Abstract
Per-ARNT-Sim (PAS) domains constitute a typically dimeric, conserved α/β tertiary fold of approximately 110 amino acids that perform signalling roles in diverse proteins from all kingdoms of life. The amino terminal PAS1 domain of NifL from Azotobacter vinelandii accommodates a redox-active FAD group; elevation of cytosolic oxygen concentrations result in FAD oxidation and a concomitant conformational re-arrangement that is relayed via a short downstream linker to a second PAS domain, PAS2. At PAS2, the signal is amplified and passed on to effector domains generating the ‘on’ (inhibitory) state of the protein. Although the crystal structure of oxidised PAS1 reveals regions that contribute to the dimerisation interface, 21 amino acids at the extreme N-terminus of NifL, are unresolved. Furthermore, the structure and function of the linker between the two PAS domains has not been determined. In this study we have investigated the importance to signalling of residues extending beyond the core PAS fold. Our results implicate the N-terminus of PAS1 and the helical linker connecting the two PAS domains in redox signal transduction and demonstrate a role for these flanking regions in controlling the oligomerisation state of PAS1 in solution.
Highlights
PAS (Per-ARNT-Sim) domains represent highly conserved a/b folds of approximately 110 amino acids that perform diverse signalling functions in proteins from all kingdoms of life [1]
The crystal structure of the oxidised PAS1 domain of NifL from A. vinelandii is derived from an expression construct that includes residues 1–140
Removal of residues 2–14 generates a ‘‘locked on’’ phenotype, possibly as a consequence of rearrangement of a putative a-helix that extends from position 15–19. Truncations extending through this helix gave rise to defects in redox signalling, perhaps reflecting disruption to the A9a-helix of the PAS1 domain, which we have shown previously to be important for redox signal transduction [10]
Summary
PAS (Per-ARNT-Sim) domains represent highly conserved a/b folds of approximately 110 amino acids that perform diverse signalling functions in proteins from all kingdoms of life [1]. Ligand binding PAS domains typically accommodate their co-factor within a spatially conserved cleft formed by the inner surface of the b-sheet and two a-helices termed Ea and Fa. In addition to the core fold, PAS domains commonly possess flanking regions at their N- and C-termini. Sequences that flank PAS domains are predicted to adopt an a-helical structure and where structures have been determined, the majority adopt an a-helical conformation [2]. Overall, these structures reveal that flanking regions either extend outwards from the PAS domain or pack against the conserved b-sheet of the core structure. The native arrangement of these elements may be dependent upon other regions of the protein that may or may not be present in a particular structure
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