptor Protein, Class-3, 23-Nov-09 Mode 13 for 1G6N chain B

30. Mapping Conformational Transitions in the Cyclic AMP Receptor Protein, Class-3, 23-Nov-09 Mode 16 for 3H3U chain B

31. Mapping Conformational Transitions in the Cyclic AMP Receptor Protein, Class-3, 23-Nov-09

32. Normal Mode Analysis of 1G6N and E.coli model based on 3H3U structure as a reference. Mode 13 shows 57.9 % collectivity and 43.9 % overlap.

33. Mapping Conformational Transitions in the Cyclic AMP Receptor Protein, Class-3, 23-Nov-09

34. NMA of mode 13 shows significant change in C- helix when cAMP binding domain is aligned.

35. Mapping Conformational Transitions in the Cyclic AMP Receptor Protein, Class-3, 23-Nov-09 34 Å 42 Å 45 Å Comparison of 1O3T, 1G6N and 3H3U 1O3T 1G6N Mtb CRP

36. Mapping Conformational Transitions in the Cyclic AMP Receptor Protein, Class-3, 23-Nov-09

37. Summary of Overall Conformational Changes Effected by cAMP-binding 1.In absence of cAMP, the cAMP-binding and DNA-binding domains interact closely with each other, reducing mobility of the DNA-binding domain 2.The reduced mobility prevents sequence specific recognition of DNA 3.Binding of cAMP triggers reorientation of side chains (especially Arg 123) in the binding pocket of CRP 4.The cAMP-binding domain is drawn towards the C-helix closing over the bound cAMP 5.Conformational change in the cAMP-binding domain forces out the DNA-binding domain away from the C-helix 6.The DNA-binding domain remains sufficiently flexible, poised for sequence- specific DNA recognition