Rearrangements of α‐helical structures of FlgN chaperone control the binding affinity for its cognate substrates during flagellar type III export

Abstract

The bacterial flagellar type III export chaperones not only act as bodyguards to protect their cognate substrates from aggregation and proteolysis in the cytoplasm but also ensure the order of export through their interactions with an export gate protein FlhA. FlgN chaperone binds to FlgK and FlgL with nanomolar affinity and transfers them to FlhA for their efficient and rapid transport for the formation of the hook-filament junction zone. However, it remains unknown how FlgN releases FlgK and FlgL at the FlhA export gate platform in a timely manner. Here, we have solved the crystal structure of Salmonella FlgN at 2.3 Å resolution and carried out structure-based functional analyses. FlgN consists of three α helices, α1, α2 and α3. Helix α1 adopts two distinct, extended and bent conformations through the conformational change of N-loop between α1 and α2. The N-loop deletion not only increases the probability of FlgN dimer formation but also abolish the interaction between FlgN and FlgK. Highly conserved Asn-92, Asn-95 and Ile-103 residues in helix α3 are involved in the strong interaction with FlgK. We propose that the N-loop coordinates helical rearrangements of FlgN with the association and dissociation of its cognate substrates during their export.

Structural characterization of FlgE2 protein from Helicobacter pylori hook

“…In addition, the interaction of HpFlgE2 with the cap protein HpFlgD with a dissociation constant comparable to the value observed for the interaction of FlgN and FlgK in S. typhimurium (K D = 76 nM) [30,31] suggests that the two proteins can co-participate to the assembly of the flagellar hook. Moreover, the role of HpFlgE2 in the hook assembly can be proposed since the fold of the D3 in HpFlgE2 is similar enough to the fold of the D3 in FlgE/FlgE1 from other species.…”

Hierarchical protein targeting and secretion is controlled by an affinity switch in the type III secretion system of enteropathogenic Escherichia coli

“…Such interaction would also rationalize the low‐affinity binding component of CesAB/EspA for EscV (i.e., Δ sepL , Fig J). In the flagellum system, the C‐terminal region of substrate loaded‐chaperones targets the chaperone/secretory protein complex to the C‐domain of the EscV homologue, FlhA with higher affinity than the chaperone alone (Bange et al , ; Minamino et al , ; Kinoshita et al , , ; Furukawa et al , ). Defining the structural basis of such interactions with EscV is a major challenge due to its multimeric and membrane‐embedded nature.…”

“…This effect could be mediated Fig 3J). In the flagellum system, the C-terminal region of substrate loaded-chaperones targets the chaperone/secretory protein complex to the C-domain of the EscV homologue, FlhA with higher affinity than the chaperone alone (Bange et al, 2010;Minamino et al, 2012;Kinoshita et al, 2013Kinoshita et al, , 2016Furukawa et al, 2016).…”

The role of intrinsically disordered C‐terminal region of FliK in substrate specificity switching of the bacterial flagellar type III export apparatus

“…It has been reported that deletion of domain D4 of FlhA C allows filament-type substrates to be exported into the periplasm prior to hook completion . The FlgN/FlgK, FlgN/ FlgL, FliT/FliD and FliS/FliC chaperone/filament-type substrate complexes all bind to well-conserved Asp-456, Phe-459 and Thr-490 residues of FlhA C , promoting unfolding and translocation of the filament-type substrates into the lumen of the growing structure Kinoshita et al, 2013Kinoshita et al, , 2016Furukawa et al, 2016). This raises the possibility that domain D4 of FlhA C suppresses the interactions of these conserved residues with the flagellar chaperone/ filament-type substrate complexes during hook assembly.…”