www.nature.com/scientificreports OPEN FliS/fagellin/FliW heterotrimer couples type III secretion and fagellin homeostasis Received: 3 May 2018 Florian Altegoer 1, Sampriti Mukherjee2, Wieland Steinchen1, Patricia Bedrunka1, Accepted: 20 July 2018 Uwe Linne1, Daniel B. Kearns2 & Gert Bange1 Published: xx xx xxxx Flagellin is amongst the most abundant proteins in fagellated bacterial species and constitutes the major building block of the fagellar flament. The proteins FliW and FliS serve in the post-transcriptional control of fagellin and guide the protein to the fagellar type III secretion system (fT3SS), respectively. Here, we present the high-resolution structure of FliS/fagellin heterodimer and show that FliS and FliW bind to opposing interfaces located at the N- and C-termini of fagellin. The FliS/fagellin/FliW heterotrimer is able to interact with FlhA-C suggesting that FliW and FliS are released during fagellin export. After release, FliW and FliS are recycled to execute a new round of post-transcriptional regulation and targeting. Taken together, our study provides a mechanism explaining how FliW and FliS synchronize the production of fagellin with the capacity of the fT3SS to secrete fagellin. Te bacterial fagellum is among the most-studied bacterial nanomachines and represents one of the most power- ful motors in the biosphere. Flagellar architecture is highly conserved among bacterial species and can be divided into: a membrane-embedded basal body including a fagella-specifc type III secretion system (fT3SS), the cyto- plasmic C-ring and the rod, extracellular hook and flament structures. Flagella biogenesis follows a highly conserved sequence of assembly steps that is tightly regulated in time and space (reviewed in1–4). Dozens of assembly factors and chaperones orchestrate fagella assembly at the transcrip- tional, post-transcriptional, translational or post-translational level5–8. Te most abundant fagellar building block is the fagellin protein that assembles in a helical pattern of more than 20,000 copies to form the extracellular fl- ament9. Successful assembly of the fagellar flament relies on the FliS protein that prevents cytoplasmic aggrega- tion of fagellin10 and guides fagellin to the cytoplasmic side of the fT3SS. At the fT3SS, the fagellin/FliS complex interacts with the cytoplasmic domain of the fT3SS transmembrane protein FlhA (FlhA-C)11–13. Terefore, FliS acts as targeting factor directing its fagellin client to the export gate of the fT3SS for subsequent secretion. In a wide range of bacterial species, production of fagellin is post-transcriptionally regulated by the proteins CsrA and FliW14–18. In Bacillus subtilis, CsrA inhibits translation of fagellin by binding to two sites that are present within the 5′untranslated region (UTR) of the hag mRNA14,19. FliW allosterically antagonizes CsrA in a noncompetitive manner by excluding the 5′ UTR from the CsrA–RNA binding site, allowing the translation of fagellin17,18. FliW, which can also bind to fagellin, is sequestered as cytoplasmic levels of fagellin rise, allowing CsrA again to inhibit translation of the hag mRNA. Tis cycle is thought to enable homeostasis of cytoplasmic fagellin concentrations over a low and narrow threshold20. While our knowledge on the functional roles of FliS, FliW and CsrA is steadily increasing, structural infor- mation is lagging behind. We present the high-resolution structure of FliS bound to full-length fagellin showing the FliS/Flagellin interaction is more complex than indicated by previous partial structures21,22. Moreover, we show that FliW and FliS bind to opposing interfaces located at the N- and C-termini of fagellin, respectively. Te heterotrimeric complex of fagellin, FliS and FliW is competent to interact with FlhA-C, although FliW is not required for efcient interaction. Tis fnding suggests to us that FliW remains sequestered until fagellin export by the fT3SS. Afer release, free FliW would bind and antagonize CsrA to enable further production of fagellin. Taken together, our data suggest a mechanism explaining how FliW and FliS might synchronize the production of fagellin with fT3SS secretion. 1LOEWE Center for Synthetic Microbiology & Dep. of Chemistry, Philipps University Marburg, Hans-Meerwein- Strasse 6, 35043, Marburg, Germany. 2Department of Biology, Indiana University, 1001 East 3rd Street, Bloomington, IN, 47405, USA. Correspondence and requests for materials should be addressed to G.B. (email: gert.bange@ synmikro.uni-marburg.de) SCIENTIFIC REPORTS | (2018) 8:11552 | DOI:10.1038/s41598-018-29884-8 1 www.nature.com/scientificreports/ Figure 1. Crystal structure of the fagellin/FliS heterodimer. (a) Domain organization of fagellin. (b) Crystal structure of fagellin/FliS as a cartoon representation in two orientations. fagellin and FliS are shown in orange and blue, respectively. ‘N’ and ‘C’ indicate N- and C-termini, respectively. Te disordered N-terminus of fagellin is shown as dashed line. Contact areas between fagellin and FliS are encircled in grey. (c) Electrostatic surface potential of FliS within the contact area of fagellin to FliS. Te fagellin-binding pocket is of hydrophobic and polar nature (lef side), whereas the opposite site of FliS is highly charged (right side). Results Crystal structure of FliS in complex with full-length fagellin. We frst wanted to determine a struc- ture elucidating the complex of the FliS chaperone in complex with its client fagellin. Tus far, the only structural information that was available was of FliS bound to a C-terminal fragment of fagellin (residues 478–518; pdb: 1ORY, 21, 4IWB,22). To obtain the complete structure, the fagellin/FliS complex from B. subtilis was co-expressed in E. coli BL21(DE3) and co-purifed by nickel-ion afnity chromatography (IMAC) followed by size exclusion chromatography (SEC). Multi-angle light scattering analysis indicated that the B. subtilis fagellin/FliS complex forms a heterodimer (Fig. S1a). Crystals of the fagellin/FliS complex were obtained in two spacegroups, the primitive monoclinic space group P21 at 1.5 Å resolution and the primitive orthorhombic spacegroup P22121 at 2.1 Å resolution. Te fagellin/FliS heterodimer shows overall dimensions of 95 Å, 40 Å and 40 Å (Fig. 1b, Table S1) and could be built to complete- ness except for the frst 45 highly disordered residues at the N-terminus of fagellin (Fig. 1a,b, dashed line). SCIENTIFIC REPORTS | (2018) 8:11552 | DOI:10.1038/s41598-018-29884-8 2 www.nature.com/scientificreports/ Te structure of B. subtilis fagellin is similar to the fagellin of other bacterial species23 and can be divided into D0-N, D1 and D0-C domains (Fig. 1a, S1b). Te crystal structure suggests that the fagellin/FliS complex forms a rather compact particle detectable also in solution by small angle X-ray scattering (SAXS). Te two proteins interact by an extensive network of hydrophobic and electrostatic interactions with an interface area of 2600 Å2 which can be divided into contact areas 1, 2 and 3. Contact area 1 involves the C-terminus of fagellin (D0-C) wrapped around the surface of FliS in an extended horseshoe like conformation with a mainly hydrophobic interaction area of 1600 Å (Contact area 1; Fig. 1b,c). Contact area 1 is highly similar to that in an earlier structure of FliS bound to the C-terminus of fagellin21 with a Cα r.m.s.d of 2.2 Å over 140 amino acid residues (Fig. S1c). Contact area 2 aligns FliS to the D1 core domain of fagellin, a region that was not present in previous crystal structures24–26. Interactions within this interface are of more electrostatic nature (Contact area 2, Fig. 1b,c). Notably, residues 46 to 57 are kinked by 45° in the orthor- hombic structure, while the helix is straight in the crystal structure derived from monoclinic crystals (Fig. S2). Apart from this diference, both structure superimpose well with a r.m.s.d of 1.5 over 329 Cα-atoms. Taken together, our crystal structure shows that the interaction between fagellin and FliS involves a complex interface at fagellin including the D1 and D0C domains. Flagellin/FliS and FlhA-C interact via a complex interface. With the structure of full-length fagellin bound to FliS in hand, we wanted to understand how fagellin/FliS interacts with FlhA-C. Hence, we performed hydrogen-deuterium exchange (HDX) mass spectrometry (HDX-MS). Tis method allows monitoring con- formational changes within proteins and the determination of interaction sites27. FlhA-C, fagellin/FliS and the FlhA-C/fagellin/FliS complex were incubated in deuterated bufer for diferent times, digested with pepsin and the resulting peptic fragments analyzed by electrospray ionization-mass-spectrometry (Fig. S3c,d). When bound to FlhA-C, a signifcant decrease in HDX of fagellin/FliS was detected in three regions of fagellin at D1-N, D1-C and D0-C (termed: F1-F3). Te region within D1-N includes residues 61–72 (Fig. 2a,F1), but slight protection is also visible from residue 123–142. Te most prominent HDX protection can be observed in the D1/D0-C of fagellin ranging from residues 236–249 and 275–300 (Figs 2a,F2,F3, S3c). While the frst patch is within the D1-C domain of fagellin, residues of the latter are arranged in two short α-helices connected by a loop and wrap around FliS (Fig. 2a). We now inspected regions at FlhA-C that become protected from HDX upon binding of fagellin/FliS. In total, the binding interface covers four regions (termed: A1-A4). Firstly, a small patch ranging from residues 371–390 within the D1a domain shows protection (Figs 2b,A1, S3d). Further residues within the D1b domain (416–428 and 460–471) exchange less in the presence of fagellin/FliS (Figs 2b,A2, S3d). Lastly, residues 562–571 (A3) and 579–596 (A4) show a dramatic decrease in deuteron incorporation, indicating stabilization or shielding (Figs 2b, S3d).