Stabilizing Effect of Inherent Knots on Proteins Revealed by Molecular Dynamics Simulations

Abstract

A growing number of proteins have been identified as knotted in their native structures, with such entangled topological features being expected to play stabilizing roles maintaining both the global fold and the nature of proteins. However, the molecular mechanism underlying the stabilizing effect is ambiguous. Here, we combine unbiased and mechanical atomistic molecular dynamics simulations to investigate how a protein is stabilized by an inherent knot by directly comparing chemical, thermal, and mechanical denaturing properties of two proteins having the same sequence and secondary structures but differing in the presence or absence of an inherent knot. One protein is YbeA from Escherichia coli, containing a deep trefoil knot within the sequence, and the other is the modified protein with the knot of YbeA being removed. Under certain chemical denaturing conditions, the unknotted protein fully unfolds whereas the knotted protein does not, suggesting a higher intrinsic stability for the protein having a knot. Both proteins unfold under enhanced thermal fluctuations but at different rates and with distinct pathways. Opening the hydrophobic core via separation between two a-helices is identified as a crucial step initiating the protein unfolding, which, however, is restrained for the knotted protein by topological and geometrical frustrations. Energy barriers for denaturing the protein are reduced by removing the knot, as evidenced by mechanical unfolding simulations. Finally, yet importantly, no obvious change in size or location of the knot was observed during denaturing processes, indicating that YbeA may remain knotted for a relatively long time during and after denaturation.

Knotted proteins: Tie etiquette in structural biology

“…Furthermore, it was also observed that kinetic stability of lattice proteins increases considerably with the complexity of knot type (98). An enhanced kinetic stability for knotted proteins was also reported in offlattice Molecular Dynamics simulations (26,134). A reduction of the intrinsic unfolding rate due to the presence of a knot in the native structure was previously reported by Yeates and co-workers through in vitro experiments that also compared an engineered knotted model system with its unknotted counterpart (84).…”

T54R mutation destabilizes the dimer of superoxide dismutase 1T54R by inducing steric clashes at the dimer interface

“…29 Since the effects of this previously reported mutation at SOD1 dimer interface is not known in detail, we analyzed what effects T54R mutation has upon the monomeric and dimeric structures of SOD1 T54R . Because molecular dynamics simulation (MDS) can be an invaluable tool to study the stability of dimeric proteins, 30,31 we used this method and other computational as well as experimental techniques in this study. In the wild-type SOD1 protein, threonine-54 (T54) residue is situated in an unstructured loop (T54-loop) that connects the fourth and h beta-sheets of the eight-stranded beta barrel structure.…”

Revealing Topological Barriers against Knot Untying in Thermal and Mechanical Protein Unfolding by Molecular Dynamics Simulations

“…Combining single-molecule fluorescence resonance energy transfer and MD simulations, the knot in TrmD was found to slide towards the C-terminal during unfolding, and the knot size kept nearly unchanged [31]. Our previous simulations showed that the knot can restrict surrounding domains to retard opening of the hydrophobic core, which was defined as a crucial step of protein unfolding [11]. Moreover, the knotted conformation was found to cooperate with dimerization to further enhance the protein stability [12].…”

“…By contrast, the dynamic information about how proteins fold into their native states is relatively less understood [7]. In particular, our past knowledge on protein folding has been challenged since identification of knotted topologies, which play essential roles in protein functioning, such as providing additional stability for maintaining the global fold and catalytic properties [8][9][10][11][12][13]. Elucidation of the folding mechanisms of knotted proteins represents an important new challenge in the protein-folding field.…”