FIG 6.

Synergistic effects of the NagB protein-protein interactions (PPIs) is possible as a result of simultaneous binding of protein partners. (A and B) Structural models of simultaneous binding are shown for NagB plus HPr plus NanE (A), as well as NagB plus HPr plus U-PII (B). The binding of HPr to NagB does not sterically obstruct the binding of either NanE (A) or U-PII (B) in the model, but the binding of NanE to NagB does sterically obstruct the binding of U-PII (and vice versa). Although the figure shows that NanE and HPr are together before binding to NagB in panel A and U-PII and HPr are together before binding to NagB in panel B, there is no evidence that this actually occurs. (C) Binding sites for HPr (green), NanE (blue), and U-PII (orange) on the surface of NagB. Note that the majority of the NanE and U-PII interfaces on NagB overlap each other (shown in gray). Residues indicated by black lines are shared among all three NagB partners. (D) Functional NagB is a hexamer in which the cavity interacts with solvent (red); the intersubunit trimer contacts, a loop spanning residues 216 to 223, are indicated by heavy dashed lines, while the intertrimeric contacts (residues 244 to 250) are indicated by light dashed lines. The orientation of the NagB monomer with respect to the trimer and hexamer is indicated. (E) Overlay of the HPr and NanE with a hexamer of NagB. HPr (green) and NanE (blue) attach at trimer and subunit interfaces, respectively, with very little steric hindrance, at highly conserved residues (red) on both themselves and NagB. Green and blue arrows indicate how HPr and NanE, respectively, rotate from position shown to position on NagB hexamer (45° clockwise from top). (F) Overlay of U-PII with a hexamer of NagB (two NagB subunits removed for clarity). U-PII binds within the hexameric cage of NagB, attaching using highly conserved residues. A loop of U-PII (top right) sterically hinders one of the six NagB subunits (not shown). The orange arrow indicates how U-PII rotates from position shown to position in NagB hexamer (45° counterclockwise from top). PDB identification codes and chains used for NagB, HPr, and U-PII were 1FS5 chain A (open R conformer), 3CCD chain A, and 5L9N chain A (uridylylated PII), respectively. For NanE, the full-length Swiss Model Repository model based on the template 3IGS chain A (79.7% sequence identity) was used. For the NagB hexamer, PDB 1CD5, chains A to F, was used and six 1FS5 A chains were aligned onto the hexamer, proving a close approximation of the open, R conformer NagB hexamer.