Abstract
The knot is one of the most remarkable topological features identified in an increasing number of proteins with important functions. However, little is known about how the knot is formed during protein folding, and untied or maintained in protein unfolding. By means of all-atom molecular dynamics simulation, here we employ methyltransferase YbeA as the knotted protein model to analyze changes of the knotted conformation coupled with protein unfolding under thermal and mechanical denaturing conditions. Our results show that the trefoil knot in YbeA is occasionally untied via knot loosening rather than sliding under enhanced thermal fluctuations. Through correlating protein unfolding with changes in the knot position and size, several aspects of barriers that jointly suppress knot untying are revealed. In particular, protein unfolding is always prior to knot untying and starts preferentially from separation of two α-helices (α1 and α5), which protect the hydrophobic core consisting of β-sheets (β1–β4) from exposure to water. These β-sheets form a loop through which α5 is threaded to form the knot. Hydrophobic and hydrogen bonding interactions inside the core stabilize the loop against loosening. In addition, residues at N-terminal of α5 define a rigid turning to impede α5 from sliding out of the loop. Site mutations are designed to specifically eliminate these barriers, and easier knot untying is achieved under the same denaturing conditions. These results provide new molecular level insights into the folding/unfolding of knotted proteins.
Highlights
Understanding how proteins fold into their native states is of essential importance for tackling diseases via elucidation of pathology caused by protein unfolding or misfolding [1].We have learned from decades of experimental and computational studies that both protein folding and functioning are essentially determined by the unique information encoded in their amino acid sequences [2,3,4]
Simulations presented in this work were conducted based on the all-atom molecular dynamics (MD) method, which has been widely used for studies of biological systems, including proteins, DNA and plasma membranes [35,36,37,38,39,40,41,42]
Dimerization provi most α/β-knotted proteins, including YbeA considered in our simulations, more stability viaincooperating with the knotted as revealed by our previ exist and function nature as homodimers, previous conformation, studies have demonstrated stability simulations
Summary
Understanding how proteins fold into their native states is of essential importance for tackling diseases via elucidation of pathology caused by protein unfolding or misfolding [1]. We have learned from decades of experimental and computational studies that both protein folding and functioning are essentially determined by the unique information encoded in their amino acid sequences [2,3,4]. Based on this theory, the artificial intelligence model of AlphaFold was developed, which can accurately predict static structures of most proteins given only their amino acid sequences [5,6].
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