Ultraweak interactions drive cap-mediated positioning and elongation of the bacterial flagellar filament

Domain: biology
Published: 2025-12-26
Editorial outcome: minor_revision

Abstract
The bacterial flagellum, essential for motility and pathogenesis, requires the filament cap (FliD) to polymerize flagellin (FliC). However, the mechanisms governing the transition from the hook junction to the filament elongation remain elusive, obscured by stoichiometric mismatches and barely detectable interactions. To resolve this, we deploy solution NMR to characterize ultra-weak interactions, quantify affinities (Kd ≈ 0.1 mM for junction protein FlgL; 1.65 mM for FliC). These data enable rational complex stabilization for cryo-EM structure determination of Salmonella FliD pentamers complexed with FlgL or FliC, revealing that both substrates engage an identical conserved surface in a 5:5 stoichiometry. Integrating these structures into native flagellar tip densities reveal a 5:11 FliD:FlgL/FliC architecture, where six additional subunits barely detected by NMR dock at secondary sites. Mutations that disrupt or enhance these interfaces impair motility and filament integrity, while disulfide-locked FliD pentamers confirm that cap rigidity is crucial for elongation. These findings support a rotary cap mechanism where ultra-weak binding and structural fidelity of the cap ensure efficient flagellin polymerization. Our study resolves the long-standing paradox of stoichiometric mismatch in flagellar filament biogenesis, providing a blueprint for the assembly of dynamic macromolecular machines.

Introduction
The bacterial flagellum is a highly regulated macromolecular rotary nanomachine. It functions as a motor equipped with a propeller, enabling bacterial mobility and playing a critical role in bacterial survival, reproduction, colonization, and pathogenicity. This complex apparatus is comprised of more than 25 conserved proteins exported through a dedicated type III secretion system (T3SS), organized into three main regions: the basal body, the proximal hook, and the distal helical filament. The basal body acts as a motor and protein export apparatus, driven by proton-motive force. The hook, a universal joint in a curved structure, connects the basal body to the filament and transmits the torque from the stator to the filament tail. Assembly of the ∼55 nm long hook needs ~120 copies of the hook protein FlgE. The long helical filament, composed mainly of polymerized flagellin (FliC), functions as a propeller to generate thrust and adapts to different environments through various super-helical geometries.

Assembly of the flagellum proceeds through a highly ordered process, beginning with the basal rod, followed by the hook, hook-filament junction, filament cap, and ultimately the filament. The filament elongates starting from the interface between the hook-filament junction and the filament cap. The hook-filament junction consists of two layers of morning glory-like structures, built by the junction protein FlgK and junction protein FlgL. In the junction, a ring-shaped FlgK oligomer is in contact with the hook and the FlgL ring. After the assembly of the hook junction, the filament cap protein FliD is secreted to the distal end of the hook junction. The secreted FliD docks at the FlgL ring at the tip of the hook and forms a cap-shaped oligomer that nucleates filament elongation. Despite these evidence implicating interactions between FliD and other flagellar components, direct biochemical characterization of these interactions in vitro remains scarce.

Beyond interaction features, a comprehensive understanding of filament assembly demands structural elucidation of flagellar components at the growing distal end. Advances in cryo-electron microscopy (cryo-EM) and X-ray crystallography have resolved high-resolution structures of individual components. FliD, the cap protein, forms a homo-tetramer to homo-hexamer stool-shaped complex; FlgL, the hook-filament junction component directly involved in filament assembly, remains mysterious except for its truncated and monomeric structures till very recent; FliC, the primary flagellin protein, polymerizes into a helical filament and its structure has been determined at near-atomic resolution, which contributes to the filament rigidity and motility. Pioneering work during the last three decades, based on elegant electron microscopy, proposed the influential hypothesis of a rotating FliD cap acting as a dynamic polymerization chaperone and rotating incrementally to sequentially position incoming flagellin subunits for processive filament elongation. This model provided a crucial conceptual framework, yet the underlying molecular mechanics—how the pentameric cap dynamically interfaces with the flagellum’s prevailing 11-fold symmetry—remained a structural and biophysical paradox. While a recent near-atomic structure provided key insights into the FliD cap atop the FlgL ring, the accommodation, the atomic details of subunit binding, and the functional necessity of precisely tuned interaction affinities have remained elusive. This lack of clarity has hindered a mechanistic understanding of how the flagellar cap discriminates the hook-filament junction from flagellin to mediate rapid filament assembly.

Here, we address these challenges through an integrated approach combining solution-state nuclear magnetic resonance (NMR) spectroscopy to characterize ultraweak FliD-substrate interactions, with cryo-EM structural analysis of transient complexes and native flagellar assemblies. Our structural and biochemical results reveal how finely tuned binding affinities and cap architecture coordinate filament assembly via a rotary mechanism, resolving the paradox of symmetry mismatch and ultraweak interactions in the assembly of this essential macromolecular machine.

Results
Interaction of FliD with FlgL and FliC
The flagellar filament cap protein FliD of Salmonella enterica serovar Typhimurium is composed of 467 residues and harbors four major domains: D0, D1, D2, and D3. The D0 domain features an N-terminal signal peptide region (SPR, residue 1–44) and a C-terminal chaperone binding region (CBR, residues 411–467), which are crucial for protein secretion through the T3SS component. The D1 domain (residues 44–72, 268–411) facilitates flagellin targeting and filament extension, while the D2 (residues 72–103, 223–268) and D3 (residues 103–223) (Polymerization domain, PD) domains intrinsically oligomerize to form the cap structure of the flagellar filament. Our initial trial shows that spontaneous oligomerization of the full-length FliD would form high levels of aggregation rather than a well-defined cap-like oligomer. Subsequently, we designed and screened FliD truncations to form a homogeneous oligomer. The FliD D0 domain truncation successfully yielded a homopentamer, and its cryo-EM structure was solved at 3.06 Å resolution. This structure showed significant orientational differences compared to previously reported X-ray structures of the FliD D2-D3 domains. The structure shows that the FliD pentamer is stabilized by the intermolecular interactions between D2 and D3 domains, including several hydrophobic interactions involving residues I234 and I236 from D2 domain, V172, and V174 from D3 domain, as well as a salt bridge between E237 in D2 domain and K175 in D3 domain. Especially, the high conservation of these hydrophobic residues across species suggests their functional importance. Substitution of individual residues at the interface destabilized the pentamer, as measured by a shift in the monomer-pentamer equilibrium using size-exclusion chromatography, highlighting the critical role of these interactions. The FliD pentamer maintains a stable core, evidenced by 7100 Å² of buried hydrophobic interface. This buried surface area (~7100 Å²) falls within the range of stable oligomeric interfaces—comparable to molecular chaperone SecB (Homo-tetramer, 8373 Å²), yet significantly larger than transient signaling complexes like EIIA-EIIB (702 Å²) in phosphotransferase system, underscoring the inherent stability of the FliD pentameric core. However, its solvent-exposed surface lacks large hydrophobic or charged patches, indicating an absence of strong, specific binding sites for other flagellar subunits. This non-specific binding landscape suggests FliD engages FlgL and FliC through weak, transient interactions—a design essential for its role as a rigid platform that enables rapid subunit exchange during processive filament elongation.

To gain atomic interaction of FliD with other filament-associated proteins, we used the solution NMR spectroscopy. Given the large size of FliD, this protein was segmented into three constructs based on its domain boundaries using the “divide and conquer” strategy. The interaction between each segmented domain and FliC/FlgL was analyzed using NMR titration experiments. NMR analysis revealed that FliC and FlgL bind specifically to the D1 domain of FliD. Significant line broadening observed during titration indicates chemical exchange on the microsecond-to-millisecond time-scale, which is characteristic of weak, transient protein–protein interactions. While only minimal chemical shift perturbations were observed for the D2-D3 domains, indicating substantially even weaker or nonspecific binding compared to the interactions at the D1 domain.

The 1H-15N correlated spectra of FliDd1 showed well-dispersed peaks, enabling near-complete resonance assignment. Upon the addition of FlgL and FliC to labeled FliD-D1, significant line broadening was observed in the 1H-15N correlated spectra, indicating interactions between these proteins and the D1 domain. Further NMR analysis revealed that FlgL and FliC share an overlapping binding site on the D1 domain, located on the outer surface of the leg region within the stool-like cap structure. The location of this binding site is consistent with its position during flagellar filament assembly and is situated in the most conserved region of FliD, underscoring the functional importance of this interface.

Structure of the flagellar filament cap FliD in complex with FlgL
FlgL, the hook-filament junction protein, represents the smallest flagellar propeller component. Unlike multidomain FliD, FlgL comprises only D0 and D1 domains. Since it was found that FliD-D1 could interact with FlgL, the affinity between these two proteins was quantified using NMR titration experiments. The dissociation constant (Kd) between FliD-D1 and FlgL was determined to be ~100 μM, consistent with the established classification of an ultra-weak interaction (Kd in the high micromolar to millimolar range), which is distinct from conventional “weak” binding events. This ultra-weak binding precluded stable complex formation for structural studies. Initial attempts to stabilize the native complex using GraFix (gradient fixation) were unsuccessful due to complex dissociation, prompting the use of interface-guided fusion constructs to elevate the complex ratio by mimicking proteins in infinite high concentrations. We designed several fusion constructs linking FlgL to the N- or C-terminus of FliD with flexible linkers. The construct yielding a homogeneous, monodisperse complex suitable for high-resolution cryo-EM analysis was the one with the FlgL D1 (resi 40–280) domain fused to the N-terminus of full-length FliD. This fusion construct allowed for successful structure determination. The cryo-EM structure of the FliD-FlgL complex was resolved at a resolution of 3.30 Å.

The overall structure of the FliD-FlgL complex reveals a 5:5 stoichiometry, where each FlgL monomer binds independently to a single FliD subunit. The single FliD-FlgL interface buries a total surface area of 955 Å², with two major helices from FlgL interacting with a flat, elongated region on the D1 domain of FliD. This binding interface is in agreement with our NMR titration data and provides structural validation for earlier hypothetical models that were based on low-resolution electron microscopy images. Structural analysis of the interface reveals a highly hydrophilic and complementary surface, suggesting that polar interactions and van der Waals forces collectively contribute to stabilizing the complex.

Three major interaction motifs were identified at the interface. First, a salt bridge forms between FlgL E92 carboxyl group and FliD R326 side chain, complemented by a hydrogen bond with FliD T340. Second, a series of hydrophobic contacts occur between FliD Y296, A298, and L313, and FlgL L254 and L75. Third, another salt bridge forms between FliD R29 and FlgL D266, along with additional hydrophobic interactions between FliD P32 and FlgL L269 and L273. Notably, the structural data confirmed that there is no direct interaction between FlgL monomers. Mutagenesis studies, in which key residues involved in these interactions were substituted, resulted in decreased stability of the FliD-FlgL complex, further confirming the importance of these specific interactions in this transient complex formation. To further validate our structure, we engineered disulfide bonds between four residue pairs at the interface (Cα distance < 5 Å). Oxidation-induced crosslinking of these cysteine mutants confirmed the accuracy of our structure.

Structure of the flagellar filament cap FliD in complex with FliC
The flagellin FliC exhibits a domain architecture highly similar to that of full-length FliD. Although there is extensive evidence for the important role of FliD in flagellin assembly, there is no publication reporting direct interaction between FliD and other filament components in vitro. This suggests that these FliD-containing complexes are highly transient and dynamic. Benefiting from the solution NMR in characterizing ultra-weak and transient interactions, we quantified the dissociation constant (Kd) of the FliD-FliC interaction at 1.65 mM with stoichiometry 1:1, confirming the ultra-weak binding affinity. To stabilize this ultra-transient complex for structure analysis, we employed the fusion approach as FlgL-FliD complex stabilization. Since full-length FliC has a strong filamentation propensity, we first identified that D0 domain truncation yielded monodisperse protein. After screening multiple fusion constructs, the optimal construct for structural studies is covalently linked the N-terminus of FliC D1D2D3 (residues 45–455) to the C-terminus of full-length FliD. Using this construct, we identified two distinct complexes and determined their cryo-EM structures - one with 5:5 stoichiometry at 3.30 Å resolution and another with 10:10 stoichiometry at 3.27 Å resolution.

The structures reveal that each FliC subunit binds independently to one FliD subunit, with FliD oligomerizing in a C5 or D5 symmetry. In the C5 symmetry structure, the oligomerization interface mirrors that of the FliD pentamer. However, in the D5 symmetry conformation, an additional interface was observed where the C-terminal of FliD forms an antiparallel helix bundle. This inter-pentamer interaction is mediated by hydrophobic contacts involving residues Y436, F440, L446, M446, and L450. Mutating these hydrophobic residues into polar amino acids restores aggregation in full-length FliD. Although this interface is visualized and confirmed in our experiments, it is likely non-physiological, as previously suggested. Instead, we propose that these hydrophobic residues contribute to chaperone-mediated substrate anti-aggregation and in recognizing newly translocated FliC subunits.

All intermolecular contacts within the FliD-FliC complex occur either between FliD-FliD, or FliD-FliC, with no direct interaction between FliC subunits. The FliD-FliC interface, although burying a surface area of 1296 Å² and showing shape complementarity, features only two key interactions that contribute to the ultra-weak binding affinity. One is a salt bridge between FliD K361 and FliD E94, while the other involves FliD I72 inserting into a hydrophobic cleft in FliD formed by residues Y296, A298, and L313. Unlike the FliD-FlgL complex, no significant third interface was observed. Notably, the overall structure of the FliD pentamer remains unchanged upon FliC binding, with a root-mean-square deviation of 1.109 Å for the backbone atoms between the apo and FliC-bound states. Substitutions of amino acids at the FliD-FliC interface, particularly those disrupting key interactions, markedly reduced the binding affinity between FliD and FliC. Disulfide bond crosslinking further validates the transient structure of the complex in solution.

Structural models of the hook-filament junction tip and filament tip
In the native flagellum, the filament and hook are formed by repeating arrays of 11 subunits of FliC or FlgE per turn. Though without direct and precise evidence, the hook-filament junction is also expected to contain 11 copies of FlgK as well as FlgL. This indicates the stoichiometry of the FliD-FliC or FliD-FlgL complex should be 5:11 rather than the 5:5 stoichiometry we reported here. To reconcile this apparent stoichiometric discrepancy, we sought to determine the structure of the native hook-filament junction tip and the filament tip directly from bacteria flagella.

To obtain the native hook-filament junction tip, we knocked out the fliC gene to arrest flagellar assembly before filament elongation, preserving the flagellum in the pre-elongation state at the hook-filament junction. The native flagellar junction tip, as well as the filament tip as control, were mechanically shaved from the modified Salmonella strain and assessed by negative staining electron microscopy. The resulting cryo-EM density map revealed a clear structural arrangement, showing a FliD pentamer embedded within an 11-fold symmetric FlgL ring. Despite the moderate resolution of ~6.2 Å, our determined FliD-FlgL structure at 5:5 stoichiometry, fits confidently within the density map. Additionally, six extra FlgL subunits could be docked onto the complex. The structure model of the hook-filament junction tip is depicted. 11 FlgL subunits are arranged in a pattern of five up-and-down pairs, with one extra subunit positioned at a downer step. The extra FlgL does not interact with or occupied by FliD, indicating the presence of an accessible site primed for the incorporation of the first flagellin subunit.

Using a similar approach, we obtained a cryo-EM density map of the native filament tip. This map, resolved at 6.97 Å, revealed a comparable architecture for the FliD-FliC assembly at a 5:11 stoichiometry, akin to the FliD-FlgL complex. Identically, five additional FliC subunits bind to the FliD-FliC 5:5 complex at a secondary binding site, adopting a parallel down conformation. One additional FliC subunit is positioned in an even lower conformation adjacent to one of the five FliC subunits. This concave subunit exhibits no direct interaction with neighboring FliD molecules, leaving its binding surface exposed. This distinct configuration suggests that this subunit may serve as the site for newly translocated FliC during another round of flagellar elongation.

The in vitro complexes we characterized were observed at a 5:5 stoichiometry, rather than the 5:11 stoichiometry seen in native structures. We propose that the six additional subunits are stabilized through a combination of lateral interactions with adjacent FlgL/FliC subunits and weaker, dynamic contacts with secondary sites on FliD, collectively enabling the stable 5:11 architecture observed in native flagella. Despite attempts to stabilize this minor conformation through the design and screening of FliD-FlgL as well as FliD-FliC fusion constructs, we all failed likely due to the extremely transient nature of this interaction.

The transient complexes formation is required for flagellar assembly and bacteria motility
Structural dissection and comparison of the resolved complexes reveal key features of the dynamic, weak-affinity interactions governing flagellar filament components positioning by the filament cap FliD. FlgL and FliC occupy a shared binding site on FliD, revealing a conserved mechanism for substrate recognition by the filament cap. Although the residues involved in FliD binding differs significantly between FlgL and FliC, one key interaction that dominantly contributes to the complex stability shows the same form and physicochemical property. Specifically, the hydrophobic side chains of FlgL L254 and FliC I72 interact similarly with hydrophobic groove forms by FliD Y296, A298, and L313. The hydrophobic property of these residues is highly conserved in FliD, FlgL, and FliC. Noteworthy, polar and neutral residues distributed across the interface of both binary complexes form extended, complementary flat-strip interfaces that stabilize these ultra-weak interactions via van der Waals forces. In the native configurations, a second docking site was observed. Distinguished from the major docking site, no specific electrostatic or hydrophobic interactions were confirmed in the structural models, suggesting the significant contribution of shape complementarity and van der Waals interactions in mediating this interaction.

To evaluate the functional significance of the observed interactions, we introduced key mutations that disrupt complex formation, as identified by our binding experiments, into corresponding Salmonella strains. In addition, residues located at the interface and conserved in bacteria species were also mutated. The impact of these mutations on motility was quantified by measuring the colony diameter in motility assays. Mutations targeting key interaction residues, such as FliD Y296G, FliD L313G, FlgL L254G, and FliC I72G, exhibited statistically significant reductions in motility. Additionally, we assessed mutations at the second interface, where residues are relatively conserved. Mutations of these residues also led to a reduction in motility.

Conventional motility assays provide only a rough estimation of flagellar assembly. To directly and precisely visualize the effects of disrupted interaction on flagellum elongation, transmission electron microscopy with negative staining was used to image the flagella of wild-type and mutant Salmonella strains. Quantitative analysis revealed a significant reduction of fully assembled flagella in the mutant strains, highlighting the critical role of proper positioning of flagellar components by the flagellar cap in assembly and motility.

Structural plasticity governs FliD-mediated filament assembly
Our structural and biochemical analyzes establish that ultraweak interactions are essential for filament elongation and bacterial motility. To test whether modulating binding affinity affects assembly, we engineered structure-guided mutations intended to strengthen the FliD-FlgL and FliD-FliC interfaces. Quantitative pull-down assays confirmed up to a 3.5-fold enhancement in binding affinity for the most effective FliD-FlgL interface mutant. Beyond mutations designed to enhance hydrophobic or electrostatic interactions (e.g., V299I, D349R), we also identified key residues where substituting long side chains with glycine enhanced binding. This suggests these wild-type residues serve as stereospecific blockades that help maintain the weak interaction. Applying the same strategy to the FliD-FliC interface yielded affinity-enhancing mutants. Notably, all such mutants—whether targeting FliD-FlgL or FliD-FliC interactions—impaired bacterial motility to varying degrees, demonstrating that finely tuned, low-affinity binding is critical for efficient filament assembly and function.

Given the dynamic nature of the FliD-FliC/FlgL complex during assembly, disulfide bond crosslinking was used to examine the rigidity of the FliD pentamer cap in filament extension. Structural analysis revealed A91 and A168 at adjacent subunit interfaces (Cβ-Cβ distance: 4.3 Å). We generated a disulfide-locked FliD variant (FliD2C: A91C/A168C) that formed covalent pentamers upon oxidation. SDS-PAGE under non-reducing conditions confirmed >40% crosslinking efficiency, while negative-stain EM showed intact cap structures indistinguishable from wild-type. Critically, Salmonella expressing FliD2C exhibited no significant changes of flagellar ultrastructure and bacteria motility. These results demonstrate that the pentameric conformation remains static during elongation and that oligomer rigidity—not dynamic reorganization—enables filament in vivo assembly.

Methods
Bacterial strains, plasmids, and media
The bacterial strains utilized in this study are derived from Salmonella enterica serovar Typhimurium strain LT2 (also known as SGSC1412/ATCC 700720). Detailed information on the plasmids and primers used is provided in Supplementary Table 2. Bacteria were cultivated aerobically at 37°C in liquid cultures as Luria-Bertani (LB) media, Terrific-Broth (TB) media, and the minimum media (M9). LB media contains Baco-Tryptone 10g, yeast extract 5g and NaCl 10g per liter. One liter TB contains Baco-Tryptone 12g, yeast extract 24g, glycerol 4mL, KH2PO4 2.3g, and K2HPO4 12.5g. M9 media contains the minimum necessary nine kinds of substances for bacteria growth, which are NH4Cl (or U-15N- NH4Cl) 1g/L, Na2HPO4 6.78g/L, KH2PO4 3.0g, NaCl 0.5g/L, MgSO4 0.24g/L, CaCl2 0.01g/L, glucose (or U-13C-glucose) 2g/L, vitamin B1 0.01g/L and H2O 1L. Antibiotic selection for plasmid maintenance, with ampicillin at 100µg/mL, was applied to the cultures immediately before the bacterial transfer.

The λ-RED system was carried out to obtain scarless gene knockout from bacteria chromosomes through chromosomal recombination. This process was conducted according to the methods reported in the literature. Mutant genes were introduced into bacteria strains with pBAD, or pTrc plasmid, and gene expression was induced with 0.1% arabinose or 50 µM IPTG, respectively.

Protein expression and purification
All the genes of the flagellar proteins were derived from Salmonella Typhimurium STM1913/700720 (ATCC). The DNA sequences encoding FlgL, FliD, FliC, as well as domains of these three proteins, were amplified with PCR and then inserted into pET16b vector with an N-terminal 8XHis-MBP-TEV (Tobacco etch virus protease site) tag or 8XHis-TEV tag. The fusion constructs for structure determination were prepared by fusing FlgL or FliC to the N-terminus of FliD with a highly flexible and disordered linker, GGS-linker. The amino acid sequence of the GGS-linker is GGSGSGSLVPRGGSGGSGGS. All mutants were made by site-directed single-point mutagenesis with PhusionR high-fidelity and efficiency DNA polymerase (NEB). Primers related to making all the plasmids are collected and listed in Supplementary Table 1. Non-isotopically labeled proteins were expressed with BL21(DE3) cells grown in Terrific-Broth (TB) or LB medium in the presence of ampicillin (100mg/ml−1). Protein expression was induced at 16 °C with 0.5mM isopropyl-beta-D-1-thiogalactopyranoside (IPTG) at OD600 ≈0.6 for about 36h. Cells were collected at OD600 ≈1.8 by centrifugation at 6000×g for 30min at 4°C and stored at −20 °C until purification.

For protein purification, cells were thawed and resuspended in lysis buffer (50mM Tris-HCl pH 8.0, 150mM NaCl, and 10mM imidazole) and disrupted by a high-pressure homogenizer till the solvent became clear. The lysed cells were centrifuged at 20,000×g for 1h to remove the precipitant. The supernatant was passed through a 5ml Ni Sepharose 6 Fast Flow column (GE Healthcare) pre-washed with lysis buffer. Non-specific bound proteins were removed by washing the Ni column with above 25ml wash buffer (50mM Tris-HCl pH 8.0, 500mM NaCl, and 30mM imidazole). The target proteins were eluted with about 20ml elution buffer (50mM Tris-HCl pH 8.0, 150mM NaCl, and 500mM imidazole). The eluted proteins were subjected to TEV protease cleavage for tag removal by overnight dialysis with TEV cleavage buffer (10mM Tris-HCl, pH 8.0, 5mM beta-mercaptoethanol), followed by a second round of Ni column purification to remove the His-MBP or His tag from the supernatant. The flow-through was concentrated to less than 5ml and injected into a HiLoad 26/600 Superdex 200 size exclusion column (GE Healthcare). The size exclusion buffer contains 20mM Tris-HCl, pH 8.0, and 150mM NaCl. The peak fraction was further purified by another round of size exclusion with analytical columns, including Superdex Increase 200 10/300 column (GE Healthcare) and SuperoseTM 6 column (GE Healthcare) with the same buffer. Purified proteins were assessed by SDS-PAGE analysis and concentrated in Amicon filters (Millipore). Protein concentration was spectrophotometrically determined with Lambert-Beer law using the UV absorbance at 280nm and the corresponding extinction coefficient. The purified proteins were subject to Cryo-EM grid preparation or stored at −80°C. All proteins were purified by the same methods.

Protein isotope labeling for solution NMR characterization
15N labeled FliDd1, FliDd2d3 and FliDd1 mutants were expressed in BL21(DE3) cells and grown in minimal medium (M9) supplied with 1g/L U-[15N]-NH4Cl. 15N-13C double labeled FliDd1 sample was prepared by supplementing the M9 medium with 1g/L U-[15N]-NH4Cl and 2g/L U-[13C]-glucose. Cells were induced at around OD600 = 0.9 with 0.5mM IPTG at 16°C for about 24h. Typically, isotope-supplied cells were harvested at OD600 above 1.2 and stored at −20°C till purification. Purification of isotope-labeled proteins was conducted the same as unlabeled protein samples except for storage buffer (50mM NaPi, pH 6.8, 150mM NaCl, 0.05% NaN3). U-[15N]-NH4Cl and U-[13C]-glucose were purchased from Cambridge Isotope Laboratories and Isotec.

NMR spectroscopy of triple resonance and NMR titration
The NMR triple resonance and NMR titration experiments were carried out on Bruker AVANCE III 600 MHz and Bruker AVANCE III 800 MHz instruments, respectively, equipped with cryogenic probes. All the NMR spectra were recorded at 25°C and processed with NMRPipe and further analyzed with Sparky. The protein samples for NMR experiments were prepared in 50mM NaPi, pH 6.8, 150mM NaCl, 0.05% NaN3, and 10% deuterated D2O. Backbone resonance assignment of FliDd1 was achieved using a 15N-13C labeled sample at 0.8mM with a series of triple resonance experiments, including HNCA, CBCACONH, and HNCACB all with the gpwg3d pulse program. Each three-dimensional (3D) experiment was performed with 64 scans with d1=1.5s and 2D experiments were conducted with 12 scans with the same d1 time. The single-point NMR titration experiments were performed with 0.2mM 15N labeled protein (FliDd1, FliDd2d3, or FliDd1 mutant) titrated with 0.2mM unlabeled proteins (FlgL, FliC or FliC mutant). Residues located at the binding interface were screened and confirmed by comparing the peak intensities described by recording and analyzing the 1H-15N-HSQC spectra.

To determine the binding affinity of interaction between FliD and FlgL as well as FliC, we performed NMR titrations using 15N-labeled FliDd1 (0.2mM) with increasing amounts of FlgL (0mM, 0.05mM, 0.10mM, 0.15mM, 0.2mM, 0.3mM, 0.4mM, 0.6mM) or FliC (0mM, 0.1mM, 0.3mM, 0.5mM, 1.0mM, 1.5mM, 2.0mM). 2D-HSQC spectrum was applied to acquire the intensity value of each peak corresponding to substrate adding. The Kd values for these titrations were obtained by fitting the data with a one-site-specific binding equation. Origin 8.5 was used for data fitting and figure presentation.

Negative stain electron microscopy
Negative staining was used to check protein sample homogeneity and visualize bacteria and flagellum in situ. Protein samples were diluted to 10μg/ml and loaded onto glow-discharged carbon-film 400 mesh copper grids. In comparison, bacteria samples were diluted to OD600 (optical density at 600nm wavelength) ≈ 0.05. After 90s of incubation at room temperature, the extra solution on the grid surface was dried with filter paper. The sample-loaded grid was then stained and dried gradually with 5μL saturated uranyl acetate for 10s, 10s, and 30s. Finally, the grids were air-dried for 10min and loaded to a 120keV transmission electron microscope (FEI Talos 120).

Cryo-EM sample preparation, data collection, and process
Proteins for cryo-EM characterization were purified and concentrated to above 8mg/mL. Before making the Cryo-EM grids, protein samples were diluted to 1mg/ml, 2mg/ml, 4mg/ml, and 8mg/ml. Grid screening was performed by making four Cryo-EM grids corresponding to the different protein concentrations. Cryo-EM grids were prepared using a Vitrobot Mark IV system (FEI), by adding a 3μL protein sample to the glow-discharged Quantifoil R 1.2/1.3 holey carbon gold grids. Grid freezing was conducted with a blot time of 1.5s, a blot force of -5, and a wait time of 15s under 95% humidity at 4°C.

Cryo-EM data was collected using a 300keV Titan Krios electron microscope (FEI) equipped with a K3 direct electron detector (Gatan). Cryo-EM images were recorded in super-resolution mode with EPU66 at a nominal magnification of 105,000× in a physical pixel size of 0.85 Å/pixel and a defocus range from −1.0μm to −2.5μm. A dose rate of about 30e/Å2·s and an exposure time of 2.5s were applied with 40 movie frames. A total of 3000, 600, and 900 movie stacks were collected for FliD, FlgL-FliD, FliC-FliD, respectively. The datasets were processed in CryoSPARC (version 4.2.1). Briefly, the raw movie stacks were imported to CryoSPARC, aligned with Patch Motion Correction, followed by contrast transfer function (CTF) estimation using Patch CTF Estimation and Exposure Curation to remove bad movies. Detailed overflow of the cryo-EM data processing for each sample was shown in Supplementary Figs. 2, 7 and 11 and summarized in Supplementary Table 1.

Model building and refinement
The initial models of salmonella FliD, FlgL, and FliC were obtained from the Alphafold web server with the ID: AF-P16328-F1 [https://alphafold.ebi.ac.uk/entry/P16328], AF-P16326-F1 [https://alphafold.ebi.ac.uk/entry/P16326] and AF-P06179-F1 [https://alphafold.ebi.ac.uk/entry/P06179], respectively. Buried surface areas were calculated using the PISA server (https://www.ebi.ac.uk/msd-srv/prot_int/cgi-bin/piserver). UCSF ChimeraX was used to dock the initial models into the cryo-EM maps. From this starting point, manual rebuilding was conducted in Coot. Further refinements were performed with several cycles of real space refinement in Phenix and Coot iteratively. Final structures were uploaded to Molprobility server for quality confirmation. Figures for structure presentation were prepared by PyMOL, UCSF Chimera, and UCSF ChimeraX. Statistical data of structural refinement was summarized in Supplementary Table 2.

Pull-down assay
To determine the effect of mutations on FlgL-FliD stability, we performed pull-down assays to test the relative binding affinity change between wild-type proteins and their mutants. Protein samples used for this experiment were desalted into the binding buffer (25mM Tris-HCl and 150mM NaCl at pH 8.0). Briefly, His-FlgL or its mutants was incubated with untagged FliD or its mutants at a 1:1 concentration ratio (0.03mM, 4mL) for 0.5h at 4°C. The mixtures were injected into a 1mL His Trap HP column (Cytiva) pre-washed with binding buffer. Then the column was washed with wash buffer containing 50mM Tris-HCl and 150mM NaCl at pH 7.4 for 10 times column volume. Finally, the column was eluted with 1mL elution buffer (50mM Tris-HCl, 150mM NaCl, 500mM imidazole, pH 7.4). 5μL of the elution mixed with loading buffer were loaded into a 15% SDS-PAGE gel. All the pulldown assays were repeated three parallel times. Quantitative analysis of the pulldown assay was performed by ImageJ and the figures were presented with Prism 10.0.

Preparation of the flagellar Hook-Junction-Cap and Filament-Cap
Salmonella △flic and wild-type strains were used to prepare flagellar Hook-Junction-Cap and Filament-Cap, respectively. Bacterial cells were grown in 5L of M9 media at 30°C with shaking at 30×g overnight until the OD600 reached around 1.0. Cells were harvested and collected by centrifugation at 3000×g for 30min at 4°C. For Hook-Junction-Cap preparation, the cell pellet was suspended with 50–100ml lysis buffer (50mM Tris-HCl, 150mM NaCl, pH 7.4) and then loaded into a homogenizer to lys the bacteria. The supernatant was collected by centrifugation at 20,000×g for 30min at 4°C. To purify the flagellar hook-junction-cap component, the supernatant was desalted into the low salt buffer (50mM Tris-HCl, pH 8.0) and injected into a DEAE Sepharose fast flow anion exchange column (GE Healthcare). A gradient from 0% to 100% elution buffer (50mM Tris-HCl, 1M NaCl, pH 8.0) was applied to separate impurities and collected elution for 2ml per tube. Negative staining was conducted to check the samples tube by tube. Further purification was performed by using cation exchange with a column packed with source S resin (GE Healthcare), followed by negative staining to check the sample tube by tube. The final step was to concentrate the target sample and load it into a Superose 6 Increase 40 size exclusion column pre-washed with buffer containing 20mM Tris-HCl, 150mM NaCl, pH 7.4. The sample purity of each fraction under visible peaks was checked by negative staining. The purified Hook-Junciton-Cap sample was concentrated to around 8mg/ml and prepared to make Cryo-EM grids. For Filament-Cap preparation, collected cell pellets were gently suspended with 50–100ml fresh M9 culture and stirred overnight at 16°C to cut the flagella tip from the bacteria surface. The bacteria cells were removed by centrifugation at 6000×g for 30min. 50% (NH4)2SO4 was added to the supernatant to precipitate the flagellar filament from soluble impurities. The precipitant was collected by another round of centrifugation at 20,000×g for 30min and resuspended with buffer containing 20mM Tris-HCl, 150mM NaCl, pH 7.4. The final step of purification was the same as the Hook-Junction-Cap sample. Cryo-EM grids of the Filament-Cap sample were prepared as others and stored in liquid nitrogen till use.

Motility assay
Motility assays were conducted following established protocols with some modifications. In brief, Salmonella strains with specific gene knockout (flid-, flgl-, flic-) were transformed with pBAD plasmid carrying wild-type genes or their mutants. Fresh overnight cultures (OD600 = 0.5, 1μL) were stabbed in 0.2% LB-agar plates (0.3% agar, 1% Tryptone, 0.5% Nacl, ampicillin 100μg/mL, 0.125% arabinose and 0.6% glucose). The plates were incubated for 6h at 37°C. After incubation, images of the plates were captured using a LAS 4000 imaging system (GE Healthcare), and the colony diameters were measured with ImageJ software. Each strain was tested in at least three independent replicates. The data were analyzed and presented graphically using Prism 10.

Flagella length measurements
Salmonella strains containing mutant plasmids were grown overnight in LB media containing the appropriate anti-biotics and 0.125% arabinose and shaken at 30rpm, at 37°C. Cells were collected by centrifugation at 1000×g for 10min, and the pellet was resuspended in 500µl of M9 medium. Next, 5µl of bacterial suspension were placed on a freshly glow-discharged carbon-covered electron microscopic support grid for 1min. The grid was washed twice with PBS buffer and stained for 1min with an aqueous solution of 0.75% uranyl acetate. The grids were operated on a 120keV FEI Tecnai 20 electron microscope (Thermo Fisher Scientific). Flagella were counted for 20–40 cells in each sample as indicated in the Figure legends. Use image software to count the number and length of flagella in the sample. Each analysis was repeated a minimum of three times.

Statistics and reproducibility
Pearson’s correlation coefficient (P value) of statistical analysis was conducted with GraphPad Prism 10.0 software. The P-values were determined with one-way ANOVA followed by Dunnett’s multiple comparisons test using wild-type protein or strains as control. Experiments related to statistics data were repeated in at least three independent preparations. Error bars indicate the standard error of the mean (n=3, **P<0.001, ***P<0.0001). Source data are provided as a Source Data file. Each statistical analysis was performed on data obtained from at least three independent biological replicates. For representative electron microscopy images, more than five randomly selected micrographs were acquired per condition from each independent replicate.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "This study focusses on how the flagellum filament cap protein, FliD, mediates extension of FliC filament units from the hook\njunction protein FlgG. The authors present cryo-EM structures of pentameric FliD, a pentameric FliD/FlgG complex, and a\npentameric FliD/FliC complex, which is supported by NMR data and in vitro and cellular mutational analysis. A major output\nfrom the study is the analysis of the very low-affinity interactions that define these complexes and techniques that have been\ndeveloped to overcome this for these high-resolution structural studies. The authors\nalso validate and extend these\nstructures through fitting them into maps derived from native hook tip and filament tip structures purified from Salmonella\nmutant strains. This is important work and expands our understanding of flagellum biogenesis across prokaryotes. The\ntechnical basis of the work is sound, and the conclusions are clear and are in the main supported by the results. The work is\ncarried out with good attention to detail and is generally described clearly, although the gramma and sentence phrasing\nshould be carefully checked throughout (some examples are listed). Please see below for my recommendations to improve\nthe study.\nMajor\n1) Line 47 - please be more specific as to which “in vitro” experiments are being referred to here.\n2) Line 147-150 – this sentence needs re-phrasing as it is not clear (e.g. “while no left extensive ..”). They authors should\nexplain why they anticipate a “weak interaction” (e.g. electrostatically driven interactions can still be strong). They should\nalso comment on the low total buried surface area within the FliD pentamer. A stable complex usually consists of 1500-3000\nÅ². Why should this be so low in the FliD pentamer?\n3) Line 158 – the authors state that based on NMR,” no observations” are observed with the D2-3 domain. Based on the\nspectra this is not the case, although chemical shift perturbations (CSP) are significantly reduced. This should be modified.\nAlso, throughout the manuscript there is a lack of detail in how the NMR data is presented and these needs updating:\na. Fig S4+5+6 – assignments have been made, and these should be used to show CSP per residue in a bar graph and\nhighlight a cut-off for significant changes (and explain rational for the cut-off).\nb. Fig. S6+10A – state molar excess FlgL or FliC rather than concentration.\nc. In the main text and methods no information on the assignment quality has been provided (what % assigned, which\nregions). Do regions where no assignments are made infer anything about the sample?\nd. Assignments should be deposited in an appropriate repository.\ne. Fig S14+15 – quantify the data – e.g. what are the average intensity changes over all peaks for WT vs mutants?\nf. Fig 2j – the authors need to define the criteria for how they included residues here with significant CSP. They should also\npresent the FlgL and FliC binding sites separately (or colour each separate, and overlapping regions another colour etc)\ng. What was the rational for using only the peaks indicated in Fig. 3B and 4B rather than all peaks above a specific cut-off for\ncalculating the Kd’s?\n4) Line 181 (and similar for line 222) – “The optimized fusion construct suitable for structure determination turns out to be the\nFlgL D1 domain …” - it is not clear why this turns out to be suitable as no other constructs are described. What happened\nwhen it was fused at the other terminus? As outlined above, based on the current presented NMR data, this was not\nvalidated by NMR.\n5) For all plots the number of repeats and type of error bar should be defined. Statistical analysis should also be carried\nwhen comparing relative changes in all plots.\n6) Discussion – the authors describe ultra-weak interactions between the FliD pentamer and FlgL or FliC, which bind as\nmonomers. However, in the context of the intact flagellum assembly, there will be significant avidity affects stabilising these\ninteractions – this should be discussed in relation to how FliD is displaced during\naddition of incoming subunits of FliC.\nUsing the fused FliD-FlgL construct the authors should also test this displacement model – e.g. using NMR and titrating\nunlabelled FliD-FlgL against 15N labelled FliC to assess whether CSP is linear with increased concentration or whether\nthere is a “jump” at defined concentrations.\nMinor\n1) Line 72 – change “comprises above 25” to “is comprised of more than 25”.\n2) Line 108 – change “is typically low-order oligomeric” to “typically exists as a lower-order oligomer”.\n3) Line 125 – change “emphasizes” to “is composed of”.\n4) Line 128 – change “through T3SS” to “through the T3SS component”.\n5) Line 137 – Supplementary Table 1 is missing.\n6) Line 176 – change “proteins is quantified” to “proteins was quantified”.\n7) Line 193 – change “model” to “models”.\n8) Lines 271, 274 – Fig. ?\n9) Line 577 – Fig XXX and Table XXX?\n10) Line 580 – provide AF database codes for these models", "reviewer_2": "This manuscript is well-written by Chen L et al. It presents unique insights into the assembly of the Salmonella flagellar\nfilament through cryo-electron microscopy and NMR structural studies of the sub-complex proteins. It proposes a molecular\nmechanism for filament elongation driven by the cognate filament cap protein, with a focus on the ultra-weak interaction\nbetween component proteins. These findings are beneficial for the research community of the bacterial flagellum.\nOverall, some references cited in the introduction section of the main text are not suitable. For example, the hook length\ncontrol in Salmonella, as described by Hirano et al. (1994) in J. Bacteriology, should be cited as a reference. Additionally, the\nhook length and number of copies may vary among species. A careful inspection of the reference cited is strongly\nrecommended before publication.\nKeeping a unique style and format for the Expression of amino acid residues is desired. A three-letter code or a single-letter\ncode is mixed in the main text. Especially, “D2-D245” or “D3-K173” are confusing for general readers. The authors need to\nclarify such numbering of residues throughout the manuscript.\nA proper technical method for calculating the surface area would help general readers better understand the content and\nsignificance of the value. It is necessary to discuss specific numerical evidence, comparing it with others, and providing\nexamples.\nStructural analysis of fusion or truncated constructs may lead to a biased interpretation of the original structures or\nstoichiometry. As a primary cross-linking technique, Grafix by Stark, H. could be applied to cryoEM studies of such full-length\ncomponents.\nNone of the information for plasmids used in the manuscript is available in Supplementary Table 2. Primers are only\nincluded in it. DNA lists for the construction of scarless gene knockouts are also missing, for example, when creating the\nSalmonella fliC deletion for this study.\nIrrelevant PDB accession IDs are described in the main text. Careful inspection of these mistakes would be appreciated\nbefore manuscript submission.\nIn summary, these scientific findings and structures are expected to be shared with the broader community of researchers\nstudying the bacterial flagellum.", "reviewer_3": "This very thorough study provides strong evidence for interactions between FliD and its partners that are relatively weak,\nand provides a solid basis for understanding its action in assisting the delivery of flagellin subunits to their site of assembly\nat the tip of the growing bacterial flagellum. The data are extensive, comprising EM, NMR, mutational analysis, and some\nphysiology. On the whole, I think it is a quite impressive study and makes an important contribution to the field of flagellar\nassembly. The writing is a bit difficult to understand, or at least non-conventional, in several places (detailed in the\ncomments below). A more important concern is whether or not the novelty of the results is overstated; I recall that a similar\nmodel for FliD's action has been proposed before by Namba and coworkers. The present work certainly adds a great deal,\nbut I wonder whether it would be appropriate to describe more fully the previously proposed models for FliD's action, either\nto highlight the novelty of the present model, or to give credit where it is due, whichever is appropriate.\nSome specific (mostly minor) comments and suggestions:\n1. In what sense is FlgL directly involved in filament assembly? This description would seem to suggest an ongoing role in\nfacilitating FliC incorporation, but isn't it the case that it just attaches to the base of the filament, while (after the first ring of\nflagellins is installed) assembly is occurring away from FlgL?\n2. line 104. What is meant by \"polymerization at near-atomic resolution...\"?\n3. Do the other components form 11-fold symmetric rings? I thought they formed 11-start helices; i.e. the symmetry is not\naxial but helical. And it's not only FliD that forms low-order oligomers; FlgK and FlgL form complexes of comparatively few\nsubunits, and I think FliE as well (with only about six subunits).\n4. line 110. Some of the usages here seem non-conventional. Is 'differs' intended to mean 'discriminates'? Similarly the use\nof 'unclarity.'\n5. At the end of the intro, I wondered whether this 'paradox resolution' is really very different from what has been proposed\nbefore--is the present model different from the model described by Namba and coworkers?\n6. line 125 this seems an inappropriate use of 'emphasizes'\n7. fig 2 legend. please describe briefly (in the legend) how the monomer-multimer equilibrium was measured\n8. line 144. K175, not K173; also, this residue seems not to be very well conserved, based on the sequence LOGO shown.\n9. line 146. Destabilized the pentamer by what measure?\n10. line 148. use of 'left' is non-conventional; similarly line 152, use of \"to gain atomic interaction\" .\n11. lines 175-176. use of present tense here is non-conventional.\n12. line 190. Does total surface area here refer to the area of a single D/L interface, or the sum for all five interfaces in the 5:5\ncomplex?\n13. line 193. please elaborate on the previous 'theoretical' model. What theory was the basis for the previous model?\n14. In the discussion of D/L interactions, I'd recommend not giving priority to van der Waals interactions (priority in the sense\nof being the first and only type of interaction mentioned in the paragraph where the interface is first described). The more\ndetailed description in the following paragraph suffices, in my opinion.\n15. line 213. I'm not sure what is meant here by 'scaffold structure.'\n16. line 217. The usages here seem distracting--is the NMR actually state of the art and if so, in what sense? I think it\nsuffices to say that solution state NMR was used and provided important information on the affinities and sites of interaction.\nSimilarly, use of \"ultra-weak\" seems unnecessary, and hard to justify in any rigorous sense. What exactly qualifies as \"ultra\"\nweak? (I have the same feeling about 'ultra-transient' just below.)\n17. line 220. suggest 'with stoichiometry' (instead of 'in stoichiometry')\n18. line 223. use of \"most potential\" is non-conventional. line 222: this seems to be missing something--it might benefit by\nadding \"was done for\" to make it read \"as was done for the FlgL-FliD complex...\"\n19. two different stoichiometries are introduced without preamble. I can guess what's going on, but it would be useful to\ninclude a simple statement first, explicitly indicating that two different stoichiometries were observed.\n20. Fig. 4 panel F. \"inaggregates\" is non-conventional; does it mean soluble, or monodisperse?\n21. line 258. The word 'axial' here is unnecessary and a little confusing. In the following line, I think 'or' is better than 'and.'\nAnd in line 260, use of 'to compose' is non-conventional.\n22. line 280. I didn't understand 'flexibility in the first flagellin insertion'\n23. line 284. What is the basis for a stoichiometry of 11 for FliC here, if it's part of the filament, which contains many (typically\nthousands of) subunits? Is there a clear structural feature that demarcates the 11 most distal subunits?\n24. Fig. 5 panel J. There seems to be some mismatch between the model and the observed map; i.e. some parts of the\ngreen subunits seem to fall outside the density. Am I mis-reading this figure?\n25. I'm not expert in cryo-EM and was left wondering how much confidence we should have in the model-fitting in Figure 5.\nThe density doesn't (to the untrained eye) seem to have clearly diagnostic features to allow confident fitting. What are the\ncriteria?\n26. line 295. Is it necessary to assume that the six additional subunits of L or C bind to FliD? Could they not bind primarily to\nthe five subunits (of L or C) already present?\n27. line 305. Use of 'transient.' The binding is weak, as shown by measurements here. 'Transient' suggests that the\ntimescale for binding is known and is short, but the measurements here, at least as described to this point, don't provide\nrates. Does NMR line-broadening, or NMR relaxation experiments, provide the timescale of the individual FliD/partner\nbinding events? They might; if they do, it's worth describing/discussing.\n28. line 307. I didn't understand this at first; please more explicitly state that the two different components bind to FliD in very\nsimilar ways, showing close overlap in their docked positions.\n29. fig 6 panel C. Do all three parts of this panel refer to FliD? If so, please state in the legend.\n30. line 319. What is meant here by statistics?\n31. line 324. mutations, not mutants; use of 'transient' here again seems unnecessary and distracting. And the mutations\nwere ones at the interface (this is what we know), but might or might not be ones that disrupt the complex (only known after\nthe measurement)\n32. if the flic mutations disrupt the FliD/FliC interface, why do those in FliC have a much stronger effect than those in FliD?\n33. line 346. it isn't made clear at first that these mutations were made with the intention of stabilizing the interface. Please\nmake this more clear. Also, the following lines have a number of typos and non-conventional usages.\n34. line 354. 'ultra-transient' takes the intensifying language to an even higher level, which again seems unnecessary and\ndistracting.\n35. 'strikingly' also seems unnecessary. (The reader is either struck or not, according to their own lights)\n36. line 380. Here, the novelty of the present contribution is highlighted. I recall seeing a beautiful movie from Namba's lab\nthat describe this same process. Was that earlier model incorrect? Or less fully grounded in structural details? Did it capture\nany part of the present model? If so, perhaps credit is due. If not, then the contrast with previous ideas strengthens the case\nfor the importance of this new advance.\n37. line 424. 'downer' (here and in a few other places) could be just 'down,' I think.\n38. lines 436 and surrounding. The FliD cap rotates in this model, and I believe that it also rotates in the earlier model from\nNamba and coworkers.\n39. Re transience of the complexes. Is the FliD complex transient as it functions in cells? Isn't it the case that it is always a\npentamer? And is its complexation with FlgL, or with the growing FliC filament, transient? Doesn't it remain always bound,\nbut in a way that allows it to shift its binding mode (to walk, in effect) so that it rotates? To draw an analogy with another\nrotating device: would we say that the gamma subunit of ATP synthase forms a transient complex with the rest of the\nenzyme, because it rotates relative to much of the rest of the enzyme? Maybe, but I think I'd describe it otherwise."}, "author_response": "Response to referees.\nReviewer #1 (Remarks to the Author):\nThis study focusses on how the flagellum filament cap protein, FliD, mediates\nextension of FliC filament units from the hook junction protein FlgG. The authors\npresent cryo-EM structures of pentameric FliD, a pentameric FliD/FlgG complex, and\na pentameric FliD/FliC complex, which is supported by NMR data and in vitro and\ncellular mutational analysis. A major output from the study is the analysis of the very\nlow-affinity interactions that define these complexes and techniques that have been\ndeveloped to overcome this for these high-resolution structural studies. The authors\nalso validate and extend these structures through fitting them into maps derived from\nnative hook tip and filament tip structures purified from Salmonella mutant strains. This\nis important work and expands our understanding of flagellum biogenesis across\nprokaryotes. The technical basis of the work is sound, and the conclusions are clear\nand are in the main supported by the results. The work is carried out with good attention\nto detail and is generally described clearly, although the gramma and sentence\nphrasing should be carefully checked throughout (some examples are listed). Please\nsee below for my recommendations to improve the study.\nRe: We sincerely thank you for your thoughtful and constructive comments, which have\nsignificantly helped us improve the manuscript. We have carefully addressed each point\nraised, as detailed below. All changes in the manuscript are highlighted and colored\nblue in the revised manuscript.\nMajor\n1) Line 47 - please be more specific as to which “in vitro” experiments are being\nreferred to here.\nRe: Thanks for this suggestion. The sentence has been revised for clarity. It now reads:\n\" Integration of these structures into native flagellar tip densities (6.7–7.0 Å) revealed\na 5:11 FliD:FlgL/FliC architecture, where six additional subunits barely detected by\nsolution NMR dock at secondary sites.\"\n2) Line 147-150 – this sentence needs re-phrasing as it is not clear (e.g. “while no left\nextensive ..”). They authors should explain why they anticipate a “weak interaction”\n(e.g. electrostatically driven interactions can still be strong). They should also comment\non the low total buried surface area within the FliD pentamer. A stable complex usually\nconsists of 1500-3000 Å². Why should this be so low in the FliD pentamer?\nRe: We apologize for the unclear phrasing. This expression has been rewritten as\nfollows: \" The FliD pentamer maintains a stable core, evidenced by 7100 Å² of buried\nhydrophobic interface (Supplementary Fig. 3d). This buried surface area (~7100 Å²)\nfalls within the range of stable oligomeric interfaces—comparable to molecular\nchaperone SecB (Homo-tetramer, 8373 Å²)43,44, yet significantly larger than transient\nsignaling complexes like EIIA-EIIB (702 Å²) in phosphotransferase system45,\nunderscoring the inherent stability of the FliD pentameric core. However, its solvent-\nexposed surface lacks large hydrophobic or charged patches (Fig. 2i), indicating an\nabsence of strong, specific binding sites for other flagellar subunits. This non-specific\nbinding landscape suggests FliD engages FlgL and FliC through weak, transient\ninteractions—a design essential for its role as a rigid platform that enables rapid subunit\nexchange during processive filament elongation. \"\n3) Line 158 – the authors state that based on NMR,” no observations” are observed\nwith the D2-3 domain. Based on the spectra this is not the case, although chemical shift\nperturbations (CSP) are significantly reduced. This should be modified. Also,\nthroughout the manuscript there is a lack of detail in how the NMR data is presented\nand these needs updating:\nRe: Thank you for this careful reading and valuable critique. We have revised the\nstatement to more accurately reflect the NMR observations: \"NMR analysis revealed\nthat FliC and FlgL bind specifically to the D1 domain of FliD (Supplementary Fig. 4a-\nc), while only minimal chemical shift perturbations were observed for the D2-D3\ndomains (Supplementary Fig. 4d-f), indicating substantially weaker or nonspecific\nbinding compared to the interactions at the D1 domain.\"\nAdditionally, we have thoroughly updated our NMR data presentation throughout the\nmanuscript and addressed each sub-point as follows:\na. Fig S4+5+6 – assignments have been made, and these should be used to show CSP\nper residue in a bar graph and highlight a cut-off for significant changes (and explain\nrational for the cut-off).\nRe: Thank you for this suggestion. We have created bar graphs displaying chemical\nshift perturbations (CSPs) per residue for titrations of FliD-D1 with FlgL and FliC\n(Supplementary Fig. 5d). A significance threshold was set at the mean CSP value,\nclearly indicated in the figure. Residues exhibiting CSPs above this threshold for both\nFlgL and FliC are highlighted with transparent magenta boxes, revealing their shared\nbinding site on FliD. The corresponding figure legend now reads: \"(d) Chemical shift\nperturbations (CSPs) plotted against residue number for FlgL (dark orange) and FliC\n(teal) binding to FliD-D1. Unassigned residues are shown in grey. Dashed lines indicate\nthe mean CSP values (dark orange for FlgL, teal for FliC). Residues with intensity\nreductions above the mean for both binding partners are highlighted with transparent\nmagenta boxes.\"\nb. Fig. S6+10A – state molar excess FlgL or FliC rather than concentration.\nRe: Thank you for this suggestion. We have updated Figures S6b and S10b to indicate\nmolar excess in addition to concentrations. The revised figure legends now state: For\nFigure S6: \"Overlay of ¹H-¹⁵N HSQC spectra of 0.2 mM ¹⁵N-labeled FliD-D1 titrated\nwith unlabeled FlgL at molar ratios from 1:0 to 1:3 (0-0.6 mM). Spectra are color-coded\nfrom red to violet with increasing FlgL concentration, showing progressive line\nbroadening.\". For Figure S10B: \"Overlay of ¹H-¹⁵N HSQC spectra of 0.2 mM ¹⁵N-\nlabeled FliD-D1 titrated with unlabeled FliC at molar ratios from 1:0 to 1:10 (0-2.0\nmM). Spectra are color-coded from red to violet with increasing FliC concentration,\nshowing progressive line broadening.\"\nThese revisions provide both concentration and molar ratio information for clearer\ninterpretation of the titration data.\nc. In the main text and methods no information on the assignment quality has been\nprovided (what % assigned, which regions). Do regions where no assignments are made\ninfer anything about the sample?\nd. Assignments should be deposited in an appropriate repository.\nRe: Thank you for these important points regarding our NMR assignments. We have\nnow provided detailed information about assignment quality in the figure legend of\nsupplementary figure 5, which presents the assignment quality of our NMR data. “The\nbackbone assignment for the FliD-D1 domain is >70% complete (43 unassigned\nresidues out of 164 total).”; “The unassigned residues are distributed throughout the\nstructure rather than clustered in specific regions suggesting they correspond to\nflexibility rather than indicating sample quality issues.”. While not complete, this level\nof assignment provides robust coverage of the structured regions and has proven\nsufficient to confidently identify the binding interface, as confirmed by both previous\ntheoretical models and our subsequent complex structures. To ensure data accessibility,\nwe have deposited the assignment data in the BMRB database under accession number\n53375 and updated the Data Availability section accordingly.\ne. Fig S14+15 – quantify the data – e.g. what are the average intensity changes over\nall peaks for WT vs mutants?\nRe: Thank you for this suggestion. We have quantified the NMR titration data in\nSupplementary Figures 14-15 and presented the analysis in main text Figure 4h.\nFor quantification, we focused specifically on residues located at the binding interface\nfor several important reasons. First, this approach maintains consistency with our\nmethodology for determining the K values in Figures 3B and 4B, where we used well-\nD\nbehaved interface residues that exhibited clear, monotonic chemical shift perturbations\nwithout significant broadening. Second, by tracking intensity changes of these same\ninterface residues across different mutants, we can directly calculate complex formation\nratios and compare them to wild-type, providing statistically robust measurements of\naffinity changes.\nThis focused analysis on interface-proximal reporters ensures reliable quantification\nwhile maintaining direct relevance to the functional binding event. The consistent\nbehavior observed across multiple interface residues, together with the global averaging\nresults, provides solid evidence for the effects of mutations on complex stability and\nbinding affinity.\nf. Fig 2j – the authors need to define the criteria for how they included residues here\nwith significant CSP. They should also present the FlgL and FliC binding sites\nseparately (or colour each separate, and overlapping regions another colour etc)\nRe: Thank you for this valuable suggestion. We have now clearly defined our criteria\nfor identifying residues with significant CSPs in the revised manuscript. We added a\nsentence in the figure legend of Suplementary Fig. 5d as “The significant binding\ninterface residues were identified as those showing chemical shift perturbations or\nintensity reductions greater than the mean reduction of all measured changes.”\nAdditionally, we have updated Fig. 2j as recommended to separately visualize the FlgL\nand FliC binding sites and the corresponded figure legend as “(j) The FlgL and FliC\nbinding site on FliD determined by NMR titration is rendered as surface mode and\ncolored in dark orange and cyan, respectively. Notably, all residues implicated in FliC\nbinding fall within the FlgL-binding region; thus, the overlapping interface is\nrepresented by the cyan-colored surface. Unassigned residues located at the potential\ninterface are colored in light grey.”. This revised presentation clearly distinguishes\nshared versus unique interaction surfaces while maintaining visual clarity.\ng. What was the rational for using only the peaks indicated in Fig. 3B and 4B rather\nthan all peaks above a specific cut-off for calculating the Kd’s?\nRe: Thank you for this question regarding our NMR analysis. The peaks selected for\nK calculation in Figures 3B and 4B were chosen based on established criteria for\nD\nreliable quantification in NMR titrations. These specific residues met three key criteria:\n(1) All of them exhibited well-dispersed peaks that remained resolvable throughout the\ntitration series without totally broadening to disappear, enabling accurate chemical shift\ntracking; (2) they are located within the structurally defined binding interface and\nshowed consistent, monotonic chemical shift perturbations; and (3) they represent a\nvalidated subset of responsive peaks, as global fitting of all significantly perturbed\nresidues produced comparable K values. This focused approach ensures we derive\nD\naffinity measurements from the most reliable, interface-proximal reporters while\nmaintaining consistency with the complete dataset.\n4) Line 181 (and similar for line 222) – “The optimized fusion construct suitable for\nstructure determination turns out to be the FlgL D1 domain …” - it is not clear why\nthis turns out to be suitable as no other constructs are described. What happened when\nit was fused at the other terminus? As outlined above, based on the current presented\nNMR data, this was not validated by NMR.\nRe: Thank you for this valid point. We have carefully revised the text to provide a\nclearer rationale: \" Initial attempts to stabilize the native complex using GraFix\n(gradient fixation)47 were unsuccessful due to complex dissociation, prompting the use\nof interface-guided fusion constructs to elevate the complex ratio by mimicking\nproteins in infinite high concentrations48,49. We designed several fusion constructs\nlinking FlgL to the N- or C-terminus of FliD with flexible linkers. The construct\nyielding a homogeneous, monodisperse complex suitable for high-resolution cryo-EM\nanalysis was the one with the FlgL D1 (resi 40-280) domain fused to the N-terminus of\nfull-length FliD (Supplementary Fig. 6c-f). This fusion construct allowed for successful\nstructure determination. The cryo-EM structure of the FliD-FlgL complex was resolved\nat a resolution of 3.30 Å (Supplementary Table 1, Supplementary Fig. 7).\"\n5) For all plots the number of repeats and type of error bar should be defined. Statistical\nanalysis should also be carried when comparing relative changes in all plots.\nRe: We agree entirely. We have now meticulously reviewed all plots (e.g., pull-down\nassays, motility assays, flagella length measurements) to ensure that the number of\nbiological replicates (n ≥ 3) and the type of error bars (SEM) are explicitly stated in the\nfigure legends. Furthermore, appropriate statistical analyses (one-way ANOVA with\nDunnett's test) have been applied and are now indicated directly on the figures where\ncomparisons are made.\n6) Discussion – the authors describe ultra-weak interactions between the FliD\npentamer and FlgL or FliC, which bind as monomers. However, in the context of the\nintact flagellum assembly, there will be significant avidity affects stabilising these\ninteractions – this should be discussed in relation to how FliD is displaced during\naddition of incoming subunits of FliC.\nUsing the fused FliD-FlgL construct the authors should also test this displacement model – e.g. using NMR and titrating\nunlabelled FliD-FlgL against 15N labelled FliC to assess whether CSP is linear with increased concentration or whether\nthere is a “jump” at defined concentrations.\nRe: This is an excellent point that strengthens the biological relevance of our model.\nWe have added a paragraph to the Discussion addressing the role of avidity: \"While we\ncharacterize the interactions between monomeric FliD substrates as ultra-weak, it is\nimportant to consider the context of the native flagellum. The pentameric nature of the\nFliD cap likely creates a significant avidity effect, multiplying the overall binding\nstrength for the undecameric FlgL or FliC ring. This avidity would provide the\nnecessary stability for the cap to remain positioned at the growing tip, while the\nindividual monomer-monomer interactions remain weak enough to allow rapid, low-\nenergy subunit exchange during processive filament elongation.\"\nWe greatly appreciate your suggestion to test the displacement model using NMR\ntitration with the FliD-FlgL fusion construct. In practice, we considered several\ntechnical challenges: (1) The interaction of FlgL-FliC, correspond to FliD-FlgL or FliD-\nFliC binding, adds complexity to interpreting competitive displacement by using the\nfusion construct for titration; (2) The large size of the FliD-FlgL pentameric complex\n(~350 kDa) will cause severe signal broadening when titrated into 15N-labeled FliC,\neven at moderate concentrations; (3) The oligomerization tendency of FliC itself (~50\nkDa) or its domains could exhibit concentration-dependent oligomerization that\ncompromises spectral quality. The structure of FlgL-FliC complex could provide\ncrucial evidence for this model. We previously did attempt to obtain structural insights\ninto FlgL-FliC interactions through both recombinant co-expression and native\ncomplex purification. While a FlgL D1-FliC fusion construct yielded monodisperse\nprotein, cryo-EM analysis revealed well-defined FliC filament density without\ncorresponding FlgL features, suggesting either conformational flexibility or other\nproblems under our experimental conditions.\nWe believe this important question deserves further investigation with specialized\napproaches and have noted it as a key direction for future research.\nMinor\n1) Line 72 – change “comprises above 25” to “is comprised of more than 25”.\n2) Line 108 – change “is typically low-order oligomeric” to “typically exists as a\nlower-order oligomer”.\n3) Line 125 – change “emphasizes” to “is composed of”.\n4) Line 128 – change “through T3SS” to “through the T3SS component”.\n5) Line 137 – Supplementary Table 1 is missing.\n6) Line 176 – change “proteins is quantified” to “proteins was quantified”.\n7) Line 193 – change “model” to “models”.\n8) Lines 271, 274 – Fig. ?\n9) Line 577 – Fig XXX and Table XXX?\n10) Line 580 – provide AF database codes for these models\nRe: Thanks very much for your careful reading. All minor grammatical and\ntypographical errors have been corrected throughout the manuscript (Lines 72, 108, 125,\n128, 176, 193). The missing references to supplementary figures and tables (Lines 137,\n271, 274, 577) have been inserted. The AlphaFold model database codes have been\nupdated in the revised version upon deposition.\nReviewer #2 (Remarks to the Author):\nThis manuscript is well-written by Chen L et al. It presents unique insights into the\nassembly of the Salmonella flagellar filament through cryo-electron microscopy and\nNMR structural studies of the sub-complex proteins. It proposes a molecular\nmechanism for filament elongation driven by the cognate filament cap protein, with a\nfocus on the ultra-weak interaction between component proteins. These findings are\nbeneficial for the research community of the bacterial flagellum.\nRe: We are grateful to your assessment of our work and your valuable, specific\nsuggestions, which have significantly strengthened our manuscript. We have addressed\neach comment in detail as outlined below.\nOverall, some references in the introduction section of the main text are not suitable.\nFor example, the hook length control in Salmonella, as described by Hirano et al. (1994)\nin J. Bacteriology, should be cited as a reference. Additionally, the hook length and\nnumber of copies may vary among species. A careful inspection of the reference cited\nis strongly recommended before publication.\nRe: Thanks for this critical suggestion. We have thoroughly re-examined all references\nin the introduction and throughout the manuscript. Specifically, we have added the\nseminal work by Hirano et al. (1994) on hook-length control and have updated other\ncitations to ensure they are the most appropriate and authoritative sources for the\nstatements made.\nKeeping a unique style and format for the Expression of amino acid residues is desired.\nA three-letter code or a single-letter code is mixed in the main text. Especially, “D2-\nD245” or “D3-K173” are confusing for general readers. The authors need to clarify\nsuch numbering of residues throughout the manuscript.\nRe: We sincerely apologize for this inconsistency. We have now standardized the\nformat throughout the entire manuscript. All amino acid residues are now referred to\nusing the single-letter code (e.g., D245, K173). The domain designation has been\nclarified in the text and figures to avoid confusion (e.g., \"residue K173 in the D3\ndomain\").\nA proper technical method for calculating the surface area would help general readers\nbetter understand the content and significance of the value. It is necessary to discuss\nspecific numerical evidence, comparing it with others, and providing examples.\nRe: This is an excellent point. We have now added a sentence in the Model building\nand refinement section detailing the software and parameters used for surface area\ncalculation, as \"Buried surface areas were calculated using the PISA server\n(https://www.ebi.ac.uk/msd-srv/prot_int/cgi-bin/piserver)\". Furthermore, in the main text\n(Result section), we now provide context by stating: \" This buried surface area (~7100\nÅ²) falls within the range of stable oligomeric interfaces—comparable to molecular\nchaperone SecB (Homo-tetramer, 8373 Å²)43,44, yet significantly larger than transient\nsignaling complexes like EIIA-EIIB (702 Å²) in phosphotransferase system45,\nunderscoring the inherent stability of the FliD pentameric core. \"\nStructural analysis of fusion or truncated constructs may lead to a biased interpretation\nof the original structures or stoichiometry. As a primary cross-linking technique, Grafix\nby Stark, H. could be applied to cryoEM studies of such full-length components.\nRe: We totally agree with your advice that stabilizing native complexes is ideal.\nActually, we did several attempts to use the GraFix method with different crosslinking\nagents such as GA and DSSG; however, the ultra-weak nature of the FliD-FlgL/FliC\ninteractions led to very less crosslinked and heterogeneous complex. The fusion\nstrategy was therefore a necessary and rational alternative, guided by our NMR-derived\ninterface mapping, to \"trap\" the complex for structural insights. We have explicitly\nstated this in the revised manuscript in line 184: \" Initial attempts to stabilize the native\ncomplex using GraFix (gradient fixation)40 were unsuccessful due to complex\ndissociation, prompting the use of interface-guided fusion constructs to elevate the\ncomplex ratio by mimicking proteins in infinite high concentrations.\" We emphasize\nthat our final models were validated by disulfide bond crosslinking, binding affinity\nand motility analysis, their excellent fit into the native flagellar tip densities, confirming\nthat the fusion approach did not introduce major structural biases relevant to the\nbiological assembly mechanism.\nNone of the information for plasmids used in the manuscript is available in\nSupplementary Table 2. Primers are only included in it. DNA lists for the construction\nof scarless gene knockouts are also missing, for example, when creating the\nSalmonella fliC deletion for this study.\nRe: We apologize for this oversight. We have now completely updated Supplementary\nTable 2 to include a comprehensive list of all plasmids used in this study, detailing the\nbackbone, insert, and purpose for each.\nIrrelevant PDB accession IDs are described in the main text. Careful inspection of these\nmistakes would be appreciated before manuscript submission.\nRe: Thank you for spotting this error. We have carefully checked and corrected all PDB\naccession IDs throughout the manuscript to ensure they accurately reference the\nintended structures.\nIn summary, these scientific findings and structures are expected to be shared with the\nbroader community of researchers studying the bacterial flagellum.\nRe: We sincerely appreciate your encouraging summary and for your insightful\nsuggestions. Your constructive feedback has been invaluable in enhancing the clarity,\nrigor, and accessibility of our manuscript.\nReviewer #3 (Remarks to the Author):\nThis very thorough study provides strong evidence for interactions between FliD and\nits partners that are relatively weak, and provides a solid basis for understanding its\naction in assisting the delivery of flagellin subunits to their site of assembly at the tip\nof the growing bacterial flagellum. The data are extensive, comprising EM, NMR,\nmutational analysis, and some physiology. On the whole, I think it is a quite impressive\nstudy and makes an important contribution to the field of flagellar assembly. The\nwriting is a bit difficult to understand, or at least non-conventional, in several places\n(detailed in the comments below). A more important concern is whether or not the\nnovelty of the results is overstated; I recall that a similar model for FliD's action has\nbeen proposed before by Namba and coworkers. The present work certainly adds a\ngreat deal, but I wonder whether it would be appropriate to describe more fully the\npreviously proposed models for FliD's action, either to highlight the novelty of the\npresent model, or to give credit where it is due, whichever is appropriate.\nRe: We sincerely thank you for your positive assessment of our work and for the critical\nsuggestion to better contextualize our findings. We fully agree that properly crediting\nprior models is essential. Therefore, we have carefully revised the Introduction to\nexplicitly acknowledge the seminal \"rotating cap\" hypothesis proposed by Namba and\ncoworkers (Maki et al., J. Mol. Biol., 1998; Yonekura et al., Science, 2000). This\nfoundational work provided the crucial conceptual framework for understanding\nfilament elongation. We have revised the text to frame our work as the mechanistic\nvalidation and structural elucidation of a long-hypothesized model, rather than\npresenting the core idea as entirely new. This, we believe, strengthens the significance\nof our findings by grounding them in the field's history while highlighting our\nsubstantial advancements. The revised introduction part is as: “Pioneering work during\nlast three decades, based on elegant electron microscopy, proposed the influential\nhypothesis of a rotating FliD cap act as a dynamic polymerization chaperone and\nrotating incrementally to sequentially position incoming flagellin subunits for\nprocessive filament elongation. This model provided a crucial conceptual framework,\nyet the underlying molecular mechanics—how the pentameric cap dynamically\ninterfaces with the flagellum's prevailing 11-fold symmetry—remained a structural and\nbiophysical paradox. While a recent near-atomic structure provided key insights into\nthe FliD cap atop the FlgL ring, the fundamental principles governing the symmetry\nmismatch accommodation, the atomic details of subunit binding, and the functional\nnecessity of precisely tuned interaction affinities have remained elusive. This lack of\nclarity has hindered a mechanistic understanding of how the flagellar cap discriminates\nthe hook-filament junction from flagellin to mediate rapid filament assembly.”\nSome specific (mostly minor) comments and suggestions:\n1. In what sense is FlgL directly involved in filament assembly? This description would\nseem to suggest an ongoing role in facilitating FliC incorporation, but isn't it the case\nthat it just attaches to the base of the filament, while (after the first ring of flagellins is\ninstalled) assembly is occurring away from FlgL?\nRe: Thank you for your insightful questions. FlgL's direct role is primarily in initiating\nthe first step of filament assembly by forming the foundation for FliD docking and the\nincorporation of the very first 11 FliC subunits. We have rephrased this throughout the\nmanuscript to avoid implying an ongoing role in later elongation stages.\n2. line 104. What is meant by \"polymerization at near-atomic resolution...\"?\nRe: Sorry for the confusion. We have changed it to: \"...FliC, polymerizes into a helical\nfilament and its structure has been determined at near-atomic resolution...\"\n3. Do the other components form 11-fold symmetric rings? I thought they formed 11-\nstart helices; i.e. the symmetry is not axial but helical. And it's not only FliD that forms\nlow-order oligomers; FlgK and FlgL form complexes of comparatively few subunits,\nand I think FliE as well (with only about six subunits).\nRe: Sorry for the unprecise expression. The components that comprise the propeller of\nthe flagellum, including FlgE, FlgK, FlgL and FliC, all forms 11-fold helical rings. The\nonly exception is the cap protein FliD (filament cap). Components that build the base\npart are basically not 11-fold symmetry, such as you mentioned FliE, FlgG, FlgI and so\non. We have corrected this throughout the text to refer to \"11-start helices\" or \"helical\nsymmetry\" rather than \"rings.\"\n4. line 110. Some of the usages here seem non-conventional. Is 'differs'\nintended to mean 'discriminates'? Similarly the use of 'unclarity.'\nRe: Thank you for catching this. We have replaced 'differs' with 'discriminates' and\n'unclarity' with 'lack of clarity'.\n5. At the end of the intro, I wondered whether this 'paradox resolution' is really very\ndifferent from what has been proposed before--is the present model different from the\nmodel described by Namba and coworkers?\nRe: Thank you for this important question regarding the relationship between our\nmodel and the foundational work from Namba and coworkers. Our study does not\npropose a fundamentally different process but provides the crucial mechanistic\nvalidation and structural elucidation that transforms their brilliant hypothesis into a\ndetailed, near atomic-level understanding. While the rotating cap hypothesis correctly\npredicted the overall mechanism, our work provides several key advancements as : 1)\nQuantitative measurement of ultraweak binding affinities that enable rapid subunit\nexchange and make rotation thermodynamically feasible; 2) Identify the precise\ninterfaces through which FliD engages both FlgL and FliC; 3) Directly visualize the\nnative hook and filament tip architecture, resolving the stoichiometric paradox; 4)\nExperimental demonstration of FliD pentamer rigidity during filament elongation,\nconfirming a key requirement for rotation. Thus, we have moved the field from a\ncompelling theoretical model to a structurally and biophysically grounded mechanism.\nWe have revised our introduction to more clearly frame our contribution and giving full\ncredit to the pioneering work that inspired our study.\n6. line 125 this seems an inappropriate use of 'emphasizes'\nRe: Thank you for pointing out this error. We have change it to ‘is composed of ’\n7. fig 2 legend. please describe briefly (in the legend) how the monomer-multimer\nequilibrium was measured\nRe: Thank you for your advice. We have already added: \"The equilibrium was assessed\nby analytical size-exclusion chromatography, with the monomer and pentamer peaks\nquantified by integrated peak area.\" In the legend of Fig 2.\n8. line 144. K175, not K173; also, this residue seems not to be very well conserved,\nbased on the sequence LOGO shown.\nRe: Thank you for your corrections. We have corrected it to K175. We agree the\nsequence logo shows variability of the salt bridge between K175 and E237. So, we\nchanged our expression more precisely as this ‘Especially, the high conservation of\nthese hydrophobic residues across species suggests their functional importance’\n9. line 146. Destabilized the pentamer by what measure?\nRe: Sorry for the explicit expression. We added this information to illustrate how to\nmeasure the equilibrium \"as measured by a shift in the monomer-pentamer equilibrium\nusing size-exclusion chromatography.\"\n10. line 148. use of 'left' is non-conventional; similarly line 152, use of \"to gain atomic\ninteraction\" .\nRe: Thanks for pointing out the wrong expressions. We have already rewritten these\ntwo sentence like this. ‘The FliD pentamer maintains a stable core, evidenced by 7100\nÅ² of buried hydrophobic interface (Supplementary Fig. 3d). However, its solvent-\nexposed surface lacks large hydrophobic or charged patches (Figure 2i), indicating an\nabsence of strong, specific binding sites for other flagellar subunits. This non-specific\nbinding landscape suggests FliD engages FlgL and FliC through weak, transient\ninteractions—a design essential for its role as a rigid platform that enables rapid subunit\nexchange during processive filament elongation.’\n11. lines 175-176. use of present tense here is non-conventional.\nRe: Sorry for the unprecise expression. We have already revised it to past tense.\n12. line 190. Does total surface area here refer to the area of a single D/L interface, or\nthe sum for all five interfaces in the 5:5 complex?\nRe: Yes, it refers to a single D/L interface. We have revised this sentence to “The\nsingle FliD-FlgL interface buries a total surface area of 955 Å²” for clarity.\n13. line 193. please elaborate on the previous 'theoretical' model. What theory was the\nbasis for the previous model?\nRe: Thank you for this question. The earlier model was primarily derived from low-\nresolution 2D electron microscopy images of the flagellar filament tip. At the time,\ntechnical limitations in cryo-EM and 3D reconstruction prevented determination of near\natomic-level details, making the proposed cap architecture a well-informed but\nhypothetical model. We have revised the text to state: \"This binding interface agrees\nwith our NMR titration data (Fig. 1j) and provides structural validation for earlier\nhypothetical models that were based on low-resolution electron microscopy images.\"\n14. In the discussion of D/L interactions, I'd recommend not giving priority to van der\nWaals interactions (priority in the sense of being the first and only type of interaction\nmentioned in the paragraph where the interface is first described). The more detailed\ndescription in the following paragraph suffices, in my opinion.\nRe: Thank you for this suggestion. We have revised the text as recommended,\nreordering the description to state: \" Structural analysis of the interface reveals a highly\nhydrophilic and complementary surface, suggesting that polar interactions and van der\nWaals forces collectively contribute to stabilizing the complex (Supplementary Fig. 8).\n\" This modification better reflects the balanced role of different interaction types at the\ninterface.\n15. line 213. I'm not sure what is meant here by 'scaffold structure.'\nRe: Sorry for this confusion. We revised this expression to \"a domain architecture\".\n16. line 217. The usages here seem distracting--is the NMR actually state of the art and\nif so, in what sense? I think it suffices to say that solution state NMR was used and\nprovided important information on the affinities and sites of interaction. Similarly, use\nof \"ultra-weak\" seems unnecessary, and hard to justify in any rigorous sense. What\nexactly qualifies as \"ultra\" weak? (I have the same feeling about 'ultra-transient' just\nbelow.)\nRe: Thank you for your thoughtful critique regarding terminology. We have removed\nthe phrase \"state-of-the-art\" as suggested. Concerning the term \"ultra-weak,\" we have\nrespectfully retained it based on its established usage in biophysical literature to\ndescribe specific protein-protein interactions with exceptionally low affinity, typically\ncharacterized by dissociation constants (K ) in the high micromolar to millimolar\nD\nrange—precisely the affinity regime we have measured here (0.1–1.65 mM). This\nterminology is well-supported in the field to distinguish such interactions from\nconventional \"weak\" binding events (e.g., Methods, 2012, 57: 467; Angew. Chem. Int.\nEd., 2014, 53: 11501), and we believe it most accurately conveys the unique biophysical\nproperty that is central to our proposed assembly mechanism.\n17. line 220. suggest 'with stoichiometry' (instead of 'in stoichiometry')\nRe: We have changed it to with stoichiometry.\n18. line 223. use of \"most potential\" is non-conventional. line 222: this seems to be\nmissing something--it might benefit by adding \"was done for\" to make it read \"as was\ndone for the FlgL-FliD complex...\"\nRe: Thank you for this suggestion. We have revised the text to clarify our approach. “Since full-\nlength FliC has a strong filamentation propensity, we first identified that D0 domain truncation\nyielded monodisperse protein. After screening multiple fusion constructs, the optimal construct for\nstructural studies is covalently linked the N-terminus of FliC D1D2D3 (residues 45-455) to the C-\nterminus of full-length FliD (Supplementary Fig. 10c-f).”\n19. two different stoichiometries are introduced without preamble. I can guess what's\ngoing on, but it would be useful to include a simple statement first, explicitly indicating\nthat two different stoichiometries were observed.\nRe: Thank you for this suggestion. We have revised the text to provide clearer context: \"Using this\nconstruct, we identified two distinct complexes and determined their cryo-EM structures - one with\n5:5 stoichiometry at 3.30 Å resolution and another with 10:10 stoichiometry at 3.27 Å resolution\n(Supplementary Table 1, Supplementary Fig. 11).\"\n20. Fig. 4 panel F. \"inaggregates\" is non-conventional; does it mean soluble, or\nmonodisperse?\nRe: Sorry for the confusion. We have change it to non-aggregates, which stands for\nmonodisperse.\n21. line 258. The word 'axial' here is unnecessary and a little confusing. In the following\nline, I think 'or' is better than 'and.' And in line 260, use of 'to compose' is non-\nconventional.\nRe: Thank you for your suggestions. We have revised all the expressions. ‘to compose’\nwas replaced with ‘to contain’\n22. line 280. I didn't understand 'flexibility in the first flagellin insertion'\nRe: Sorry for the confusion. We have revised the sentence to read: \"The extra FlgL\nsubunit does not interact with or occupied by FliD (Fig. 5e), indicating the presence of\nan accessible site primed for the incorporation of the first flagellin (FliC) subunit.\" This\nclarifies that the structural configuration reveals a vacant binding site ready for flagellin\ndocking.\n23. line 284. What is the basis for a stoichiometry of 11 for FliC here, if it's part of the\nfilament, which contains many (typically thousands of) subunits? Is there a clear\nstructural feature that demarcates the 11 most distal subunits?\nRe: Thank you for this insightful question. It is well-established that the flagellar\nfilament is a helical assembly composed of thousands of FliC subunits, which consist\nof 11 protofilaments in longitudinal view, and presumably each layer contains 11 FliC\nsubunits. Our cryo-EM density map of the native filament tip—though at moderate\nresolution (∼6.97 Å)—clearly reveals a FliD pentamer capping a single underlying\nlayer of 11 FliC subunits. While the limited resolution precludes atomic-level detail of\nthe interaction interfaces, the overall architecture and stoichiometry are unambiguously\nresolved: five FliD subunits are positioned atop 11 FliC subunits in the most distal turn\nof the growing filament. This 5:11 arrangement is consistent with the known 11-fold\nsymmetry of the filament and directly visualizes the structural basis for the symmetry\nmismatch resolution. We hope this clarification adequately addresses your question.\n24. Fig. 5 panel J. There seems to be some mismatch between the model and the\nobserved map; i.e. some parts of the green subunits seem to fall outside the density.\nAm I mis-reading this figure?\n25. I'm not expert in cryo-EM and was left wondering how much confidence we should\nhave in the model-fitting in Figure 5. The density doesn't (to the untrained eye) seem to\nhave clearly diagnostic features to allow confident fitting. What are the criteria?\nRE: Thank you for the careful inspection of Figure 5 and the opportunity to clarify this\npoint. The mismatch between the model and the map at the distal region of the native\nfilament tip (Fig. 5j) is indeed real, but it reflects intrinsic biological flexibility rather\nthan an inaccuracy in model fitting. Firstly, the D2–D3 domains of FliC are highly\nmobile relative to the D0–D1 core. Even in our near-atomic resolution reconstruction\nof the isolated filament, the D2–D3 regions appear less well resolved, confirming their\nintrinsic conformational variability. This flexibility results in lower local resolution and\na smeared or discontinuous density for these domains in the native tip map. Secondly,\nthe distal region of the tip, where the FliD cap contacts FliC, is reconstructed from\nfewer effective particles than the rigid filament shaft, leading to a natural resolution\ngradient. To visualize the weaker, flexible D2–D3 densities, we display the map at\nlower contour levels, which can make some portions appear outside the main density\nenvelope when overlaid with the model. Last but not least, the D2–D3 domains do not\nform stable interfaces with FliD or adjacent FliC subunits. Their poor definition\nsupports the conclusion that these domains are not locked into a single conformation\nbut remain dynamically mobile—likely facilitating filament flexibility during rotation\nand propulsion.\nThus, our fitting of the well-resolved components—the FliD pentamer and the D0–D1\ncores of FliC—is robust. These regions were rigid-body docked into high-resolution\nfeatures of the map. The resulting model shows good cross-correlation. The clear,\ncontinuous density of the D0–D1 regions and the 6 FliC subunits in “down”\nconformation further support the accuracy of our placement.\nIn summary, the apparent “mismatch” of the D2–D3 domains is not an artifact of model\nfitting. We have added a clarifying note to the Figure 5 legend to guide readers\nunfamiliar with the variable resolution typical of flexible regions in cryo-EM maps.\n“The smeared density corresponding to the D3 domain of FliC in the \"up\" conformation\nresults from a combination of limited particle numbers in this specific state and the\nintrinsic structural flexibility of the D3 domain itself.”\n26. line 295. Is it necessary to assume that the six additional subunits of L or C bind to\nFliD? Could they not bind primarily to the five subunits (of L or C) already present?\nRe: Thank you for this insightful question. Our proposal that the six additional subunits\ninteract with FliD is based on structural evidence from our native tip density maps,\nwhich show close proximity between these subunits and specific regions of the FliD\ncap. This interpretation is further supported by our mutational data, where altering\nresidues at these potential FliD contact sites significantly impaired bacterial motility.\nHowever, we fully agree with you that these additional subunits primarily engage in\ncrucial lateral interactions with adjacent FlgL/FliC subunits (which is also explicit in\nthe structural model from the native tips), which is essential for maintaining the\nstructural integrity of the junction and filament. Therefore, we have revised our\nmanuscript to present a more balanced interpretation, stating that the additional subunits\nlikely engage in a cooperative network of interactions with both the FliD cap and\nneighboring subunits. The corresponding text in the manuscript now reads: \"We\npropose that the six additional subunits are stabilized through a combination of lateral\ninteractions with adjacent FlgL/FliC subunits and weaker, dynamic contacts with\nsecondary sites on FliD, collectively enabling the stable 5:11 architecture observed in\nnative flagella.\"\n27. line 305. Use of 'transient.' The binding is weak, as shown by measurements here.\n'Transient' suggests that the timescale for binding is known and is short, but the\nmeasurements here, at least as described to this point, don't provide rates. Does NMR\nline-broadening, or NMR relaxation experiments, provide the timescale of the\nindividual FliD/partner binding events? They might; if they do, it's worth\ndescribing/discussing.\nRe: Thank you for this important clarification. We agree that \"transient\" properly refers\nto binding kinetics rather than affinity. We have revised the text to more accurately\nreflect our experimental evidence. The sentence now reads: \"Structural dissection and\ncomparison of the resolved complexes reveal key features of the dynamic, weak-\naffinity interactions governing flagellar filament component positioning by the filament\ncap FliD.\" This revision acknowledges that while our NMR line-broadening data are\nconsistent with exchange on the microsecond-to-millisecond timescale - characteristic\nof transient interactions - our primary measurements quantify binding affinity rather\nthan direct kinetic rates.\n28. line 307. I didn't understand this at first; please more explicitly state that the two\ndifferent components bind to FliD in very similar ways, showing close overlap in their\ndocked positions.\nRe: Sorry for the implicit expression. We have carefully revised this sentence and\nchanged it to this: “FlgL and FliC occupy a shared binding site on FliD, revealing a\nconserved mechanism for substrate recognition by the filament cap.”\n29. fig 6 panel C. Do all three parts of this panel refer to FliD? If so, please state in the\nlegend.\nRe: Sorry for the confusion. The three parts with frame in different color refer to FliD,\nFlgL and FliC, respectively. We have revised and clarified in the legend as this :\n“Sequence conservation analysis of key interfacial residues. Panels from left to right\nshow the conservation of residues in FliD (magenta), FlgL (orange), and FliC (teal) that\nare involved in the shared binding site.”\n30. line 319. What is meant here by statistics?\nRe: Sorry for the typo. We have corrected it as “electrostatic”\n31. line 324. mutations, not mutants; use of 'transient' here again seems unnecessary\nand distracting. And the mutations were ones at the interface (this is what we know),\nbut might or might not be ones that disrupt the complex (only known after the\nmeasurement)\nRe: Thanks for these suggestions. We have rewritten this sentence to make it clearer as\nthis ‘To evaluate the functional significance of the observed interactions, we introduced\nkey mutations that disrupt complex formation, as identified by our binding experiments\n(Fig. 3f and 4h), into corresponding Salmonella strains.’\n32. if the flic mutations disrupt the FliD/FliC interface, why do those in FliC have a\nmuch stronger effect than those in FliD?\nRe: Thank you for raising this insightful question. You are correct that our in vitro NMR\ntitrations show similarly severe binding defects for disruptive mutations in both FliD\nand FliC. The more pronounced in vivo motility defect observed for the FliC mutants\nlikely stems from the multifunctional nature of these residues. While the mutated FliC\nresidues are critical for the initial interaction with the FliD cap, they may also play\nadditional, essential roles in subsequent steps of filament assembly. For instance, these\nsame residues could be involved in FliC-FliC interactions during the polymerization\nprocess or in maintaining the structural integrity of the growing filament. Since the\nfilament comprises thousands of FliC subunits, even a subtle defect at the monomer\nlevel could be dramatically amplified, leading to a severe failure in filament assembly\nor function in vivo. In contrast, FliD functions primarily as a pentameric cap, potentially\nmaking it more tolerant to single-point mutations at the interface.\n33. line 346. it isn't made clear at first that these mutations were made with the intention\nof stabilizing the interface. Please make this more clear. Also, the following lines have\na number of typos and non-conventional usages.\nRe: We have clarified the text to explicitly state: \"To test whether modulating binding\naffinity affects assembly, we engineered structure-guided mutations intended\nto strengthen the FliD-FlgL/FliC interface.\"\n34. line 354. 'ultra-transient' takes the intensifying language to an even higher level,\nwhich again seems unnecessary and distracting.\n35. 'strikingly' also seems unnecessary. (The reader is either struck or not, according\nto their own lights)\nRe: Thanks for your advices. We have carefully rewritten the whole paragraph and the\nrevised one was updated in the manuscript. It is now like this: “Our structural and\nbiochemical analyses establish that ultraweak interactions are essential for filament\nelongation and bacterial motility. To test whether modulating binding affinity affects\nassembly, we engineered structure-guided mutations intended to strengthen the FliD-\nFlgL and FliD-FliC interfaces (Fig. 7a-b). Quantitative pull-down assays confirmed up\nto a 3.5-fold enhancement in binding affinity for the most effective FliD-FlgL interface\nmutant (Fig. 7b, Supplementary Fig.20a). Beyond mutations designed to enhance\nhydrophobic or electrostatic interactions (e.g., V299I, D349R), we also identified key\nresidues where substituting long side chains with glycine enhanced binding. This\nsuggests these wild-type residues serve as stereospecific blockades that help maintain\nthe weak interaction. Applying the same strategy to the FliD-FliC interface yielded\naffinity-enhancing mutants (Fig. 7c-d, Supplementary Fig.20b). Notably, all such\nmutants—whether targeting FliD-FlgL or FliD-FliC interactions—impaired bacterial\nmotility to varying degrees (Fig. 7e, Supplementary Fig.21), demonstrating that finely\ntuned, low-affinity binding is critical for efficient filament assembly and function. ”\n36. line 380. Here, the novelty of the present contribution is highlighted. I recall seeing\na beautiful movie from Namba's lab that describe this same process. Was that earlier\nmodel incorrect? Or less fully grounded in structural details? Did it capture any part\nof the present model? If so, perhaps credit is due. If not, then the contrast with previous\nideas strengthens the case for the importance of this new advance.\nRe: Please see our response to the major concern. We have expanded the Discussion to\nproperly credit the prior model and explicitly list the new evidence our study provides,\nfrom a compelling hypothesis to a structurally and biophysically defined mechanism.\n37. line 424. 'downer' (here and in a few other places) could be just 'down,' I think.\nRe: Yeah, we have revised it.\n38. lines 436 and surrounding. The FliD cap rotates in this model, and I believe that it\nalso rotates in the earlier model from Namba and coworkers\nRe: Thank you for this important clarification. We have revised the text to more\naccurately acknowledge the foundational work while highlighting our specific\ncontributions. The relevant section now reads:“These structural insights support a\nstepwise mechanism for filament elongation at the hook-filament junction (Fig. 7i),\nproviding structural and biophysical validation for the rotating cap model first proposed\nby Namba and coworkers.”\n39. Re transience of the complexes. Is the FliD complex transient as it functions in cells?\nIsn't it the case that it is always a pentamer? And is its complexation with FlgL, or with\nthe growing FliC filament, transient? Doesn't it remain always bound, but in a way that\nallows it to shift its binding mode (to walk, in effect) so that it rotates? To draw an\nanalogy with another rotating device: would we say that the gamma subunit of ATP\nsynthase forms a transient complex with the rest of the enzyme, because it rotates\nrelative to much of the rest of the enzyme? Maybe, but I think I'd describe it otherwise.\nRe: We appreciate this insightful comment and agree that clarification is warranted.\nIndeed, the FliD pentamer itself is a stable structural unit both in vitro and in vivo, and\nwe do not imply that the pentamer dissociates during function. Rather, our use of the\nterm transient specifically refers to the intermolecular interactions between the stable\nFliD pentamer and its binding partners—FlgL and FliC—during filament elongation.\nOur NMR titration experiments demonstrated ultraweak affinities (K values of ~100\nD\nµM for FliD–FlgL and ~1.6 mM for FliD–FliC), accompanied by extensive line\nbroadening of NMR peaks consistent with exchange on the µs–ms timescale. These\nobservations indicate that while FliD remains pentameric, its interfaces with FlgL and\nFliC undergo rapid association–dissociation cycles rather than forming stable, long-\nlived complexes. Structurally, our cryo-EM analyses revealed that each FliD subunit\nbinds independently to one subunit of FlgL or FliC through small, hydrophilic, and van\nder Waals–dominated interfaces (~955–1296 Å² buried area), further supporting a\ndynamic binding mode. In native flagellar tips, we observed additional subunits at\nsecondary binding sites lacking specific pairwise interactions, consistent with a highly\ndynamic exchange process. Thus, we distinguish structural stability of the FliD\npentamer from functional transience of its interactions with substrates. This property is\nlikely essential for processive filament elongation: weak, short-lived interactions allow\nrapid subunit turnover without the need for active dissociation. While the analogy with\nATP synthase rotation is valuable, we note that in the flagellar cap, the “rotation” arises\nnot from rigid mechanical rotation of a subunit within a fixed complex, but rather from\nsequential binding and release of subunits at multiple equivalent sites—a process\ndependent on transient interactions. To avoid confusion, we have revised the text to\nclarify this point and replaced “transient complex” with “weak and short-lived\ninteraction” where appropriate. The revised passage now reads: “The FliD pentamer\nforms a stable structural platform, but its interactions with FlgL and FliC are weak and\nshort-lived, consistent with exchange on the µs–ms timescale. Such dynamic binding\nlikely enables rapid subunit incorporation during processive filament elongation.”"}, {"round": 2, "reviewer_comments": {"reviewer_1": "I am happy that the authors have addressed all of my comments.\nI have assessed the responses to reviewer #3. Please see the attached file.\n[Editorial Note: Please find the attachment at the end of this file]", "reviewer_2": "This revised version of the manuscript, \"Ultraweak interactions drive cap-mediated positioning and\nelongation of the\nbacterial flagellar filament\" by Chen et al., is significantly improved and provides a clear, feasible framework for\nunderstanding the molecular mechanism of bacterial filament elongation assisted by the cognate filament cap protein\nunderpinned by structural biology approaches. After the author's revision, the reviewer found no comments or further claims.\nThis manuscript would be accepted without any additional corrections. The reviewer strongly suggests publishing it in\nNature Communications."}, "author_response": "2nd round response to referees.\nReviewer #1 (Remarks to the Author):\n1. As a general point it would be useful if the authors can reference changes in the\nupdated manuscript using the new line numbers as it is difficult finding changes\nwhen only the original line numbers are referred to.\nRe: We have updated our responses to reference line numbers from the revised\nmanuscript to make it easier to locate the changes.\n2. Line 104: Change “Pioneering work during last three decades, based on elegant\nelectron microscopy, proposed the influential hypothesis of a rotating FliD cap act\nas” to “Pioneering work during the last three decades, based on elegant electron\nmicroscopy, proposed the influential hypothesis of a rotating FliD cap acting as”\nRe: Thank you for the correction. The sentence has been revised to: \"Pioneering work\nduring the last three decades, based on elegant electron microscopy, proposed the\ninfluential hypothesis of a rotating FliD cap acting as...\" (now line 104).\n3. line 217. The usages here seem distracting--is the NMR actually state of the art and\nif so, in what sense? I think it suffices to say that solution state NMR was used and\nprovided important information on the affinities and sites of interaction. Similarly, use\nof \"ultra-weak\" seems unnecessary, and hard to justify in any rigorous sense. What\nexactly qualifies as \"ultra\" weak? (I have the same feeling about 'ultra-transient' just\nbelow.)\nRe: Thank you for your thoughtful critique regarding terminology. We have removed\nthe phrase \"state-of-the-art\" as suggested. Concerning the term \"ultra-weak,\" we have\nrespectfully retained it based on its established usage in biophysical literature to\ndescribe specific protein-protein interactions with exceptionally low affinity, typically\ncharacterized by dissociation constants (KD) in the high micromolar to millimolar\nrange—precisely the affinity regime we have measured here (0.1–1.65 mM). This\nterminology is well-supported in the field to distinguish such interactions from\nconventional \"weak\" binding events (e.g., Methods, 2012, 57: 467; Angew. Chem. Int.\nEd., 2014, 53: 11501), and we believe it most accurately conveys the unique biophysical\nproperty that is central to our proposed assembly mechanism.\nThe authors have provided a good definition of what “ultra-weak” refers to in their\nresponse, but the manuscript would be improved if they can also include this in the\ntext to inform the general reader.\nRe: Thank you for this suggestion. We have updated the main text to include a definition\nof the term \"ultra-weak\" for clarity. The relevant sentence in line 197-199 now reads\n“The dissociation constant (K ) between FliD-D1 and FlgL was determined to be ~100 uM\nd\n(Fig. 3b), consistent with the established classification of an ultra-weak interaction (K in\nd\nthe high micromolar to millimolar range), which is distinct from conventional 'weak'\nbinding events53,56,57.”.\n4. 27. line 305. Use of 'transient.' The binding is weak, as shown by measurements here.\n'Transient' suggests that the timescale for binding is known and is short, but the\nmeasurements here, at least as described to this point, don't provide rates. Does NMR\nline-broadening, or NMR relaxation experiments, provide the timescale of the\nindividual FliD/partner binding events? They might; if they do, it's worth\ndescribing/discussing.\nRe: Thank you for this important clarification. We agree that \"transient\" properly refers\nto binding kinetics rather than affinity. We have revised the text to more accurately\nreflect our experimental evidence. The sentence now reads: \"Structural dissection and\ncomparison of the resolved complexes reveal key features of the dynamic, weak-affinity\ninteractions governing flagellar filament component positioning by the filament cap\nFliD.\" This revision acknowledges that while our NMR line-broadening data are\nconsistent with exchange on the microsecond-to-millisecond timescale - characteristic\nof transient interactions - our primary measurements quantify binding affinity rather\nthan direct kinetic rates.\nAlthough the authors describe the NMR timescales (usec-msec) here in their response,\nthis is not described explicitly in the text as requested by the reviewer. The authors\nshould expand on their NMR explanation in the manuscript and include the\ntimescales based on the NMR exchange regime observed and how this infers stability.\nRe: Thank you for this careful reading and valuable critique. We have revised the\nstatement to more accurately reflect the NMR observations: \" The interaction between\neach segmented domain and FliC/FlgL was analyzed using NMR titration experiments.\nNMR analysis revealed that FliC and FlgL bind specifically to the D1 domain of FliD\n(Supplementary Fig. 4a-c). Significant line broadening observed during titration\nindicates chemical exchange on the microsecond-to-millisecond timescale, which is\ncharacteristic of weak, transient protein–protein interactions. While only minimal\nchemical shift perturbations were observed for the D2-D3 domains (Supplementary Fig.\n4d-f), indicating substantially even weaker or nonspecific binding compared to the\ninteractions at the D1 domain.\" (This update is located at line 173-177 of the main text)\n1. As a general point it would be useful if the authors can reference changes in the updated\nmanuscript using the new line numbers as it is difficult finding changes when only the original line\nnumbers are referred to.\n2. Line 104: Change “Pioneering work during last three decades, based on elegant electron\nmicroscopy, proposed the influential hypothesis of a rotating FliD cap act as” to “Pioneering work\nduring the last three decades, based on elegant electron microscopy, proposed the influential\nhypothesis of a rotating FliD cap acting as”\n3. line 217. The usages here seem distracting--is the NMR actually state of the art and if so, in what\nsense? I think it suffices to say that solution state NMR was used and provided important information\non the affinities and sites of interaction. Similarly, use of \"ultra-weak\" seems unnecessary, and hard\nto justify in any rigorous sense. What exactly qualifies as \"ultra\" weak? (I have the same feeling\nabout 'ultra-transient' just below.)\nRe: Thank you for your thoughtful critique regarding terminology. We have removed the phrase\n\"state-of-the-art\" as suggested. Concerning the term \"ultra-weak,\" we have respectfully retained it\nbased on its established usage in biophysical literature to describe specific protein-protein\ninteractions with exceptionally low affinity, typically characterized by dissociation constants (K ) in\nD\nthe high micromolar to millimolar range—precisely the affinity regime we have measured here (0.1–\n1.65 mM). This terminology is well-supported in the field to distinguish such interactions from\nconventional \"weak\" binding events (e.g., Methods, 2012, 57: 467; Angew. Chem. Int. Ed., 2014, 53:\n11501), and we believe it most accurately conveys the unique biophysical property that is central to\nour proposed assembly mechanism.\nThe authors have provided a good definition of what “ultra-weak” refers to in their response, but\nthe manuscript would be improved if they can also include this in the text to inform the general\nreader.\n4. 27. line 305. Use of 'transient.' The binding is weak, as shown by measurements here. 'Transient'\nsuggests that the timescale for binding is known and is short, but the measurements here, at least as\ndescribed to this point, don't provide rates. Does NMR line-broadening, or NMR relaxation\nexperiments, provide the timescale of the individual FliD/partner binding events? They might; if they\ndo, it's worth describing/discussing.\nRe: Thank you for this important clarification. We agree that \"transient\" properly refers to binding\nkinetics rather than affinity. We have revised the text to more accurately reflect our experimental\nevidence. The sentence now reads: \"Structural dissection and comparison of the resolved complexes\nreveal key features of the dynamic, weak-affinity interactions governing flagellar filament\ncomponent positioning by the filament cap FliD.\" This revision acknowledges that while our NMR\nline-broadening data are consistent with exchange on the microsecond-to-millisecond timescale -\ncharacteristic of transient interactions - our primary measurements quantify binding affinity rather\nthan direct kinetic rates.\nAlthough the authors describe the NMR timescales (usec-msec) here in their response, this is not\ndescribed explicitly in the text as requested by the reviewer. The authors should expand on their\nNMR explanation in the manuscript and include the timescales based on the NMR exchange regime\nobserved and how this infers stability."}]

Decision letter