Table 1.
Knotted proteins representatives list.
Figure 1.
SOTCase and homologous proteins: phylogenetic tree and structural alignment core.
(a) The phylogenetic tree was obtained by applying a neighbor joining algorithm [38] to the CLUSTALW multiple sequence alignment of SOTCase and its sequence homologs. The branches' length reflects the percentage sequence dissimilarity (5% gauge shown at the top). The numbers at the nodes, calculated by the bootstrap algorithm, indicate the percent robustness of the separation of two bifurcating branches. The two branches involving knotted proteins (all trefoils) are highlighted in green. (b) Two orthogonal views of the MISTRAL alignment core of six representatives of the SOTCase homologous proteins, namely 2fg6C (knotted), 2i6uA, 2g68A, 2at2A, 1pg5A and 1ortA. These proteins are 313 amino acids long on average. Their alignment core consists of 212 amino acids at an average RMSD of 1.9Å. The color scheme redwhite
blue follows the N to C sequence directionality. The rendering of PDB structures was carried out using the VMD [56] software.
Figure 2.
Structural alignment of knotted and unknotted proteins.
SOTCase (a) is shown in cartoon representation; the knot-promoting loop segments are highlighted in orange and purple. The MISTRAL alignment with unknotted entry 1ortA is shown in panel (b): aligned residues are colored in blue and red, respectively, while non aligned residues are correspondingly colored in cyan and pink. Knotted protein TARBP1-MTd is shown in panel (c) with the knot-promoting loop segment highlighted in purple. The MISTRAL alignments of TARBP1-MTd with the unknotted proteins 1b93A and 1hdoA are shown in panels (d) and (e), respectively.
Figure 3.
Two-dimensional schematic diagrams.
The secondary and tertiary organization of the knotted TARBP1-MTd (PDBid 2ha8A) (a) and unknotted protein chain 1hdoA (b), which admit a significant structural superposability, see Fig. 2. The color-coding of the aligned and non-aligned secondary elements and of the knot-promoting loop follows the one in Fig. 2. The overall correspondence of the secondary elements is manifest, despite noticeable differences in their “wiring” which reflect in (i) a different fold organization and (ii) different knotted state.
Figure 4.
Knotted photosensory core module.
PaBphP (a) and its alignment with the unknotted chain 2b18A (b). In the knotted structure the knot-promoting loop is highlighted in purple, while the N-terminal domain, which threads through the loop, is shown in green. In the bottom panel, the aligned residues of knotted and unknotted proteins are colored in blue and red, respectively, while non aligned residues are correspondingly colored in cyan and pink. The N-terminal PAS domain (green) and C-terminal PHY domain (cyan) are well-separated by the aligned region, which instead covers almost completely the central GAF domain of PaBphP photosensory core module.
Figure 5.
UCH (a) and its alignment with the unknotted chain 1aecA (b). The aligned residues of the knotted and unknotted protein are colored in blue and red, respectively while unsaturated colors (cyan and pink) are used for non-aligned residues.
Figure 6.
-SAM-S (a) and unknotted protein 1b×4A (b), colored according to the residue index (red-white-blue); bottom, the structural superposition of these two entries where the aligned residues of knotted and unknotted proteins in the bottom row are colored in blue and red, respectively, while non aligned residues are correspondingly colored in cyan and pink. Panel (c) shows the whole structures, while in panel (d) two orthogonal views of the sole aligned regions are presented. In all panels catalytic residues are included in Van der Waals representation. In panel (a), the knotted topology of
-SAM-S can be readily perceived following the coloring of the chain.