Figure 1.
The topology of covalently linked loops in proteins.
Closed loops occur in proteins under oxidizing conditions when two cysteines are close enough in space to form a disulphide bridge. The size of the loop depends on the sequence separation between the cysteines forming the closed loop. The simplest knot in mathematics is called a “zero knot” (a closed circle). This occurs in proteins under oxidizing conditions when the N- and C-terminal residues are cysteines. In this case a complete circle will be formed by the polypeptide backbone. When the two cysteines are positioned in the middle of the sequence a so-called “cinch” is formed leaving the two terminals open. When one of the terminal residues is a cysteine a lasso like structure is formed. If there are any parts of the polypeptide chain piercing the covalent loop a pierced lasso is formed. The pierced lasso is colored blue in this figure. The white fragment demonstrates the threaded polypeptide chain and the red part is the terminal in front of the covalent loop.
Figure 2.
The unique topology of a Pierced Lasso Bundles (PLBs).
(A) The native structure of leptin (PDB code 1AX8 [79]). A disulphide bridge is located between residue C96 and the C-terminal cysteine (yellow), creating a covalent C-terminal loop (dark blue). Consequently, a helical hairpin of helix C and half of helix B (white), have to thread through the covalent loop to reach the native state. The N-terminal part stays in front of the covalent loop (red). Hence, the overall conformation of the protein creates a Pierced Lasso Bundle (PLB) topology [12]. (B) Four of the new PLBs that we discovered in the PDB plus three unthreaded four-helix bundles are shown in ribbon diagram format. These new PLB proteins have their covalent loop at the opposite end from leptin, creating an N-terminal lasso (light blue). The threaded element (white) is behind the loop from this view while the C-terminal end (red) is in front of the covalent loop. (C) A cartoon defining the elements of the different PLBs showing the PL in blue (dark blue for a C-terminal lasso and light blue for an N-terminal lasso). The threaded element, behind the covalent loop, is white and the terminal in front of the loop is red. The unthreaded foud-helix bundles are shown in white.
Figure 3.
Schematic view of the helical cytokine sub-family.
The cytokine sub-family (red) is divided into three families: The long-chain helical cytokines (pink), the short-chain helical cytokines (orange) and interferons/interleukin 10 (yellow). The overall topology within the cytokines is conserved with differences in length and topology, where the interferons and interleukin 10 have two extra helices outside the four-helix bundle and the long-chain and short-chain helical cytokines have one extra helix. The figure shows the proteins discussed in the text, where threaded motifs (PLBs) are circled and unthreaded proteins are boxed. We note that there are many unthreaded examples as well as a few PLB proteins in each family, not shown in this figure.
Figure 4.
Probability of the formation of helix A.
The plot shows data for Q (a measure of nativeness, between 0–1) versus q(segment) (formation of a particular secondary structure, in this case helix A). The plots display data for reduced (blue) and oxidized (red) protein in the case of the PLBs and the reduced state (black, DD) for the unthreaded proteins (see method section for a full description). The plots are grouped in three boxes according to position in sequence of the covalent loop, dark blue for the C-terminal covalent loop, light blue for the N-terminal covalent loop and grey for the unthreaded proteins. The plots show that the formation of helix A is a late event on the folding landscape as it starts to build its contacts at high Q. The diagonal dashed grey line shows where q(segment) is tracking the overall folding progress. A significant shift is seen between reduced and oxidized N-terminal PLBs where the oxidized state folds faster than the reduced state.
Figure 5.
The threading mechanism of the N-terminal PLB proteins.
(A) The threading mechanism for mIL-3 where slipknotting is the major event (black). We also observe cases where the C-terminal region remains random and no apparent lasso crossing is observed (see cartoon). Panels B-D show the predominance of the plugging mechanism for hIL-3, IFNϕ-1 and IFNϕ-2, respectively. In this mechanism the C-terminal pierces through the N-terminal loop as is shown in the cartoon. As in (A) we also observe cases where the C-terminal region remains random and no apparent lasso crossing is observed (see cartoon).
Figure 6.
Crystal structure of the leptin receptor complex.
The figure shows the leptin receptor complex (PDB code 1AX8 and 3V6O, [40]). The structure of leptin is color-coded according to changes in NSDs, where increased dynamics in the oxidized state is shown in red and increased dynamics in the reduced state is shown in blue. It is clear that there are more dynamics in the oxidized state versus the reduced state. Interestingly, the largest changes in the oxidized state occur at the receptor interface. Previously published results from in vitro activity assays in human cell lines showed that oxidized leptin is more active than reduced leptin [12]. This suggests that leptin benefits from being malleable for receptor interaction.
Figure 7.
Native state dynamics of leptin, hGH, IFNϕ-1 and hIL-3.
Structure based all-atom simulations were performed to obtain NSD. Data for reduced and oxidized protein are shown in blue and red, respectively. The overall fluctuations are shown as bar graphs and the difference between the two states is plotted as a yellow line. The protein sequence is displayed at the top of the graphs and a cartoon of secondary structures is displayed at the bottom (indicating the position of the N- versus the C-terminal loop in light blue and dark blue respectively). Leptin shows an interesting shift in dynamics where the oxidized state is more dynamic than the reduced state over the majority of the structure. The disulphide bridge, not only closes the covalent loop, but also acts as a point of tension inducing dynamics, far from the disulphide bridge. Helix A in leptin shows increased dynamics in the oxidized state, opposite to what is observed in the other PLBs. The decreased dynamics in helix A for the other PLBs is a result of the disulphide bride pining down helix A, thus restricting its dynamics. Interestingly helix A in hIL-3 completely unfolds in the native basin in the reduced state (the data dwarfs the effects on the rest of the sequence and is not included in this figure for clarity). hGH is has an “empty” covalent loop (a “cinch” like structure) of about 100 residues. The plot for hGH shows no significant change of the overall dynamics between reduced and oxidized protein, except for the expected local effect around the disulphide bridge. This implies that that the formation of the covalent loop alone has no effect on the NSD while the presence of a threaded topology piercing the lasso changes the entire protein dynamics.