Mapping Conformational Transitions in the Cyclic AMP Receptor Protein

1. 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 (KT large) • R-state has high affinity for oxygen (KR small) • All the subunits of any one molecule are either in the T-state or the R-state (concerted model)

3. The Concerted model for allosteric proteins Monod, Wyman and Changeux, MWC model

4. Koshland, Nemethy, Filmer (sequential) model • 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.

5. The sequential model for allosteric proteins Daniel Koshland Increasing O2 binding affinity

6. 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. Effect of cAMP on CRP and transcription initiation cAMP binds CRP, allosterically changing the conformation of the protein The CRP-cAMP complex binds to target 22 bp DNA Binding of CRP-cAMP complex to the DNA changes the conformation of both the protein and the DNA Altered conformation modulates transcription initiation by RNA polymerase

8. Structure of the cAMP Receptor Protein HTH motif DNA-binding domain C-helix cAMP-binding domain Kolb et al., Ann Rev Biochem, 1993

9. 34 Å 42 Å Comparison of cAMP+CRP with cAMP+CRP+DNA 1G6N Passneret al., 2000 1O3T Chen et al., 2001

10. Evidence for cAMP-mediated structural changes in CRP • Free CRP binds DNA with low affinity in absence of cAMP, with high affinity and sequence specificity in the presence of cAMP • CRP is resistant to proteolysis, but is readily digested in the presence of micromolar concentrations of cAMP • 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 • 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 • Three cAMP analogs: • Induce structural changes and activate transcription • Induce structural changes but do not promote DNA binding or transcription activation • Bind CRP but do not induce structural changes

11. Crystallographic statistics • Mtb CRP: • Rv3676 • ~22 kDa • P212121 • a=54.1, b=84.6, c= 101.2

12. Overall Structure of Apo form of Mtb CRP

13. DNA binding domain cAMP binding domain Superposition of monomers of Mtb CRP

14. E. coli CRP-Binding site of cAMP

15. M. tb CRP-Binding site

16. cAMP binding site comparison

17. Grey: cAMP-bound form Blue: cAMP-free form

18. Helix F Helix E Helix C cAMP Aligned DI

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

21. Buried surface areas between cAMP-binding and DNA-binding domains

22. Superposition of Monomers of CRP A chain and B chain Green- DNA bound CRP Grey- cAMPbound CRP Blue- cAMPfree CRP

23. Helix E: Q170 – G177 and Helix F: S179 – E181 1O3T Difference distance map between cAMP+CRP and cAMP+CRP+DNA structures Helix E: Q170 – G177 and Helix F: S179 – E181 1G6N

24. Helix E: T168 – G177 and Helix F: S180 – R181 1G6N Helix E: Q177 – V184 and Helix F: R188 – T190 Our Structue Difference distance map between apo CRP and cAMP+CRP structures

25. 3H3U 1G6N

26. 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. Dynamic Cross Correlation Map for 1G6N and model of E. coli cAMP-free CRP.

28. Dynamic Cross Correlation Map for 3H3U and MtbcAMP-bound CRP model.

29. Mode 13 for 1G6N chain B

30. Mode 16 for 3H3U chain B

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

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

35. 34 Å 45 Å 42 Å 1O3T Mtb CRP 1G6N Comparison of 1O3T , 1G6N and 3H3U

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