Probing Charge-Controlled Inter-Domain Flexibility: Integrating Experimental and Coarse-Grained Approaches

Figures

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  1. (A). Three-dimensional representation of CBD12 from CALX isoforms 1.1 (PDB ID: 3RB5) and 1.2 (PDB ID: 3RB7) in the Ca2+-bound state. , The regions shown in red highlight the sequence and structural differences between the isoforms, while the green spheres indicate the four calcium ions resolved in the X-ray crystal structure. (B). Representation of the interdomain motions that occur in the absence of calcium, with the open state depicted in gray and the closed state in burgundy. (C). Definition of the structural parameters analyzed: r (interdomain distance, Å) and θ (interdomain angle, degrees). In the gene numbering scheme P17 is P441, D126 is D550 and I268 is I692.

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  2. All-atom dual basin SBM without electrostatics (no charges, no ions). (A). Free-energy profiles as a function of the interdomain angle. The black and red curves correspond to isoforms 1.1 and 1.2, respectively. Profiles are averaged over five independent replicas, and shaded regions indicate ±2 standard deviations. (B,C). Present density plots of the radius of gyration as a function of the Interdomain angle, with B. corresponding to isoform 1.1 and C. refer to isoform 1.2.

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  3. ELViM projecton to dual-basin SBM. Projections A and B correspond to isoform 1.1, whereas projections C and D correspond to isoform 1.2. In projections A and C, the ELViM map is colored according to the Interdomain Distance: colors near blue represent closed conformations, while colors near red indicate open conformations. In projections B and D, the point-density distribution is shown; darker colors (close to black) indicate regions of higher density, whereas lighter colors (toward white) represent regions of lower local density. These analyses were performed using an all-atom structure-based model without electrostatics (no charges and no ions).

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  4. All-atom dual basin SBM with electrostatics potential. Panels A–C show the results obtained in the absence of calcium, while panels D–F include the effect of calcium. Black curves correspond to isoform 1.1, and red curves correspond to isoform 1.2. Panels A and D display the free energy as a function of the interdomain angle. Profiles are averaged over five independent replicas, and shaded regions indicate ±2 standard deviations. Panels B, C, E, and F show density plots of the radius of gyration as a function of the interdomain angle. Panels B and E correspond to isoform 1.1, whereas panels C and F correspond to isoform 1.2.

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  5. 15N-TROSY NMR spectrum of the isoforms 1.1 (blue) and 1.2 (red) of CBD12 recorded at 800 MHz. Peaks colored in cyan (CBD12 1.1) or orange (CBD12 1.2) are negative due to folding effects. Examples of peaks that exist in the spectrum of the 1.1 isoform but could not be easily traced back in the spectrum of the 1.2 isoform are indicated by green arrows, while weak CBD12 1.2 peaks are indicated by red arrows.