Cognate RNA-Binding Modes by the Alternative-Splicing Regulator MBNL1 Inferred from Molecular Dynamics

Figures

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  Figure 1. Mutual information and conservation analysis of the CCCH domain protein family. (A) Circos representation of the CCCH domain family. Square boxes indicate the KL conservation score (from red—highest values to cyan—lowest values). The cumulative mutual information (cMI) and proximity mutual information (pMI) scores are represented as histograms in the inner circle. Lines in the center connect pairs with a MI score higher than 6.5. Red lines represent the top 5%, black lines are between 95% and 70%, and gray lines indicate < 70%. (B) The KL sequence logo of selected nodes. (C) A histogram representing the conservation per residue, cMI and pMI , within 5 Å threshold. The black arrows represent the CCCH motif, and the blue arrows correspond to residues Phe188, Gly191, Gly196, and Phe202. The sequence id numbers correspond to those in the Circos representation.

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  Figure 2. Sequence and structure comparison of MBNL1 ZnFs and MBNL2 ZnF1/2 domains. (A) Three-dimensional structure of MBNL1 ZnF1/2, MBNL1 ZnF3/4, and MBNL2 ZnF1/2, including sequence alignment, conservation, quality, and consensus sequence. (B) Electrostatic potential surface representation obtained using APBS contoured at ±10 kT/e for ZnF1/2 (PDB id 3D2N). (C) ZnF3/4 of MBNL1 binding an RNA fragment (PDB id 3D2S). The ZnF3 binding site region is contoured with a yellow circle.

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  Figure 3. Global fluctuations of MBNL2 and MBNL1 ZnF1/2 computed from their backbone heavy atoms. Atomic indices correspond to residues 24 to 75 for MBNL1 (blue structure) and 26 to 77 for MBNL2 (white structure).

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  Figure 4. Global fluctuations of MBNL1, ZnF1/2, and ZnF3/4 computed from their backbone heavy atoms. Atomic indices correspond to residues 24 to 75 for ZnF1/2 (blue structure) and 185 to 236 for ZnF3/4 (red structure).

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  Figure 5. Effect of RNA binding on local fluctuations of MBNL1 ZnFs. (A) Local fluctuations extracted from the MD simulations for each domain alone (−RNA) or bonded to RNA (+RNA). Only the atomic indices of the RNA binding site are represented (ZnF1: 18 to 38; ZnF2: 52 to 72; ZnF3: 184 to 204; and ZnF4: 218 to 238). (B) Relevant fluctuating amino acids in an unbound (blue) and bound (green) state.

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  Figure 6. Results obtained from the sMD procedure on each of MBNL1’s ZnFs-RNA systems. (A) Representative snapshots of the initial (left) and final (right) coordinates of the sMD pulling process. The CCCH motif of each ZnF is colored green. (B) Cumulative work profiles for each ZnF vs. distance from the COM (r) between ZnF and RNA ligand. Black and red lines represent the mean and the 95% confidence interval. (C) Potential of Mean Force (PMF) for each ZnF extracted from the mean work profile. RP1 and RP2 correspond to the observed rupture points.

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  Figure 7. Local fluctuations extracted from the SMD simulations at four equally distributed segments of the trajectory. The binding process fluctuations (reversed SMD trajectory) are represented in light gray (unbound) to black lines (bound). Only the atomic indices of the RNA binding site are represented (ZnF1: 18 to 38; ZnF2: 52 to 72; ZnF3: 184 to 204; and ZnF4: 218 to 238). The most relevant fluctuations that increase or decrease during the binding process are indicated with an arrow. Grayed regions are not involved in the RNA-binding process.