Pulling Forces Differentially Affect Refolding Pathways Due to Entangled Misfolded States in SARS-CoV-1 and SARS-CoV-2 Receptor Binding Domain

Abstract

Single-molecule force spectroscopy (SMFS) experiments can monitor protein refolding by applying a small force of a few piconewtons (pN) and slowing down the folding process. Bell theory predicts that in the narrow force regime where refolding can occur, the folding time should increase exponentially with increased external force. In this work, using coarse-grained molecular dynamics simulations, we compared the refolding pathways of SARS-CoV-1 RBD and SARS-CoV-2 RBD (RBD refers to the receptor binding domain) starting from unfolded conformations with and without a force applied to the protein termini. For SARS-CoV-2 RBD, the number of trajectories that fold is significantly reduced with the application of a 5 pN force, indicating that, qualitatively consistent with Bell theory, refolding is slowed down when a pulling force is applied to the termini. In contrast, the refolding times of SARS-CoV-1 RBD do not change meaningfully when a force of 5 pN is applied. How this lack of a Bell response could arise at the molecular level is unknown. Analysis of the entanglement changes of the folded conformations revealed that in the case of SARS-CoV-1 RBD, an external force minimizes misfolding into kinetically trapped states, thereby promoting efficient folding and offsetting any potential slowdown due to the external force. These misfolded states contain non-native entanglements that do not exist in the native state of either SARS-CoV-1-RBD or SARS-CoV-2-RBD. These results indicate that non-Bell behavior can arise from this class of misfolding and, hence, may be a means of experimentally detecting these elusive, theoretically predicted states.

Keywords: SARS-CoV-1 RBD; SARS-CoV-2 RBD; folding pathways; lasso entanglement; protein folding; quenched force.

Figure 1

Representative native entanglements in the native structures of SARS-CoV-1 RBD and SARS-CoV-2 RBD. These structures were obtained using AlphaFold2 and MD simulations. The loops (colored red) are closed by native contacts (colored yellow). (Left) The loop is closed by the native contact between residue 99 and 136, with both N and C termini threading through the loop. Note that only the C-terminal thread is colored blue for more visibility. (Right) The loop is closed by the native contact between residue 108 and 146, with the C-terminal thread highlighted in blue.

Figure 2

The root mean square deviation (RMSD) and the radius of gyration (Rg) of the backbone from three 500 ns all-atom MD simulations for (a) SARS-CoV-1 RBD and (b) SARS-CoV-2 RBD.

Figure 3

Dependence of −ln(P) on Q, where P is the probability of sampling a particular Q value of (a) SARS-CoV-1 RBD and (b) SARS-CoV-2 RBD. An additional minimum is observed at Q~0.2, which occurs in the presence of the external force.

Figure 4

Misfolded states were observed during the folding of the SARS-CoV-1 RBD. Force may promote the folding process of the SARS-CoV-1 RBD, resulting in an increase in the percentage of trajectories that follow paths leading to correct folding. (a,b) represent −ln(P) surfaces, where P is the probability of sampling Q and G values during refolding simulations with F = 0 and F = 5 pN, respectively. (c,d) are transition networks of discrete trajectories along metastable states represented by nodes. Blue, green and red nodes correspond to folded, intermediate and misfolded states, respectively, that trajectories visited at the end of simulations. Red numbers indicate the number of transitions between states, and black numbers represent nodes.

Figure 5

Misfolded states were also observed in the folding of the SARS-CoV-2 RBD. The force effect results in 43% of trajectories remaining in the unfolded state 1 at the end of the simulation. (a,b) represent the −ln(P) surfaces, where P is the probability of sampling certain values of Q and G from refolding simulations with F = 0 and F = 5 pN, respectively. (c,d) are transition networks of discrete trajectories along metastable states represented by nodes. Blue, green and red nodes correspond to folded, intermediate and misfolded states, respectively, that trajectories visited at the end of the simulations. The black node indicates that trajectories remain in the unfolded state 1 at the end of the simulations. Red numbers indicate the number of transitions between states, and black numbers represent nodes.

Figure 6

Conformational distribution along the G parameter of (a) SARS-CoV-1 RBD and (b) SARS-CoV-2 RBD with peaks are concise with characteristics of (Q, G) log probability surfaces, indicating the presence of structures with change in entanglement at different levels, namely G~0.05–0.1, 0.15–0.2, 0.2–0.25.