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. 2015 Jan 9;4:e02777. doi: 10.7554/eLife.02777

Figure 1. Comparison of the free-energy landscapes of human lysozyme in the free state (A) and in the bound state with triNAG (B).

The bound state exhibits a ground state (the ‘locked state’) and an additional local minimum with about 13% population (the ‘unlocked state’), which represents an intermediate in the release of the product of the enzymatic reaction. Free-energy landscapes are shown as function of the ‘breathing’ angle θ and of the RMSD from the X-ray structure, which was calculated on the Cα atoms by including secondary structure regions only, of a human lysozyme variant in complex with triNAG (PDB code 1BB5); free-energy landscapes were obtained as −kBTlnH(θ, RMSD), where H(θ, RMSD) is the number of times conformations with specific θ and RMSD values was sampled during the simulations (De Simone et al., 2013b).

DOI: http://dx.doi.org/10.7554/eLife.02777.003

Figure 1.

Figure 1—figure supplement 1. Assignments of the 1H-15N HSQC spectra of the free and triNAG-bound states of human lysozyme.

Figure 1—figure supplement 1.

The x-axis represents the 1H (in ppm) dimension and the y-axis represents the 15N (in ppm) dimension.
DOI: http://dx.doi.org/10.7554/eLife.02777.004

Figure 1—figure supplement 2. Extracts of 1H-15N HSQC spectra showing the titration of triNAG to human lysozyme for selected residues showing significant chemical shift changes upon binding.

Figure 1—figure supplement 2.

Depending on the individual residues, the time scale of exchange is fast or intermediate. Blue: free state (f); yellow: 0.5 equivalents of sugar; red: 1.1 equivalents; green: 2.4 equivalents; blue: 10 equivalents, corresponding to the bound state (b). The x-axis represents the 1H (in ppm) dimension and the y-axis represents the 15N dimension (in ppm).
DOI: http://dx.doi.org/10.7554/eLife.02777.005

Figure 1—figure supplement 3. Illustration of the breathing angle θ of lysozyme (De Simone et al., 2013b), which accounts for the large-amplitude motion between the α-domain and β-domain of lysozyme and is computed from the centres of mass of Cα-atoms from three protein regions (De Simone et al., 2013b).

Figure 1—figure supplement 3.

Region 1 (in the α-domain) spans residues 28–31 and 111–114 (in red), region 2 (in the hinge region) spans residues 90–93 (in yellow), and region 3 (in the β-domain) spans residues 44, 45, 51, and 52 (in green).
DOI: http://dx.doi.org/10.7554/eLife.02777.006

Figure 1—figure supplement 4. (A, B) Experimentally measured 15N-1H residual dipolar couplings (RDCs) of human lysozyme in the free state (A) and the triNAG-bound state (B).

Figure 1—figure supplement 4.

(C, D) Representation of the regions that are mostly affected by triNAG binding in the steric (C) and electrostatic (D) RDC measurements (red indicates small changes and blue indicates large changes).
DOI: http://dx.doi.org/10.7554/eLife.02777.007

Figure 1—figure supplement 5. Validation of the RDC-refined structural ensembles determined in this work representing the free and triNAG-bound states of human lysozyme.

Figure 1—figure supplement 5.

(A) Comparison between calculated and experimental RDCs (black circles for the steric medium and red circles for the electrostatic medium); the Q factors at 0.10 in both cases. (B) Comparison between experimental (black) and calculated (red) 3J HN-Hα scalar couplings; the RMSD value is 0.49 Hz. (C-G) Comparison between experimental and calculated chemical shifts, which we obtained by using the Sparta + method (Shen and Bax, 2010): Cα (C), Cβ (D), N (E), Hα (F), and HN (G); the RMSD values (in ppm) are 0.90, 0.82, 1.90, 0.16, and 0.24 for Cα, Cβ, N, Hα, and HN, respectively. (H) Distributions of the satisfied (about 93%, in green) and violated (about 7%, in orange) NOEs in the individual structures of the ensemble.
DOI: http://dx.doi.org/10.7554/eLife.02777.008