Fig 1.
Non-covalent lasso entanglements in native and misfolded proteins.
(a) Topology diagram illustrating the non-covalent lasso, where a closed loop (red) formed by a native contact (orange) is threaded through by a segment (blue), with the crossing residue depicted as a white circle. The remaining regions of the protein are depicted in gray. (b) Native structure of CAT-III monomer (PDB 3CLA) highlighting the native entanglement (loop: 5–78; crossing: 190). (c) Topology diagram representing the native entanglement in CAT-III. (d) Misfolded structure of CAT-III state P13, obtained from the previous work [3], with the non-native entanglement highlighted (loop: 169–184; crossing: 35). (e) Topology diagram representing the non-native entanglement in CAT-III state P13. (f) Native structure of DDLB (PDB 4C5C) highlighting the native entanglement (loop: 98–146; crossing: 179). (g) Topology diagram representing the native entanglement in DDLB. (h) Misfolded structure of DDLB state P4, obtained from the previous work [3], with the non-native entanglement highlighted (loop: 117–177; crossing: 186). (i) Topology diagram representing the non-native entanglement in DDLB state P4. (j) Misfolded structure of DDLB state P8, obtained from the previous work [3], with the non-native entanglement highlighted (loop: 145–174; crossing: 141). (k) Topology diagram representing the non-native entanglement in DDLB state P8. The layout of secondary structure elements in the topology diagrams was obtained from PDBe server [8].
Fig 2.
Non-native entanglement was formed due to loss of the native entanglement.
The 80% most probable CAT-III post-translational pathways for (a) native folding and (b) misfolding are shown as a transition network with arrows indicating transitions from one state to another. Each node represents a state, and a representative structure of each state is presented, with the state ID at the top right corner. Structures without gain of non-native entanglements are shown in cyan, while the non-native entanglements are shown in red on the closed loop and blue on the threading segment, with the rest of the protein in white. Four values are presented near each node, representing 〈Q1|3〉 (orange), 〈Q2|5〉 (green), 〈Ggain〉 (red) and 〈Gloss〉 (blue). (c) Native structure of CAT-III (PDB ID: 3CLA) with five segments represented in different colors. (d) A portion of the co-translational folding pathways of DDLB starting from state C7, which is separated into a misfolding pathway (top) and a native folding pathway (bottom). All the representations are the same as in panels (a) and (b), except for the inclusion of a white surface to depict the ribosome in each state, as non-native entanglements form co-translationally for this protein [3].
Fig 3.
Simply stabilizing the native entanglement is not an effective way to rescue misfolded DDLB proteins.
(a) The 3D structure of a coarse-grained ligand model. The interaction sites are colored magenta, and the covalent bonds are colored white. The principal axes of the mass distribution tensor are indicated by dashed arrows, with the maximum bond length from the central bead listed. (b) Native DDLB protein structure highlighting the native entanglement in Domain III (green dashed circle) and the predicted ligand binding site (yellow). The CG ligand (magenta) is shown bound to the target site. Entanglements are depicted as in Fig 1. (c) An initial structure of the nascent DDLB protein of 190 residues (cyan) on the ribosome (white) in the presence of the ligand (magenta). (d) Probabilities of natively entangled structure formation at length 230 (PNative, blue) and ligand binding (PBinding, orange) vs. the interaction energy εij (see Method). Error bars represent the 95% confidence intervals (CIs) estimated by bootstrapping. (e) Ligand binding probability vs. nascent chain (NC) length (top) and the averaged fraction of native contact formed in Domain III vs. NC length with (red) and without (blue) ligand present (bottom). Transparent stripes represent 95% CIs estimated by bootstrapping. (f) Probabilities of forming misfolded entangled states P3 (orange), P4 (red), P8 (magenta), and native state P10 (blue) as a function of post-translational time for the slow DDLB variant with (right) and without (left) ligand present. Transparent stripes represent 95% CIs estimated by bootstrapping. (g) A structure of the enriched misfolded state P3 with the ligand bound (left) and the non-native entanglement topology diagram (right, loop: 98–116; crossing: 186). Entanglements are depicted as in Fig 1.
Fig 4.
Two parallel paths to forming an entanglement involving a single threading event.
Orange circles represent the two residues forming the native contact that closes the loop. The loop and threading segment are shown in red and blue, respectively. A table is presented at the bottom showing the probabilities of both paths utilized by the native and misfolding pathways, respectively. The 95% confidence intervals were estimated by bootstrapping and are presented in the bracket for the probability value less than 100%.
Fig 5.
Ligand destabilizing native closed loop rescues misfolded DDLB.
(a) Predicted non-native structure of DDLB segment 126 to 174. The structure is colored from red to blue from N-terminal tail to C-terminal tail. The predicted ligand binding site is shown in yellow, with the CG ligand presented inside. (b) Probabilities of natively entangled structure formation at the end of translation (PNative, blue) and ligand binding (PBinding, orange) vs. the interaction energy εij (see Method). Error bars represent the 95% confidence intervals (CIs) estimated by bootstrapping. (c) Ligand binding probability vs. nascent chain length (top) and the averaged fraction of native contact formed in Domain III vs. nascent chain length with (red) and without (blue) ligand present (bottom). Transparent stripes represent 95% CIs estimated by bootstrapping. (f) Probabilities of forming misfolded entangled states P3 (orange), P4 (red), P8 (magenta), and native state P10 (blue) as a function of post-translational time for the slow DDLB variant with (right) and without (left) ligand present. Transparent stripes represent 95% CIs estimated by bootstrapping.
Fig 6.
Ligand destabilizing native closed loop rescues misfolded CAT-III.
(a) Predicted non-native structure of CAT-III N-terminal region. The structure is colored from red to blue from N-terminal tail to C-terminal tail. The predicted ligand binding site is shown in yellow, with the CG ligand presented inside. (b) Starting structure used in the binding affinity scan. (c) One of the starting structures of the RNC complex with a CG ligand present used in the co-translational folding simulations. (d) Probabilities of native state formation (PNative, blue) and ligand binding (PBinding, orange) vs. the interaction energy εij (see Method). Error bars represent the 95% confidence intervals (CIs) estimated by bootstrapping. (e) Ligand binding probabilities vs. nascent chain length in the co-translational phase (left) and vs. time in the post-translational phase (right). Representative structures are presented on the top to depict the ligand binding. (e) Ligand binding probabilities vs. nascent chain length in the co-translational phase (top left) and vs. time in the post-translational phase (top right), and the averaged fraction of native contact formed between segments I and III (〈Q1|3〉) vs. post-translation time with (red) and without (blue) ligand present (bottom). Transparent stripes represent 95% CIs estimated by bootstrapping. (f) Probabilities of forming the misfolded entangled state P13 (red) and native state P14 (blue) vs. post-translational time for the fast CAT-III variant with (right) and without (left) ligand present. Transparent stripes represent 95% CIs estimated by bootstrapping.
Table 1.
Top 5 drug candidates for DDLB that showed the strongest binding affinity to the non-native structure and weaker affinity to the native structure.
Table 2.
Top 5 drugs for CAT-III that showed the strongest binding affinity to the non-native structure and no binding affinity to the native structure.