Knotted and topologically complex proteins as models for studying folding and stability

Abstract

Among proteins of known three-dimensional structure, only a few possess complex topological features such as knotted or interlinked (catenated) protein backbones. Such unusual proteins offer potentially unique insights into folding pathways and stabilization mechanisms. They also present special challenges for both theorists and computational scientists interested in understanding and predicting protein-folding behavior. Here, we review complex topological features in proteins with a focus on recent progress on the identification and characterization of knotted and interlinked protein systems. Also, an approach is described for designing an expanded set of knotted proteins.

Figure 1

Types of topological complexity observed in proteins. In each panel, a simplified view of the protein is shown on the left, with a stylized diagram of the topology of the system on the right. (a) A unique case of non-covalent catenation. The crystal structure of bovine mitochondrial peroxiredoxin III (PDB code 1zye) revealed two interlinked rings of twelve subunits each [27]. (b) A topological folding barrier [14•] in human superoxide dismutase (1hl4). The red segment of the protein backbone is threaded through a ring formed by the surrounding blue residues. (c) The crystal structure of nerve growth factor (1bet) revealed the first view of the cystine knot motif [55]. The three disulfide bonds which define the motif are shown as red bars. (d) The backbone of the RNA 2′-O-ribose methyltransferase RrmA (1ipa) contains a deep trefoil knot, colored to facilitate visualization [44]. (e) E. coli alkaline phosphatase (1alk) was recently identified as having a deeply slipknotted topology [26••]. The magenta segment of the chain is threaded through the ‘knot core’ (green), but the C-terminal portion of the chain (red) returns through the knot core to effectively unknot the protein as a whole. (f) The dimeric citrate synthase from P. aerophilum (2ibp) is topologically linked by two intramolecular disulfide bonds, shown as red bars [23].

Figure 2

Protein knot plots of four representative knotted proteins. The key (top left) associates various knot types with colors in the plots: green = right-handed trefoil (knot designation 3₁), red = left-handed trefoil, blue = figure eight knot (4₁), yellow = 5₂ knot. Within a given plot, each point in the square matrix indicates a partial structure contained within the protein of interest. The point at the lower left corner of a matrix indicates the complete protein chain, while points closer to the diagonal indicate smaller partial structures. Truncating the N-terminus of a protein corresponds to moving from the lower left corner in a horizontal direction, while truncation of the C-terminus corresponds to moving vertically upwards. White regions are unknotted and colored regions are knotted. (a) RNA 2′-O-ribose methyltransferase (PDB code 1ipa) showing a right-handed trefoil knot that is deep (about 41 residues can be truncated from the C-terminus before the knot is eliminated) and tight (the smallest knotted substructure is only about 44 residues long) [44]. (b) Acetohydroxy acid isomeroreductase (1qmg), showing a deep figure eight knot [32]. A tight trefoil, not previously noted, is also visible within the structure. (c) Alkaline phosphatase (1alk) showing a slipknot structure [26••]; a right-handed trefoil is found within the structure, but the complete protein chain is unknotted. (d) The enzyme ubiquitin hydrolase UCH-L3 (1xd3) showing a complex five-crossing knot [38•]. Note that the five-crossing knot is formed only by the last few C-terminal residues. Otherwise, the structure contains a shallow left-handed trefoil.

Figure 3

Unknotting and knotting proteins by design. (a) Schematic of the fold of a hypothetical knotted protein. Altering the connectivity of the protein chain at the two indicated crossings (* and #) results in a protein with a nearly identical core structure, but an unknotted topology. (b) The fold of the hypothetical unknotted protein generated from the knotted protein in (a). Note how the reverse operation could be applied to the unknotted protein to regenerate the knotted version. (c) Schematic of the primary and secondary structures of the knotted (top) and unknotted (bottom) proteins. The operations necessary to unknot or knot the protein are indicated by pairs of dashed arrows (middle).