Protein: DP02953 (UniProt P08572)
Region: residues 1629	to 1638
Annotation: disorder to order (Structural transition)
Evidence: x-ray crystallography evidence used in manual assertion
Supporting statements:
Here, we define three structural motifs in sheet II (Fig. 3), SM1 (hairpin b3–b4), SM2 (hairpin b6–b7) and SM3 (b9 and the preceding loop), and their homologous counterparts in sheet II0, SM1’ (hairpin b3’–b4’), SM2’ (hairpin b6’–b7’) and SM3’ (b9’ and the preceding loop) (Figs. 3a and 3b).
The comparison between the structure of the a2NC1 domain in its noncanonical homohexamer and the same domain in a121NC1 (a2NC1a121) revealed that there are conformational differences that might explain the greater number of subunits (Fig. 2, Supplementary Fig. S6).
As can be seen in the canonical structures, SM1 and SM1’ (Fig. 3b) face the central tunnel, creating a closed ‘barrel’-like arrangement of six alternating b4 and b40 strands, which allows compact intertwining of three monomers (Than et al., 2002). These hairpins (residues 37–45 in the case of SM1 and residues 144–154 in the case of SM10) were not visible in the structure of a2NC1homo (Fig. 3), as would be expected if they were disordered in this noncanonical homo-octamer. On the other hand, they were visible in a4NC1homo but with a different conformation to that in a2NC1121 (Fig. 3). These changes are expected to decrease the strength of the association of the subunits into the protomer.
Overall, a comparison of noncanonical and canonical oligomers (Figs. 3a and 3b) revealed that the SM1, SM1’ and SM2 flexible regions are key determinants in protomer formation, while the loops connecting these motifs, Lb7b8 (motif ClA), Lb70b80 and SM3’, are needed to establish the protomer–protomer interactions that generate the hexamer.
Indeed, previous work with the natural a121NC1 heterohexamer has reported structural inter-chain variability in the conformations of these loops (Sundaramoorthy et al., 2002; Than et al., 2002).
Proposed model for canonical hexamer assembly. Individual monomers (a) start to nucleate a protomer via

b-sheets I/I0 (b). Next, the SM1/1’ and SM2/SM2’ flexible regions from b-sheets II/II0 are stabilized in the nascent protomer, resulting in favoured additional intersubunit interactions within the protomer (c). Final stabilization is attained with the proper folding of SM3’ and of the ClA and ClB motifs that allow the binding of chloride ions (green spheres) (d). The two protomers in the hexamer are now ready to be joined by sulfilimine bonds (red lines).
Our findings of noncanonical assemblies for a2NC1 and a4NC1 homo-oligomers and the structural changes observed in the chains forming these noncanonical protomers in comparison to a2NC1_121 and to the chains in a1NC1homo, a3NC1homo and a5NC1homo could imply that chain folding is closely related to protomer assembly. It is unlikely that a2NC1homo and a4NC1homo could represent stable physiological assemblies, but the structures of the individual chains in these noncanonical oligomers might provide a frozen glimpse of transient conformational states in the process of NC1 folding and hexamer building.
The disordered loops are present in the noncanonical octamer (5NB2_B), while the same loops are ordered when the a2NC1 chain is assembled into the canonical a2NC1_121 hexamer (5NAX).
Cross-references: PDB:5NB2
PDB:5NAX