A. Domain organization of canonical ankyrins. The ~89 kDa membrane binding domain consists of 24 tandem ankyrin repeats (blue), a 62 kDa spectrin-binding domain (orange) that includes the ZU5A, ZU5B, and UPA domains, and a 55 kDa C-terminal flexible regulatory domain (red) containing aDeath domain (DD). Numbering in the diagram corresponds to that of human ankyrin-R. B. Comparative domain organization of Ankyrin-R and UNC5b. UNC5b contains a large transmembrane region followed by ZU5 (light green), UPA (orange) and DD (purple) domains. The arrangement of these domains is similar to the one observed in ankyrins. In the latter, the first ZU5 domain (blue), ZU5A, mediates the interaction with β-spectrin repeats 14–15. UNC5b is known to form a supramodule involving ZU5-UPA-DD domains²⁵. Based on the sequence similarity, it is expected that ankyrin forms a similar supramodule involving the ZU5B, UPA and DD domains, but not the ZU5A spectrin binding domain.

A. Native gel shift assay of the ZU5A-ZU5Bfragment of ankyrin and spectrin. The ZU5A-ZU5B fragment was mixed with repeats 13–15 (HEβ1315) or 14–15 (HEβ1415) of human erythrocyte β spectrin to ascertain whether the fragments interact. The native gel shift shows a shift in spectrin mobility due to the interaction with ankyrin. The presence of absence of ZU5A-ZU5B is shown by + or − signs. As expected, the ankyrin fragment interacts with spectrin repeats containing the ankyrin binding region. B. Binding affinity and kinetic parameters data describing the ZU5A-ZU5B interaction with β-spectrin.Binding affinity and kinetic parameters measured by SPR between the ZU5A-ZU5B fragment and repeats 12–17 (HEβ1217) or repeats 14–15 of human erythrocyte β-spectrin. For comparison, parameters for the ZU5A only fragment are shown in the final row of the table²¹. The ZU5A-ZU5B interaction with human β-spectrin is comparable to the one observed between spectrin and ZU5A alone. C and D. SPR trace of ZU5A-ZU5B interaction with HEβ1217 and HEβ1415. Surface plasmon resonance data and fits for ZU5A-ZU5B binding to biotinylated HEβ1217 or HEβ1415 at various concentrations of ZU5A-ZU5B shown in the inset.

A. Ribbon diagram of the ZU5A-ZU5B fragment. N and C-termini are as labeled on figure. The structure shows two independent, compact, and similar well-folded β-sheet rich cores, each with different numbers of loops and helices. The ZU5B domain (green) extends away from the ZU5A (turquoise) and interacts minimally with it. The ZU5A and ZU5B domains do not interact extensively.The interface between the two domains is small and involves few contacts. The contact surface area is 514 Ų³¹, emphasizing the minimal interaction between the domains. B. Electrostatic surface charge of ZU5A-ZU5B. The ZU5A-ZU5B fragment shows a positive patch on the surface on the ZU5A domain that corresponds to the spectrin binding site^{23; 24} (circled in purple). In contrast, ZU5B does not exhibit a marked charged character and does not have the equivalent positively charged patch associated with spectrin binding. C. Electrostatic surface charge of the ZU5 domain of UNC5b (PDB ID: 3G5B). The electrostatic map of the ZU5 domain of UNC5b displays a negative patch corresponding to the region of interaction with the UPA domain (circled in green). A similarly charged patch is observed on the surface of the ankyrin ZU5B domain, suggesting that the ankyrin UPA domain could make a similar interaction with ZU5B. The UNC5b domain is shown in the same orientation as the right panel of the ZU5A-ZU5B diagram for ease of comparison. The molecular surface of the molecule is shown with the equipotential electrostatic surface mapped onto it at ± 5 k_BT/e_c (blue and red, respectively)³³.

A. Superposed structures of the ankyrin ZU5A domain on the ZU5B domain. Ribbon diagram of ZU5A (turquoise) superposed on ZU5B (green). The C-terminus of the protein is labeled. The overall β-sheet rich core is similar in both structures, but ZU5B shows differences in the positioning of helices and loops. In addition, the ZU5B domain has an additional helix. B. Superposed structures of ankyrin ZU5B on the ZU5 domain of UNC5b (PDB ID: 3G5B). Ribbon diagram of ankyrin-R ZU5B (green) superposed on the ZU5 domain of UNC5b (pink). The N-terminus of ZU5B and C-terminus of ZU5 of UNC5b are labeled. The comparison shows a clear similarity in the β-sheet rich core, but ZU5B has a larger structure due to the presence of longer loops and two extra two helices. C. Superposed structures of ankyrin-R ZU5B on the ZU5 domain of ZO-1 in complex with GRINL1A peptide (PDB ID: 2KXS). Ribbon diagram of ZU5B of ankyrin-R (green) superposed on the ZU5 domain of ZO-1 in the ZO-1/GRINL1A complex (orange). The N-terminus of ZU5B and the N-terminus of ZU5/GRINL1A of ZO-1 are labeled. The ZO-1 ZU5 domain forms an incomplete ZU5 fold that is completed by a region at the N-terminus of the protein and the GRINL1A peptide at the C-terminus³². The comparison shows region that the ZO-1 ZU5 domain is structurally very similar to the ZU5 domain of ZU5A of ankyrin-R in the β core region. Extra residues (1–14) included in the ZO-1/GRINL1A structure, which are not part of the ZO-1 or GRINL1A sequence, are shown forming an extra β strand (silver). D.Structure-based sequence alignment of four known ZU5 domains: ZU5A and ZU5B of Ankyrin-R, ZU5 of UNC5b, and ZU5/GRINL1A of ZO-1. The sequence alignment is strictly based on structural similarities. Some regions of the proteins are structurally dissimilar (shown in black boxes) precluding sequence alignment. Highly-conserved residues across the ZU5 domains are shaded in red. The β-strands observed in the ZU5B domain of ankyrin-R are indicated above the sequences. The alignment of the GRINL1A peptide is also shown.

A. Superposed structures of the ZU5A domain in ZU5A-ZU5B on the ZU5A in complex with spectrin. Ribbon diagram of the ZU5A-ZU5B (turquoise, green) structure superposed on the ZU5A domain (red) of the complex with β-spectrin (blue)²⁴. The diagram shows that ZU5B is unlikely to interact with spectrin as it is found away from the spectrin/ankyrin interface. This finding is supported by biochemical experiments that show that the ZU5B domain does not alter the binding characteristics significantly. B. Superposed structures of the ZU5A-ZU5B fragment of ankyrin-R on the UNC5b supramodule. Ribbon diagram of the ZU5A-ZU5B fragment (turquoise, green) superposed on the UNC5b supramodule comprising the ZU5, UPA and DD domains²⁵. The ZU5 domain of UNC5b (pink) was superposed on the ankyrin-R ZU5B domain. The superposition shows that the ankyrin DD domain could interact in a similar manner without any major conformational changes. The UPA domain could also interact with ZU5B in a similar manner, although there are some potential clashes in the superposed structures. Some of these potential clashes may not exist as they involve a region in the UNC5b UPA domain that is not present in ankyrin. Finally, the ankyrin ZU5A domain clashes with the UNC5b UPA domain, suggesting that a small reorientation of the ZU5A domain may be needed for proper interaction. Overall, the superposition shows that a similar supramodule could be formed in ankyrin and that this supramodule is not likely to interact directly with spectrin. C. The ZU5 domain has several distinct binding surfaces. The ZU5 domain can use different regions to interact with different proteins. The known areas of interaction are marked, showing the different, non-overlapping areas of the ZU5 domain involved in different complexes. Furthermore, the incorporation of additional β strands can also serve as a mechanism for interaction, as observed in the interaction with the DD domain in UNC5b²⁵ and the GRINL1A peptide in ZO-1³².