Piecewise All-Atom SMD Simulations Reveal Key Secondary Structures in Luciferase Unfolding Pathway

Abstract

Although the folding of single-domain proteins is well characterized theoretically and experimentally, the folding of large multidomain proteins is less well known. Firefly luciferase, a 550 residue three-domain protein, has been commonly used as a substrate to study chaperone reactions and as a model system for the study of folding of long polypeptide chains, including related phenomena such as cotranslational folding. Despite being characterized by various experimental techniques, the atomic-level contributions of various secondary structures of luciferase to its fold's mechanical stability remain unknown. Here, we developed a piecewise approach for all-atom steered molecular dynamics simulations to examine specific secondary structures that resist mechanical unfolding while minimizing the amount of computational resources required by the large water box of standard all-atom steered molecular dynamics simulations. We validated the robustness of this approach with a small NI3C protein and used our approach to elucidate the specific secondary structures that provide the largest contributions to luciferase mechanostability. In doing so, we show that piecewise all-atom steered molecular dynamics simulations can provide novel atomic resolution details regarding mechanostability and can serve as a platform for novel mutagenesis studies as well as a point for comparison with high-resolution force spectroscopy experiments.

Figure 1

Change of SMD dummy atom and reference atom during piecewise pulling simulations (the black frame is the simplified sketch of the water box, which is twice as long as the unfolding extension of protein in GROMACS 2018.2). To see this figure in color, go online.

Figure 2

NI3C validation using different box sizes shows that structural details remain unchanged by box size. (a) Force extension curves from multiple piecewise pulling simulations using two different box sizes are shown. (b) Structural details of NI3C unfolding using a 25-nm water box are shown. (c) Structural details of NI3C unfolding using a 50-nm water box are shown. To see this figure in color, go online.

Figure 3

Force extension plots from coarse-grained simulations. (a) The superpositions of 100 simulations and (b) four randomly chosen simulations are shown. To see this figure in color, go online.

Figure 4

The superposition of force extension plots from four all-atom piecewise simulations. To see this figure in color, go online.

Figure 5

Unfolded secondary structures in four all-atom simulations (blue: C-terminal domain; purple: middle domain; and green: N-terminal domain). To see this figure in color, go online.

Figure 6

Unfolding process of secondary structures for three small peaks. (a) Peak 1 is shown; (b) peak 3 is shown; and (c) peak 5 is shown. The unfolded portions of firefly luciferase are not shown to improve figure clarity. To see this figure in color, go online.

Figure 7

Unfolding process of secondary structures for two large peaks. (a) The first two secondary structures unfolded in peak 4 are shown. (b) The first secondary structures unfolded in peak 6 are shown. (c) The second and the third secondary structures unfolded in peak 6 are shown. To see this figure in color, go online.