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1 Figure S1. Secondary structure of CAP (in the camp 2 -bound state) 10. α-helices are shown as cylinders and β- strands as arrows. Labeling of secondary structure is indicated. CDB, DBD and the hinge are colored blue, magenta and yellow, respectively (same color code as in Fig. 1a). 1

2 Figure S2. Structural comparison of apo-cap (orange) and CAP-cAMP 2 -DNA (blue) 9. View from the top of DNA showing the rigid-body rotation (indicated on the left subunit) that DBD undergoes upon camp binding. α-helices are shown as cylinders and DNA as green cartoon. 2

3 Figure S3. 1 H- 15 N TROSY-HSQC spectra of WT-CAP and CAP-S62F in all liganded states (apo, on the left; camp 2 -bound, in the middle; and camp 2 -DNA-bound, on the right). 3

4 Figure S4. Effect of camp and DNA binding on the structure of WT-CAP (a) and CAP-S62F (b) as assessed by chemical shift mapping. Chemical shift difference (Δω; p.p.m.) values are plotted as a function of residue number. The blue and magenta shaded regions in the graphs delineate the CBD and DBD regions, respectively. Δω for the DNA binding effect reflects the binding of DNA to the camp-bound CAP. Δω values for the camp binding effect are mapped by continuous-scale color onto the WT-CAP-cAMP 2 structure. Stepwise titration of up to 5 mm of camp to CAP-S62F shows that the nucleotide binding pocket becomes saturated at ~0.5 mm of camp. 4

5 Figure S5. camp versus cgmp binding to WT-CAP. In contrast to camp, cgmp binds to CBD but fails to elicit the DBD active, DNA-binding compatible conformation 9. The effect of cgmp is confined to CBD whereas DBD is not affected. a, Effect of camp and b, cgmp binding on the structure of WT-CAP as assessed by chemical shift mapping. Chemical shift difference (Δω) values are plotted as a function of the primary sequence and mapped by continuous-scale color onto the structure of CAP-cAMP 2 (for direct comparison both camp and cgmp effects are mapped on the structure of CAP-cAMP 2 ). c, 1 H- 15 N HSQC spectra of WT-CAP in apo, camp 2 -bound and cgmp 2 -bound states. The blue and magenta shaded regions in the graphs delineate the CBD and DBD regions, respectively. 5

6 Figure S6. Overlaid 1 H- 15 N HSQC spectra of WT-CAP and CAP-S62F in three liganded states (apo, camp 2 - bound and camp 2 -DNA-bound). Representative residues from the helix-turn-helix motif in DBD (G173 and G184) and the camp-binding site (E72, in the insert) in CBD are shown. The data show that while camp binds to CBD of CAP-S62F (saturated at ~0.5 mm camp), the DBD structure is not affected and both apo- CAP-S62F and CAP-S62F-cAMP 2 adopt the inactive, DNA-binding incompatible structure. The chemical shifts of apo-wt-cap and WT-CAP-cAMP 2 can be used as being indicative of the inactive and active state, respectively. A, C, and D refer to the apo, camp-bound state, and DNA-bound state, respectively. 6

7 Figure S7. Structural model of CAP-cAMP 2 wherein the S62F substitution has been modeled in the right subunit. camp is shown as green sticks. Substitution of Phe for Ser introduces a bulky side chain in the vicinity of the camp molecule. In CAP-S62F, the major steric clash between the aromatic side chain of Phe and the adenine base of camp will restrict camp from assuming its favorable position within the nucleotidebinding pocket. As a result the hydrogen bonds between the camp adenine base and the side-chain hydroxyls of Thr127 and Ser128 of the C-helix cannot form. These hydrogen bonds are required for the elongation of the C-helices that result in the stabilization of the active conformation of the DBD and the activation of CAP for DNA binding 9,10. Thus, the failure of camp binding to elicit the active DBD conformation in CAP-S62F is probably due to the inability of camp to form the hydrogen bonds with Thr127 and Ser128. This conclusion is strongly supported by the chemical shift analysis of the camp binding effect on CAP-S62F (Fig. 1c and Supplementary Fig. 4b): the analysis shows that camp binds to the camp binding pocket, and especially at the phosphate binding cassette (PBC), but Thr127, Ser128 and subsequently the C- helix are not stabilized by camp. 7

8 Figure S8. Overlaid 1 H- 15 N TROSY-HSQC spectra of WT-CAP-cAMP 2 -DNA (red) and CAP-S62F-cAMP 2 - DNA (blue) complexes. The data show that the mean structure of the two complexes is almost identical, especially for the DBD, but notable differences are observed for residues (indicated with *) that lie in the camp-binding pocket, close to the S62F substitution site. 8

9 Figure S9. Relaxation rates (R 1, R 2 and { 1 H}- 15 N-NOE) and N-H order parameters (S 2 ) for CAP-S62F-cAMP 2 - DNA as a function of residue number. 9

10 Figure S10. Relaxation rates (R 1, R 2 and { 1 H}- 15 N-NOE) and N-H order parameters (S 2 ) for WT-CAP-cAMP 2 - DNA as a function of residue number. 10

11 Figure S11. Relaxation rates (R 1, R 2 and { 1 H}- 15 N-NOE) and N-H order parameters (S 2 ) for CAP-S62F-cAMP 2 as a function of residue number. 11

12 Figure S12. Relaxation rates (R 1, R 2 and { 1 H}- 15 N-NOE) and N-H order parameters (S 2 ) for WT-CAP-cAMP 2 as a function of residue number. 12

13 Figure S13. Effect of DNA binding on the N-H order parameters (S 2 ) of a, WT-CAP-cAMP 2 and b, CAP-S62FcAMP 2. c, Changes in order parameters, ΔS 2, for WT-CAP-cAMP 2 (left) and CAP-S62F-cAMP 2 (right) upon DNA binding. ΔS 2 is given as S 2 (after DNA binding) S 2 (before DNA binding), so positive ΔS 2 values denote enhanced rigidity of the protein backbone upon DNA binding. The conformational entropy of DNA complex formation estimated through ΔS 2 values (Fig. 2b) is unfavorable for WT-CAP-cAMP 2 ( TΔS conf =39.2 kcal mol -1 ) and favorable for CAP-S62F-cAMP 2 ( TΔS conf = 22.3 kcal mol -1 ). 13

14 Figure S14. Activation of CAP and CAP*. a, Overlaid 1 H- 15 N-HSQC spectra of selected DBD residues of apo and camp 2 -bound WT-CAP and CAP*-G141S. The DBD in CAP*-G141S adopts the active conformation even in the absence of camp and, as a result, apo-cap*-g141s binds strongly to DNA (K d ~8 µm). b, Energetics of camp binding to WT-CAP and CAP*-G141S. Binding isotherms are also shown. c, The thermodynamic components of the allosteric structural transition undergone by DBD as it switches from the inactive to the active conformation. In a, the DBD peaks of apo-cap*-g141s fall in the line that connects the corresponding apo-cap and WT-CAP-cAMP 2 (or CAP*-G141S-cAMP 2 ) peaks. This behavior suggests that DBD in CAP*-G141S is sampling a rapid equilibrium between two conformations: the active and the inactive 54,55. The average chemical shift will be pa*δa+pb*δb, so we estimate the population of the active DBD state in apo-cap*- G141S to ~70-80%. 14

15 Figure S15. Structural vs dynamic activation of protein binding and function. a, The protein exists in an inactive ( T ) and an active ( R ) conformational state. According to the current paradigm, allosteric effector (yellow) binding to the protein induces an allosteric transition (T R) that activates the protein for binding to the ligand (magenta). b, The current paradigm can be modified to include an alternative view (dynamic activation): effector binding fails to induce (stabilize) the T R transition, but the protein becomes activated for binding as a result of changes in protein motions that give rise to favorable conformational entropy (the enhanced dynamic character of the protein is illustrated with a wavy line). 15

16 Figure S16. Characterization of the camp-binding effect on a number of CAP mutants, originally designated as CAP* mutants 14,56. Substitutions at positions 53, 62, 141, 142, 144 and 148 have been proposed to affect CAP activity. Here we characterized the following substitutions (shown in a): D53H; S62F; G141S; R142H/A144T; and, L148R. All of these substitutions are located at the interface between the two domains (CBD and DBD), with the single exception of S62F that is located in the camp-binding pocket. b, Overlaid 16

17 1 H- 15 N HSQC spectra of representative residues from the helix-turn-helix motif in DBD (G173 and G184). The color code corresponds to the one in panel a, with the spectrum of apo-wt-cap and WT-CAP-cAMP 2 in black and green, respectively. The chemical shifts of apo-wt-cap are characteristic of the inactive, DNAbinding incompatible conformation (denoted I for inactive), whereas the chemical shifts of WT-CAP-cAMP 2 are characteristic of the active, DNA-binding compatible conformation (denoted A for active). The data show that camp binding to any of the CAP-D53H, CAP-G141S, CAP-R142H/A144T or CAP-L148R derivatives elicit the active DBD conformation, as in the case of WT-CAP. Therefore, the binding mechanism to DNA of the camp-bound state of each one of these CAP derivatives appears to be very similar to the one of WT-CAP. In sharp contrast, CAP-S62F-cAMP 2 has its DBD in the inactive conformation, as explained in the main text, and binds to DNA with a distinct and unconventional mechanism that involves a large conformational entropy change. It is of interest to note that among these mutants S62F is the only one located in the camp-binding pocket; all other mutants are located at the interface between CBD and DBD. c, 1 H- 15 N HSQC spectra of the camp complex of WT-CAP and all of the CAP derivatives. 17

18 Supplementary References 54. Volkman, B. F., Lipson, D., Wemmer, D. E. & Kern, D. Two-state allosteric behavior in a single-domain signaling protein. Science 291, (2001). 55. Yao, X., Rosen, M. K. & Gardner, K. H. Estimation of the available free energy in a LOV2-J alpha photoswitch. Nat. Chem. Biol. 4, (2008). 56. Garges, S. & Adhya, S. Sites of allosteric shift in the structure of the cyclic AMP receptor protein. Cell 41, (1985). 18