Analysis of FctB3 crystal structure and insight into its structural stabilization and pilin linkage mechanisms

Analysis of FctB3 crystal structure and insight into its structural stabilization and pilin linkage mechanisms | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Analysis of FctB3 crystal structure and insight into its structural stabilization and pilin linkage mechanisms Katsuki Takebe, Mamoru Suzuki, Takeshi Sangawa, Bernd Kreikemeyer, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3233333/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 23 Nov, 2023 Read the published version in Archives of Microbiology → Version 1 posted 8 You are reading this latest preprint version Abstract Streptococcus pyogenes harboring an FCT type 3 genomic region display pili composed of three types of pilins. In this study, the structure of the base pilin FctB from a serotype M3 strain (FctB3) was determined at 2.8 Å resolution. In accordance with the previously reported structure of FctB from a serotype T9 strain (FctB9), FctB3 was found to consist of an immunoglobulin-like domain and proline-rich tail region. Data obtained from structure comparison reveled main differences in the omega loop structure and the proline-rich tail direction. In the omega loop structure, a differential hydrogen bond network was observed, while the lysine residue responsible for linkage to growing pili was located at the same position in both structures, which indicated that switching of the hydrogen bond network in the omega loop without changing the lysine position is advantageous for linkage to the backbone pilin FctA. The difference in direction of the proline-rich tail is potentially caused by a single residue located at the root of the proline-rich tail. Also, the FctB3 structure was found to be stabilized by intramolecular large hydrophobic interactions instead of an isopeptide bond. Comparisons of the FctB3 and FctA structures indicated that the FctA structure is more favorable for linkage to FctA. Additionally, the heterodimer formation of FctB with Cpa or FctA was shown to be mediated by the putative chaperone SipA. Together, these findings provide an alternative FctB structure as well as insight into the interactions between pilin proteins. Streptococcus pyogenes pilus FctB crystal structure SipA Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Streptococcus pyogenes , also referred to as group A Streptococcus (GAS), is a major pathogenic Gram-positive bacterium, with humans the only biological host (Cunningham et al. 2008). This bacterium causes localized suppurative diseases, such as pharyngitis in the upper respiratory tract and pyoderma in skin, and also immunological sequelae, including acute rheumatic fever and acute glomerulonephritis, as well as lethal streptococcal toxic shock syndrome (Stevens et al. 1989 ; Walker et al. 2014 ). Annually worldwide, it is estimated that there are more than 616 million pharyngitis and 111 million prevalent pyoderma cases (Carapetis et al. 2005 ; Bowen, et al. 2015 ), while GAS-related diseases cause more than 500 thousand deaths each year (Carapetis et al. 2005 ). Thus, deeper understanding of the molecular mechanisms related to pathogenicity is required to develop effective treatments and prevention measures. GAS is mainly classified based on M serotyping, which is determined according to the antigenicity of M protein. Currently, based on genotyping of the emm gene encoding M protein, there are more than 240 known genotypes (Sanderson-Smith et al. 2014 ). An alternative typing scheme is T serotyping (Griffith 1934 ), which is based on the antigenicity of the trypsin-resistant antigen (T antigen), the gene of which is located within the genomic region, and referred to as the FCT ( f ibronectin-binding protein, c ollagen-binding protein, and t rypsin-resistant antigen) region, encoding a variety of adhesins. The major T antigen is the backbone pilin and its antigenicity is responsible for T typing (Mora et al. 2005 ; Falugi et al. 2008 ). Based on the heterogeneity of the gene contents, the FCT genomic region has been classified to nine types (FCT-1-9). GAS pili function as an adhesin to tonsil tissue and keratinocytes, and promote biofilm formation (Abbot et al. 2007 ; Manetti et al. 2007 ), thus have important roles in the infection process. GAS pili consist of two or three types of pilins (Nakata et al. 2021). Pili of strains possessing an FCT type 1 region, such as serotype M6 strains, are composed of the tip pilin FctX and backbone pilin T6 (Nakata et al. 2011 ; Kimura KR et al. 2012 ). On the other hand, pili of strains possessing an FCT type 2–4 region consist of three types, including the tip pilin Cpa, backbone pilin FctA, and base pilin FctB (Fig. S1 ). Linkage between pilins and anchoring to the free amino group of peptidoglycan are mediated by a pilus-specific transpeptidase and the house-keeping transpeptidase SrtA, respectively. The pilus-specific transpeptidase recognizes the C-terminal LPXTG or LPXTG-like motif of pilin, and connect each pilin by forming an intermolecular isopeptide bond (Hendrickx et al. 2011 ). For example, LPXTG-like motifs of Cpa, FctA, and FctB in M1 strains are VPPTG, EVPTG, and LPLAG, respectively. In strains possessing an FCT type 2–4 region, the deduced chaperone SipA is also considered crucial for pilus assembly, though details regarding its exact role and binding partners remain unclear (Zähner et al. 2008; Young et al. 2014b ). Crystal structures have been reported for backbone pilins, including T6 (Young et al. 2014a ) and FctA (Kang et al. 2007 ; Young et al. 2019 ), the tip pilin Cpa (Pointon et al. 2010 ), and the base pilin FctB (Linke et al. 2010 ) (Fig. S1 ). Based on results of the present study, we report a newly determined crystal structure of FctB from a serotype M3 strain possessing an FCT type 3 region (FctB3) and discuss its surface properties, hydrogen bond networks, and other characteristics. In addition, this new structure was compared to the previously reported FctB9 (FctB from a serotype T9 strain) structure (Linke et al. 2010 ), as well as structures of FctA and Cpa. The results show differences in the structural features of FctB and FctA that may efficiently promote FctA polymerization as compared to FctB incorporation by the FctA polymer. Furthermore, formation of the heterodimer complex of FctB with FctA or Cpa, which is mediated by SipA, is demonstrated. Materials and Methods Strains and plasmids A Streptococcus pyogenes serotype M49 strain 591 harboring an FCT type 3 region and isogenic mutant strains were used for immunoblot analyses. The Escherichia coli strain XL10-gold (Stratagene) served as a host for plasmids and E. coli strain NiCo21 (DE3) (New England Biolabs) transformed with pREP4 (Qiagen) was used for expression of recombinant FctB3. All E. coli strains were cultured in Luria-Bertani (LB) medium (Nacalai Tesque) at 37°C with constant agitation. The S. pyogenes strains were cultured in Todd-Hewitt broth (Becton Dickinson) supplemented with 0.2% yeast extract (Becton Dickinson) (THY medium) at 25°C in an ambient atmosphere (Nakata et al. 2020 ). When appropriate, antibiotics were added to the media at the following concentrations: ampicillin, 100 µg/ml for E. coli ; spectinomycin (FUJIFILM Wako Pure Chemical), 100 µg/ml for E. coli and 100 µg/ml for S. pyogenes ; erythromycin (Sigma Aldrich), 150 µg/ml for E. coli and 1 µg/ml for S. pyogenes . All strains used in this study are listed in Table S1 . Construction of S. pyogenes mutant strains and alignment of FctB sequence Genomic DNA preparations from S. pyogenes were conducted using a DNA extraction kit (Takara). Plasmid DNA was prepared from E. coli overnight cultures with a plasmid purification kit (Macherey-Nagel). Transformation of E. coli and S. pyogenes was performed, as previously described (Nakata et al. 2011 ). All primers listed in Table S2 were designed using the reported genome sequences of strains SSI-1 (NC004606, Nakagawa et al. 2003 ) and 591 (NZ_CP077685.1, Patenge et al 2021 ). Construction of in-frame markerless deletion mutants was conducted using the temperature-sensitive shuttle vector pSET4s, as previously reported (Takamatsu et al. 2001 , Nakata et al. 2011 ). Mutated fctB was created using an overlapping PCR with primers listed in Table S2. The fragment was cloned into the shuttle vector pAT18-P gyrA (Nakata et al. 2009 ). Introduction of the mutation was confirmed by DNA sequencing. Amino acid sequences of FctB from serotypes M1, M3, M5, M18, and M49 were aligned using ClsutalW software ( http://clustalw.ddbj.nig.ac.jp ) and visualized with CLC Main workbench software (CLC bio). Cell fractionization and immunoblot analysis Extraction of cell wall and culture supernatant fractions of S. pyogenes strains was conducted, as previously reported (Nakata et al. 2011 ). For preparation of cell wall fractions, S. pyogenes cells grown to the late exponential phase were washed with PBS and suspended in protoplast buffer (0.1 M KPO 4 , pH 6.2, 40% sucrose, 10 mM MgCl 2 ) containing complete EDTA-free protease inhibitors (Roche) and 250 µg/ml of N -acetylmuramidase (Seikagaku Biobusiness). The suspension was incubated at 37˚C for 3 h, followed by centrifugation at 4˚C and 16,000 × g for 10 min, with the resultant supernatant used as a cell wall fraction. For preparation of culture supernatant fractions, the S. pyogenes overnight culture was centrifuged at 4˚C and 16,000 × g for 20 min and proteins in the supernatant were precipitated overnight at 4˚C with 10% trichloroacetic acid. After centrifugation at 20,000 × g and 4˚C for 10 min, the protein pellet was washed three times with cold ethanol, resuspended in 1 M Tris buffer (pH 8.0), and used as the culture supernatant fraction. Using a 5–12% acrylamide gel (FUJIFILM Wako Pure Chemical), proteins in those fractions were separated by sodium dodecyl sulfatepoly-acrylamide gel electrophoresis, then blotted onto a polyvinylidene difluoride membrane. The membrane was blocked overnight at 4˚C with a solution containing a casein-based blocking reagent (Megmilk Snow Brand) and then incubated at room temperature (RT) for 2 h with mouse anti-pilin antiserum diluted 1:2000 in Tris-buffered saline (TBS) containing 0.2% Tween 20 (TBST). Following washing steps, the membrane was incubated at RT for 2 h with a horseradish peroxidase-conjugated anti-mouse IgG antibody (Cell Signaling Technology). Finally, the membrane was washed three times with TBST and developed with Pierce western blotting substrate (Thermo Scientific). Signals were detected using X-ray film (FUJIFILM). Mouse antiserum was prepared by immunizing five-week-old female BALB/c mice (Japan SLC) with recombinant pilus proteins and TiterMax Gold adjuvant (CytRx), as previously described (Kubota et al. 2023 ). Purification of recombinant FctB3 The DNA fragment encoding FctB without the putative signal peptide and cell-wall sorting signal was PCR-amplified using genomic DNA of the serotype M3 strain SSI-1 and cloned into pQE30 (Qiagen). N-terminal His-tagged FctB was expressed in NiCo21 (DE3) transformed with pREP4 and purified by affinity chromatography using Ni-NTA agarose (QIAGEN). The eluted protein was further purified by gel filtration with Superdex 200 Increase 10/300GL (Cytiva) equilibrated with Tris-buffered saline (50 mM Tris, 150 mM NaCl, pH7.6). The protein concentration was measured based on 280 nm absorbance using Nanodrop 2000 (Thermo Fisher Scientific). Crystallization of FctB and X-ray diffraction data collection Initial screening was conducted with screening kits, including Crystal Screen HT, Index HT (Hampton Research), and Wizard Classic 1 & 2 HT96 (Rigaku) using a sitting-drop vapor diffusion method at 20˚C. Crystals were obtained within 1 week from solutions containing 0.1 M sodium acetate trihydrate (pH 4.5) and 2.0 M ammonium sulfate. Further optimization of the conditions was performed using hanging- and sitting-drop vapor diffusion methods with a mixture of 1 µl of protein solution (45.12 mg ml⁻¹) and 1 µl reservoir solution. Ranges of ammonium sulfate concentration (1.8–2.4 M) and pH conditions (4.2–4.6) were tested. The final crystallization condition is summarized in Table S3. The crystals were cryoprotected with reservoir solution containing 25% (vol/vol) glycerol and flash-cooled with liquid nitrogen. First, X-ray diffraction experiments were conducted in BL44XU (SPring-8, Sayo-cho, Hyogo Pref.) and the condition was confirmed. The final data were obtained in BL-1A (Photon Factory, Tsukuba city, Ibaraki Pref.), with a wavelength of 1.1 Å and 1˚ oscillation. The diffraction datasets were integrated using the XDS Package (Kabsch 2010 ) and scaled using CCP4 suite (v. 6.4.0) (Winn et al. 2011 ). Structure solution and refinement The structure of FctB3 was determined by molecular replacement using MOLREP (Vagin and Teplyakov 2010 ). A previously reported FctB structure from a serotype T9 strain harboring FCT type 7 region (Linke et al. 2010 , PDB ID:3klq, sequence identity 97.9%) was used as a model molecule. Structural refinement was carried out using REFMAC5 (Murshudov et al. 2011 ) and PHENIX package (Adams et al. 2010 ), while manual model building was performed using Coot (Emsley et al. 2004). The final model was deposited in wwPDB (PDB ID: 8K6T). Ethics statement Experiments of mouse immunization for antiserum preparation were conducted according to a protocol approved by the Animal Care and Use Committee of Osaka University Graduate School of Dentistry (authorization number 29-014-0). Results and Discussion Cell wall anchoring of FCT region type 3 pili is mediated by FctB To confirm the reported role of FctB in a strain possessing FCT region type 3, in-frame deletion mutants of each gene in the cpa operon (Fig. 1 A) and a deletion mutant of the house-keeping transpeptidase gene srtA were constructed in the background of a serotype M49 strain harboring the FCT type 3 region, then the effects of the mutation on pilus assembly and cell wall anchoring were examined by immunoblot analyses (Fig. 1 A). In a wild-type cell wall fraction (CWF), high molecular weight ladder bands (HLBs) reflecting FctA polymerization were detected using antisera against the pilus proteins FctA, Cpa, and FctB (Fig. 1 B-D). In contrast, HLBs were not detected in the CWFs of all of the mutant strains, except for ∆ fctB and ∆ srtA (Fig. 1 B-D). In concentrated supernatant fractions (SF), HLBs detected with anti-FctA were more prominent in ∆ fctB and ∆ srtA , as compared with the wild type, which indicated that greater amounts of assembled pili were released into culture supernatants of ∆ fctB and ∆ srtA (Fig. 1 E). Therefore, in a strain harboring the FCT type 3 genomic region, FctB and SrtA also participate in cell wall anchoring of pili. In the background of ∆ cpa , HLBs disappeared, while a band representing a monomeric form of FctA and a band sized ~ 47 kDa were detected with anti-FctA (Fig. 1 B). The ~ 47 kDa band was also detected with anti-FctB (Fig. 1 D), indicating an FctA and FctB complex. Also, in the fraction of ∆ fctA , in addition to a band representing a monomeric form of Cpa, a band sized approximately ~ 100 kDa was detected with anti-Cpa (Fig. 1 C), while the same sized band was also detected with anti-FctB (Fig. 1 D), indicating formation of a Cpa and FctB complex. The level of detection of bands potentially representing FctA/FctB and Cpa/FctB complexes were decreased with sipA deletion. These findings suggest that FctB possesses an ability to bind to FctA and Cpa, with that binding mediated by SipA (Fig. 1 B-D). Overall structure of FctB3 To gain insight into the underlying mechanism of FCT type 3 pilus assembly and pilin interactions, the crystal structure of recombinant FctB from a serotype M3/T3 strain (hereafter referred to as FctB3) was determined and used as a representative of FCT type 3 pili. The structure is summarized in Table 1. The crystal belonged to the space group P 4 3 22 and the unit-cell parameters were as follows; a = b = 44.47 Å, c = 181.37 Å, and α, β and γ = 90˚. Matthews coefficient (VM) was calculated to be 3.17 Å 3 Da −1 and the solvent content was 61.14%, which indicated that a monomer molecule was contained in the asymmetric unit cell. The FctB3 crystal structure was solved at 2.8 Å resolution (R factor = 0.209, R free = 0.260). FctB3 was composed of 11 β-sheets folded into an immunoglobulin (Ig)-like fold and a proline-rich tail that formed a poly proline Ⅱ helix (Fig. 2 A). The Ig-like domain was characterized by β-sandwiches, which were composed of five-stranded (β3, β4, β7, β8, β9) and four-stranded (β1, β2, β5, β6) β-sheets. In addition, another specific structure was an omega (Ω) loop, composed of the region from Arg 104 to Thr 114 in the β9-β10 loop. The inner surface of the molecules (i.e., region not exposed to external milieu) was mainly composed of hydrophobic and aromatic residues with the exception of Asn 13 and Gln 67 (Fig. 2 B-D). For example, Phe 50 on the β56 loop formed a CH-π interaction with Leu 28 and Ile 40 on the β3 and β4 strands, respectively (Fig. 2 C), while Val 84, Val 86, and Val 88, a group of valine residues on the β8 strand, formed hydrophobic interactions with Ile 9, Val 100, and Phe 55 on the β1-β2 loop, β9 strand, and β6 strand, respectively (Fig. 2 D). On the tail side of the Ig-like domain, Tyr 75 formed a π-π interaction with the C-terminal Trp 119 and Trp 119 formed a CH-π interaction with Val 121 on the C-terminal flexible linker, as discussed below. Thus, hydrophobic interactions extended from the inner surface to the proline-rich tail region. This strong hydrophobic network stabilizes the FctB structure by functioning like adhesive tape to hold β-sandwiches together. Unlike in FctA, there was no isopeptide bond in FctB3, as reported in a study of the structure of FctB from a serotype T9 strain (FctB9) (PDB ID: 3klq) (Linke et al. 2010 ), which also showed that the FctB9 structure was similar to the structure of the FctA N-terminal domain. In the present study, structures of FctB3 and FctA3 (PDB ID: 6BBW) were found to be superposed, based on the backbone Cα atoms. FctA3 Lys 15 and Asn 152, responsible for isopeptide bond formation, correspond to Asn 13 and Pro 17 of FctB3, respectively, whereas Glu 101, a catalytic residue of FctA3 for the formation, was found to be merged with Gln 67 of FctB3 (Fig. 3 A and B). FctB3 Pro 17 formed CH-π interactions with side chains of Leu 15, Phe 24, and Asp 78. Furthermore, a hydrogen bond between Asn 13 and Gln 67 was noted. These interactions together with above-mentioned hydrophobic interactions likely compensate for the absence of an isopeptide bond to stabilize FctB structure. Furthermore, findings showing that Asn 13 and Gln 67 are the only hydrophilic residues on the inner surface of FctB3 and replacement of Pro 17 of FctB3 by a glutamine residue in FctB1 (Fig. S2) suggest a relic of an isopeptide bond in FctB. Comparison of FctB3 and FctB9 structures Previously, FctB9 structure was solved at 1.9 Å (Linke et al. 2010 ) (Fig. S1 ). The sequence identity of FctB3 and FctB9 is 97.9%, and a structural comparison of FctA and FctB9 showed root-mean-square deviation (RMSD) values less than 1.5Å. Fitting of the main chain atoms revealed that the direction of the proline-rich tail was quite different, and RMSD value for Cα fit between FctB3 and FctB9 was 0.397 Å. The RMSDs of the Ig-like domain and the proline-rich tail region were 0.500Å and 0.460Å, respectively (Fig. 4 A). The intramolecular hydrophobic interaction network observed in FctB3 was also present in FctB9 and hydrophobic residues were conserved in FctB9 (Fig. 4 B-E). FctB9 Lys 110 links to the Thr residue in the FctA LPXTG-like motif via isopeptide bond formation, and Lys 110 is positioned in the omega loop (Linke et al. 2010 ). Therefore, the omega loop structure has been noted as important for recognition of the LPXTG-like motif of FctA (Linke et al. 2010 ). A comparison of the omega loop conformation between FctB3 and FctB9 showed differences in the main chain from Arg 104 to Glu 109, though the location of Cα of Lys 110 was well overlapped (Fig. 5 A-D). This is because the nitrogen atom and carbonyl group of the Ser 111 main chain form hydrogen bonds to the carbonyl group of the Ser 102 main chain and the hydroxyl group of the Ser 102 side chain, respectively (Fig. 5 A-C). Furthermore, Ser 102 and Ser 111 were conserved among FctB of various serotype strains (Fig. 5 D, Fig. S2). The hydrogen bond network in the omega loop, including the hydrogen bond between the nitrogen atom in the Arg 104 main chain and the oxygen atom in Val 81 main chain, was also noted to be conserved. Arg 104 was located on the N-terminus of the omega loop and this hydrogen bond may stabilize the loop structure by fixing the omega loop to the Ig-like domain. Another conserved hydrogen bond was formed between the hydroxyl group of the Ser 111 side chain, oxygen atom of the Ala 112 main chain, and nitrogen atom of the Thr 114 main chain. This hydrogen bond network likely stabilizes the C terminus of the omega loop in conjunction with the hydrogen bond between Ser 102 and Ser 111. Most of conserved hydrogen bonds in the omega loop were formed between main chains, thus this network is considered to be less susceptible to the surrounding environment. Although the amino acid sequences of the omega loop of FctB3 and FctB9 are identical, the structure was found to be changed. This is likely due to the hydrogen bond network formed by the Arg 103 and Arg 104 side chains. The side chain of FctB3 Arg 104 was found to be interacting with Asp 107 (Fig. 5 A), whereas that of FctB9 Arg 104 was interacting with Ser 111 (Fig. 5 B). Furthermore, the hydrogen bond interaction of the Arg 103 side chain with Glu 108 noted in FctB9 (Fig. 5 BC) was not observed in FctB3 (Fig. 5 A). Based on the high proportions of acidic and basic amino acid residues in the constituent of the omega loop, large amounts of residues are capable of forming hydrogen bonds between side chains, and switching of the hydrogen bond network by Arg 103 and Arg 104 may occur. The switching mode of the network without changing the Lys 110 position might be advantageous for linkage to FctA and subsequent anchoring of assembled pili to the cell wall. On the other hand, the proline-rich tails of FctB3 and FctB9 showed protrusions in different directions, caused by distinct hydrogen bond networks formed by Trp 119 (FctB3) and Arg 119 (FctB9) (Fig. 6 A-C). Trp 119 was conserved in FctB1, whereas Arg 119 was not conserved in FctB from other serotypes, including M5, M9, M12, M18, and M49. Trp 119 of FctB3 formed a CH-π interaction with Arg 17, Tyr 75, and Val 121, but no hydrogen bond with the main chains was formed (Fig. 6 A). Nevertheless, the side chain of Arg 119 in FctB9 formed hydrogen bonds with the main chains of Val 121 and Lys 122 in the linker region, as well as the CH-π interaction with Tyr 75 (Fig. 6 B). Val 121 and Lys 122 of FctB9 were found adjacent to the proline-rich tail. Hence, it is likely that either Arg 119 or Trp 119 is a determinant for the direction of the proline-rich tail. In support of this, another structure in the asymmetric unit of FctB9 showed that a disorder of the Arg 119 side chain caused a conformational change in the proline-rich tail, and Val 121 and Lys 122 were disordered (Fig. 6 C). Formation of FctB/FctA and FctB/Cpa heterodimer complex involves SipA FctB3 and the FctA1 N-terminal domain also showed high homology, as previously noted for FctB9 (Linke et al. 2010 ). In the crystal packing of FctA1, a state mimicking the linkage has been reported (Kang et al, 2007 ) (Fig. 7 A). The C-terminus sequence EPT, of which Thr is linked to the neighboring pilin, was not found contained in the reported structure, whereas Lys 161 responsible for interaction with other subunits, and the groove for binding to LPXTG-like motifs composed of β7, β8, and β9, were suggested to be important for the linkage (Kang et al, 2007 ) (Fig. 7 A). We modeled the FctA/FctB complex by superposition based on this structure (Fig. 7 B). Comparisons of FctA1/ FctA1 and FctA1/ FctB3 models showed structural differences in the contact area, including the groove. When the FctA1 N-terminal domain and FctB3 structures were superposed, the positions of FctB3 Lys 110 and FctA Lys 161 were shown to be overlapped (Fig. 7 C). An in vivo experiment was conducted to determine whether Lys 110 of FctB mediates formation of an isopeptide bond to growing FCT type 3 pili. Culture supernatants obtained from ∆ fctB expressing wild-type or mutant FctB were subjected to immunoblot analyses with anti-FctA and anti-FctB (Fig. 7 D, E). In addition to Lys 110, the conserved lysine residues Lys 47, Lys 116, Lys 118, and Lys 122 were chosen to be replaced with alanine residues. In the culture supernatant obtained from ∆ fctB expressing K110A mutant FctB, prominent HLBs were detected with anti-FctA (Fig. 7 D), and HLBs detected with anti-FctB completely disappeared (Fig. 7 E), confirming the linkage of FctB to growing FCT type 3 pili via Lys 110, as previously reported for FctB9 (Linke et al. 2010 ). The β67 loop, as well as β8 and β9 of FctB are most likely responsible for recognition of FctA, since those regions were found to be well overlapped with the FctA1 β56 loop, β8, and β9 (Fig. 7 C). The FctA1 β56 loop, β8, and β9, together with the β23 loop constitute the N-terminal tip of the Ig-like domain responsible for recognition of Cpa and FctA. Structural differences were observed mainly between the FctB β34 loop and corresponding FctA β23 loop, indicating that a difference in loop structure affects binding to other subunits. The shorter loop of FctB was considered extremely unfavorable for binding to other pilus subunits, as compared with the corresponding region of FctA, since the contact area for the binding is smaller and the groove of the binding pocket for LPXTG-like motifs is shallower. Therefore, as compared with FctA, FctB is not favorable in regard to association with other pilus subunits and, as in the case of FctA, it is suggested that the association may require auxiliary proteins, such as the putative chaperone SipA. Superposition of the C-terminal domains of FctA and Cpa showed a high homology (RMSD: 1.395Å). Although the C terminus sequence of VVPT in Cpa, in which Thr is linked to FctA, was also not contained in the reported Cpa structure, the binding mode was predicted based on the position of Lys161 in FctA, and the location of the C terminus of Cpa, and groove in FctA domain 1 (Fig. 8 A). In addition, the structural similarity between the C-terminal domains of Cpa and FctA allowed modeling of the Cpa/FctB complex (Fig. 8 B), which was performed using an FctA/FctB complex model. With this complex model, Lys 110 of FctB was shown to be close to the Cpa C-terminus sequence VVPT and that region could be located in the groove on the FctB surface, suggesting that FctB can be linked to Cpa (Fig. 8 B). In fact, a band potentially representing the Cpa-FctB complex was detected in immunoblot data (Fig. 1 ). To clarify whether Lys 110 is linked to Cpa and FctA, wild type FctB, K47A, or a K110A mutant was expressed in either the double-deletion mutant of cpa and fctB (∆ cpa ∆ fctB ) (Fig. 8 C) or that of fctA and fctB (∆ fctA ∆ fctB ) (Fig. 8 D). In the CWF of ∆ cpa ∆ fctB expressing wild-type fctB (Comp) or K47A fctB , a ~ 46 kDa band corresponding to the FctA/FctB complex was detected, whereas it was not detectable in empty vector-transformed ∆ cpa ∆ fctB (Mock) and ∆ cpa ∆ fctB expressing K110A (Fig. 8 C). Similarly, a ~ 100 kDa band reflecting the complex of Cpa and FctB was detected in the CWF of ∆ fctA ∆ fctB expressing either wild-type fctB (Comp) or K47A fctB , but not in that of ∆ fctA ∆ fctB expressing K136A fctB or empty vector-transformed control (Mock) (Fig. 8 D). When the cell wall sorting signal mutant of either cpa or fctA was expressed in the background of ∆ cpa ∆ fctA , no band of the complex was detected (data not shown). Therefore, Lys 110 of FctB is suggested to be linked to both FctA and Cpa. Finally, to examine the requirement of SipA in formation of heterodimers, i.e., FctA/FctB and Cpa/FctB complexes, an sipA mutation was introduced into both ∆ cpa ∆ fctB and ∆ fctA ∆ fctB , and then complemented with wild-type fctB (∆ cpa ∆ fctB ∆ sipA::fctB and ∆ fctA ∆ fctB ∆ sipA::fctB ). As a control, revertant strains (∆ cpa ∆ fctB Wr sipA::fctB and ∆ fctA ∆ fctB Wr sipA::fctB ), which arose during the mutant construction were also utilized. In the background of ∆ cpa (Fig. 8 E, lane 1, Wr sipA ) and ∆ fctA (Fig. 8 F, lane 1, Wr sipA ), FctA-FctB and Cpa-FctB complexes, respectively, were detected. Notably, those complexes were not clearly detected following the sipA deletion (Fig. 8 E and F, lane 2, ∆ sipA ). These findings indicate that SipA is required for the linkage between Cpa and FctB, as well as between FctA and FctB via FctB Lys 110. Conclusion In the present study, the FctB3 structure was determined. Comparisons with the previously reported FctB9 structure showed that the main differences are the omega loop structure and direction of the proline-rich tail. The difference in the omega loop structure is likely caused by the distinct hydrogen bond network, which also indicates that network switching without changing the position of the lysine residue responsible for the linkage of pilin via an isopeptide bond may be advantageous for FctA polymerization and subsequent anchoring of pili to the cell wall. The direction of the proline-rich tail is potentially caused by a single residue located at the root of the tail. Furthermore, it is notable that the FctB structure is stabilized by intramolecular large hydrophobic interactions instead of an isopeptide bond. It is proposed that structural features that allow polymerization of FctA to occur more efficiently than incorporation of FctB to halt FctA polymerization are present. In addition, the findings show that formation of the heterodimer complex of FctB with FctA or Cpa is mediated by SipA. Thus, the present study provides an alternative structure for FctB as well as insight into the interactions between pilins of strains possessing FCT genomic region type 3. Declarations Acknowledgments We thank T. Sekizaki (Kyoto University) and D. Takamatsu (National Institute of Animal Health) for providing the pSET4s plasmid. pAT18 was kindly provided by P. Trieu-Cuot (Institut Pasteur). This work was performed using a synchrotron beamline BL44XU at SPring-8 under the Collaborative Research Program of Institute for Protein Research, Osaka University. Preliminary diffraction data were collected at the Osaka University beamline BL44XU at SPring-8 (Harima, Japan) (Proposal No. 2017A6732, 2017B6732, and 2018A6832). Also, this work was performed under the approval of the Photon Factory Program Advisory Committee (Proposal No. 2016G609, 2017G169). Funding This work was supported by JSPS KAKENHI Grants‐in‐Aid for Scientific Research (Grant Nos. 19K22715, 19H03825, 22H03262, 22H03263). Author Contribution KT and MN contributed to the study conception and design. Material preparation, data collection, and analysis were performed by KT, MS, TS, and MN. The first draft of the manuscript was written by KT and MN, and all authors contributed to revision of the manuscript. All authors approved the final manuscript. Data Availability The protein structure data are available in the wwPDB repository under accession number 8K6T. Declarations The authors declare no conflict of interest. References Abbot EL, Smith WD, Siou GP, Chiriboga C, Smith RJ, Wilson JA, Hirst BH, Kehoe MA (2007) Pili mediate specific adhesion of Streptococcus pyogenes to human tonsil and skin. 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J Bacteriol 190:527–535. https://doi.org/10.1128/JB.01520-07 Tables Table 1 is available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files Table1.docx SupplementalData.pdf Cite Share Download PDF Status: Published Journal Publication published 23 Nov, 2023 Read the published version in Archives of Microbiology → Version 1 posted Editorial decision: Major revision 17 Aug, 2023 Reviews received at journal 15 Aug, 2023 Reviewers agreed at journal 06 Aug, 2023 Reviewers agreed at journal 06 Aug, 2023 Reviewers invited by journal 06 Aug, 2023 Editor assigned by journal 06 Aug, 2023 Submission checks completed at journal 04 Aug, 2023 First submitted to journal 04 Aug, 2023 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3233333","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":224188385,"identity":"760f4fec-8327-4bd8-8815-5681e795e050","order_by":0,"name":"Katsuki Takebe","email":"","orcid":"","institution":"Osaka University Graduate School of Dentistry","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Katsuki","middleName":"","lastName":"Takebe","suffix":""},{"id":224188386,"identity":"2ea851b0-24c9-483f-bee1-88ed010e2b8c","order_by":1,"name":"Mamoru Suzuki","email":"","orcid":"","institution":"Osaka University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Mamoru","middleName":"","lastName":"Suzuki","suffix":""},{"id":224188387,"identity":"ec0bd3ce-9e39-46b9-9a88-8fe139ef24b0","order_by":2,"name":"Takeshi Sangawa","email":"","orcid":"","institution":"Osaka University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Takeshi","middleName":"","lastName":"Sangawa","suffix":""},{"id":224188388,"identity":"a63c0ee8-9877-41d8-ab54-deb1f032c738","order_by":3,"name":"Bernd Kreikemeyer","email":"","orcid":"","institution":"University Medicine Rostock","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Bernd","middleName":"","lastName":"Kreikemeyer","suffix":""},{"id":224188389,"identity":"0acc254f-fd7a-4243-af77-6accdfb39e0d","order_by":4,"name":"Masaya Yamaguchi","email":"","orcid":"","institution":"Osaka University Graduate School of Dentistry","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Masaya","middleName":"","lastName":"Yamaguchi","suffix":""},{"id":224188390,"identity":"0256feba-1e1e-4828-8c46-983222c942a7","order_by":5,"name":"Narikazu Uzawa","email":"","orcid":"","institution":"Osaka University Graduate School of Dentistry","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Narikazu","middleName":"","lastName":"Uzawa","suffix":""},{"id":224188391,"identity":"82b1af2e-bcee-48c8-a79a-d4fcd7a9ceb8","order_by":6,"name":"Tomoko Sumitomo","email":"","orcid":"","institution":"Tokushima University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Tomoko","middleName":"","lastName":"Sumitomo","suffix":""},{"id":224188392,"identity":"62e51cd5-67bc-4f34-9500-05b0d79a5769","order_by":7,"name":"Shigetada Kawabata","email":"","orcid":"","institution":"Osaka University Graduate School of Dentistry","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Shigetada","middleName":"","lastName":"Kawabata","suffix":""},{"id":224188393,"identity":"1c2856a0-f537-462a-aa41-2cb1e0f06198","order_by":8,"name":"Masanobu Nakata","email":"data:image/png;base64,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","orcid":"","institution":"Kagoshima University Graduate School of Medical and Dental Sciences","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Masanobu","middleName":"","lastName":"Nakata","suffix":""}],"badges":[],"createdAt":"2023-08-04 05:29:13","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3233333/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3233333/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s00203-023-03727-1","type":"published","date":"2023-11-23T15:01:48+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":41378752,"identity":"2110a67c-483f-4a48-ad5a-8d11d166a530","added_by":"auto","created_at":"2023-08-10 15:08:13","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":412151,"visible":true,"origin":"","legend":"\u003cp\u003eImmunoblot analysis of FCT region type 3 pilus proteins in \u003cem\u003eS. pyogenes\u003c/em\u003e 591 and derivative strains. (A) Genetic map of pilus operon. Gene designations are presented above each arrow. Black arrow, genes encoding pilus proteins Cpa, FctA, and FctB; dark gray arrow, gene encoding pilus-specific type B transpeptidase SrtC2; light gray arrow, gene encoding putative chaperone SipA. (B-E) Results of immunoblot analysis of cell wall (B-D) and culture supernatant (E) fractions of wild type and its derivative strains. Bands in the lower panel (D) represent the monomeric form of FctB. M, protein size marker. \u003csup\u003e*\u003c/sup\u003e, Bands potentially representing the FctA and FctB complex. \u003csup\u003e#\u003c/sup\u003e, Bands potentially representing the Cpa and FctB complex.\u003c/p\u003e","description":"","filename":"Figures1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3233333/v1/eeb999ffa6e2673fcbd3b48e.jpg"},{"id":41378753,"identity":"f93c8e0c-a36d-4440-b140-0480b9dfc7d7","added_by":"auto","created_at":"2023-08-10 15:08:13","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":412984,"visible":true,"origin":"","legend":"\u003cp\u003eOverall structure of FctB and its intermolecular hydrophobicity network. (A) Crystal structure of FctB3. FctB3 was found to contain an Ig-like domain composed of 11 β sheets and a proline rich tail. An overhanging omega loop was located between the β9 and β10 sheets. (B-D) Intermolecular hydrophobicity network of FctB3. The responsible residues are shown by green sticks, and yellow dotted lines denote CH-π and π-π interactions.\u003c/p\u003e","description":"","filename":"Figures2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3233333/v1/99aa7ab53813080fea6a3bfb.jpg"},{"id":41380732,"identity":"8d3283e6-1c74-43e6-81c6-8de1a1ab1d64","added_by":"auto","created_at":"2023-08-10 15:24:13","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":318996,"visible":true,"origin":"","legend":"\u003cp\u003eIntramolecular isopeptide bond of FctA3 and corresponding region of FctB3. (A) In FctB3, Pro 17 was found to form hydrophobic interactions with Leu15, Phe24 and Asp 78. (B) In FctA3, Lys 15 and Asn152 form an isopeptide bond.\u003c/p\u003e","description":"","filename":"Figures3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3233333/v1/d8fe911e93e563c9d45b52b0.jpg"},{"id":41378758,"identity":"24355c94-aad4-41eb-ab9a-e5b34dc924dd","added_by":"auto","created_at":"2023-08-10 15:08:13","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":630737,"visible":true,"origin":"","legend":"\u003cp\u003eSuperimposition of crystal structures of FctB3 and FctB9. (A) Superimpositions of overall structures of FctB3 (green) and FctB9 (two chains in cyan and yellow). The major difference is the direction of the proline rich tail. (B-E) Intermolecular hydrophobicity network compared between FctB3 (green) and FctB9 (yellow). Amino acid residues responsible for hydrophobicity were found to be highly conserved, while a major difference was observed in the C-terminal region of the Ig-like domain.\u003c/p\u003e","description":"","filename":"Figures4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3233333/v1/581f263b3486d87d4404854a.jpg"},{"id":41379867,"identity":"20915720-3709-495a-92cc-f5678ce1ab1f","added_by":"auto","created_at":"2023-08-10 15:16:13","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":594039,"visible":true,"origin":"","legend":"\u003cp\u003eHydrogen bond network in omega loop of FctB. Yellow dotted lines denote hydrogen bond interactions in (A) FctB3, (B) FctB9 chain A, and (C) FctB9 chain B. (D) Merged image.\u003c/p\u003e","description":"","filename":"Figures5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3233333/v1/e5c1cb5131a4951e5aabee83.jpg"},{"id":41379868,"identity":"4cad9309-f173-4977-b0ca-dfdc645404b7","added_by":"auto","created_at":"2023-08-10 15:16:13","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":350373,"visible":true,"origin":"","legend":"\u003cp\u003eInteractions of base residues of proline-rich tail in FctB3 and FctB9. Yellow dotted lines denote CH-π or π-π interactions with surrounding residues by (A) Trp 119 in FctB3, (B) Arg 119 in FctB9 chain A, or (C) Arg 119 in FctB9 chain B.\u003c/p\u003e","description":"","filename":"Figures6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3233333/v1/179f68049d9fe7b6248f32ee.jpg"},{"id":41379870,"identity":"849b55c3-dbde-4bc5-8d4e-53cf5ed7bfea","added_by":"auto","created_at":"2023-08-10 15:16:13","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":527182,"visible":true,"origin":"","legend":"\u003cp\u003eHomology of FctB3 and FctA1 structures and deduced interactions of FctA and FctB. (A and B) Deduced intermolecular interactions of (A) FctA1/FctA1 and (B) FctA1/FctB3. (C) Superimposition of FctB3 (green) and FctA1 (red) structures. Region encircled in yellow indicates pocket that is responsible for recognition and linkage of LPXTG-like motif of pilin. Region encircled in blue indicates omega loop, which is enlarged in the lower panel. Relative positions of Lys 110 of FctB3 and Lys 161 of FctA1 are shown. The PDB IDs of FctA1 and FctB3 are 3B2M and 8K6T, respectively. (D and E) Culture supernatant fractions obtained from \u003cem\u003efctB\u003c/em\u003e deletion mutant (∆\u003cem\u003efctB\u003c/em\u003e) expressing wild-type FctB (Comp), or mutated FctB, where the selected Lys residue was replaced with an Ala residue (K47A, K110A, K116A, K118A, K122A), then subjected to immunoblot analyses with (D) anti-FctA or (E) anti-FctB.\u003c/p\u003e","description":"","filename":"Figures7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3233333/v1/1145cffa8e835e7033366792.jpg"},{"id":41378754,"identity":"9993bb22-7210-4ba5-8b4d-7611a305066a","added_by":"auto","created_at":"2023-08-10 15:08:13","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":612986,"visible":true,"origin":"","legend":"\u003cp\u003eDeduced structures of Cpa1/FctA1 and FctA1/FctB3, and heterodimer formation of Cpa/FctB and FctA/FctB mediated by SipA. (A and B) Deduced intermolecular interactions of Cpa1/FctA1 (A) and Cpa1/FctB3 (B) are shown. The PDB IDs of Cpa1, FctA1, and FctB3 are 2XID, 3B2M, 8K6T, respectively. The VVPT sequence (blue line) in the tail region of Cpa1 is not contained in the crystal structure. (C and D) Cell wall fractions were prepared from a double deletion mutant of \u003cem\u003ecpa \u003c/em\u003eand \u003cem\u003efctB\u003c/em\u003e (∆\u003cem\u003ecpa\u003c/em\u003e∆\u003cem\u003efctB\u003c/em\u003e) (C) or \u003cem\u003efctA \u003c/em\u003eand \u003cem\u003efctB\u003c/em\u003e (∆\u003cem\u003efctA\u003c/em\u003e∆\u003cem\u003efctB\u003c/em\u003e) (D) transformed with an empty shuttle vector (Mock), or expression vector of wild-type FctB\u003cem\u003e \u003c/em\u003e(Comp) or mutant FctB, where Lys 47 or Lys 110 was replaced with Ala (K47A and K110A). Pilus proteins were detected by immunoblot analyses using mouse antisera. Bands representing heterodimers of FctA and FctB (C), and those of Cpa and FctB (D) are indicated by arrowheads. M, protein size marker. (E) Total proteins in cell wall fraction extracted from a triple deletion mutant of \u003cem\u003ecpa\u003c/em\u003e, \u003cem\u003efctB\u003c/em\u003e, and \u003cem\u003esipA\u003c/em\u003e (∆\u003cem\u003ecpa\u003c/em\u003e∆\u003cem\u003efctB\u003c/em\u003e∆\u003cem\u003esipA\u003c/em\u003e), or double deletion mutant of \u003cem\u003ecpa\u003c/em\u003e and \u003cem\u003efctB \u003c/em\u003e(∆\u003cem\u003ecpa\u003c/em\u003e∆\u003cem\u003efctB \u003c/em\u003eWr\u003cem\u003esipA\u003c/em\u003e) were transformed with the wild-type \u003cem\u003efctB \u003c/em\u003eexpression vector, then immunoblotted with anti-FctA (left panel) or anti-FctB antisera (right panel). Bands representing the heterodimers of FctA and FctB are indicated by arrowheads. (F) Total proteins in cell wall fraction extracted from a triple deletion mutant of \u003cem\u003efctA\u003c/em\u003e, \u003cem\u003efctB\u003c/em\u003e, and \u003cem\u003esipA\u003c/em\u003e (∆\u003cem\u003efctA\u003c/em\u003e∆\u003cem\u003efctB\u003c/em\u003e∆\u003cem\u003esipA\u003c/em\u003e) or a double deletion mutant of \u003cem\u003efctA\u003c/em\u003e and \u003cem\u003efctB \u003c/em\u003e(∆\u003cem\u003efctA\u003c/em\u003e∆\u003cem\u003efctB \u003c/em\u003eWr\u003cem\u003esipA\u003c/em\u003e) transformed with the \u003cem\u003efctB \u003c/em\u003eexpression vector, were immunoblotted with anti-Cpa (left panel) or anti-FctB (right panel) antiserum. Bands representing the heterodimer of Cpa and FctB are indicated by arrowheads.\u003c/p\u003e","description":"","filename":"Figures8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3233333/v1/7cb21a191aa0b2ed2ab727e5.jpg"},{"id":47146849,"identity":"be0ef88c-cdc7-4ae0-b01d-2daf57754807","added_by":"auto","created_at":"2023-11-27 15:09:27","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1186304,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3233333/v1/8bab3902-1aed-4fd5-b844-f31f59d53453.pdf"},{"id":41378751,"identity":"e980b1e2-a4c1-41a4-ac2e-ee271d5ce1f0","added_by":"auto","created_at":"2023-08-10 15:08:13","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":16876,"visible":true,"origin":"","legend":"","description":"","filename":"Table1.docx","url":"https://assets-eu.researchsquare.com/files/rs-3233333/v1/7fe6c41a48e8ba6ace40a646.docx"},{"id":41378761,"identity":"1da19565-d204-4789-b7d7-a1b363c2a848","added_by":"auto","created_at":"2023-08-10 15:08:13","extension":"pdf","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":1091164,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementalData.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3233333/v1/7aef72723b8d913876d01e97.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Analysis of FctB3 crystal structure and insight into its structural stabilization and pilin linkage mechanisms","fulltext":[{"header":"Introduction","content":"\u003cp\u003e \u003cem\u003eStreptococcus pyogenes\u003c/em\u003e, also referred to as group A \u003cem\u003eStreptococcus\u003c/em\u003e (GAS), is a major pathogenic Gram-positive bacterium, with humans the only biological host (Cunningham et al. 2008). This bacterium causes localized suppurative diseases, such as pharyngitis in the upper respiratory tract and pyoderma in skin, and also immunological sequelae, including acute rheumatic fever and acute glomerulonephritis, as well as lethal streptococcal toxic shock syndrome (Stevens et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e1989\u003c/span\u003e; Walker et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Annually worldwide, it is estimated that there are more than 616\u0026nbsp;million pharyngitis and 111\u0026nbsp;million prevalent pyoderma cases (Carapetis et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Bowen, et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), while GAS-related diseases cause more than 500 thousand deaths each year (Carapetis et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). Thus, deeper understanding of the molecular mechanisms related to pathogenicity is required to develop effective treatments and prevention measures.\u003c/p\u003e \u003cp\u003eGAS is mainly classified based on M serotyping, which is determined according to the antigenicity of M protein. Currently, based on genotyping of the \u003cem\u003eemm\u003c/em\u003e gene encoding M protein, there are more than 240 known genotypes (Sanderson-Smith et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). An alternative typing scheme is T serotyping (Griffith \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e1934\u003c/span\u003e), which is based on the antigenicity of the trypsin-resistant antigen (T antigen), the gene of which is located within the genomic region, and referred to as the FCT (\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003ef\u003c/span\u003eibronectin-binding protein, \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003ec\u003c/span\u003eollagen-binding protein, and \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003et\u003c/span\u003erypsin-resistant antigen) region, encoding a variety of adhesins. The major T antigen is the backbone pilin and its antigenicity is responsible for T typing (Mora et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Falugi et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). Based on the heterogeneity of the gene contents, the FCT genomic region has been classified to nine types (FCT-1-9). GAS pili function as an adhesin to tonsil tissue and keratinocytes, and promote biofilm formation (Abbot et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Manetti et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2007\u003c/span\u003e), thus have important roles in the infection process.\u003c/p\u003e \u003cp\u003eGAS pili consist of two or three types of pilins (Nakata et al. 2021). Pili of strains possessing an FCT type 1 region, such as serotype M6 strains, are composed of the tip pilin FctX and backbone pilin T6 (Nakata et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Kimura KR et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). On the other hand, pili of strains possessing an FCT type 2\u0026ndash;4 region consist of three types, including the tip pilin Cpa, backbone pilin FctA, and base pilin FctB (Fig.\u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Linkage between pilins and anchoring to the free amino group of peptidoglycan are mediated by a pilus-specific transpeptidase and the house-keeping transpeptidase SrtA, respectively. The pilus-specific transpeptidase recognizes the C-terminal LPXTG or LPXTG-like motif of pilin, and connect each pilin by forming an intermolecular isopeptide bond (Hendrickx et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). For example, LPXTG-like motifs of Cpa, FctA, and FctB in M1 strains are VPPTG, EVPTG, and LPLAG, respectively. In strains possessing an FCT type 2\u0026ndash;4 region, the deduced chaperone SipA is also considered crucial for pilus assembly, though details regarding its exact role and binding partners remain unclear (Z\u0026auml;hner et al. 2008; Young et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2014b\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eCrystal structures have been reported for backbone pilins, including T6 (Young et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2014a\u003c/span\u003e) and FctA (Kang et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Young et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), the tip pilin Cpa (Pointon et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2010\u003c/span\u003e), and the base pilin FctB (Linke et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2010\u003c/span\u003e) (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Based on results of the present study, we report a newly determined crystal structure of FctB from a serotype M3 strain possessing an FCT type 3 region (FctB3) and discuss its surface properties, hydrogen bond networks, and other characteristics. In addition, this new structure was compared to the previously reported FctB9 (FctB from a serotype T9 strain) structure (Linke et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2010\u003c/span\u003e), as well as structures of FctA and Cpa. The results show differences in the structural features of FctB and FctA that may efficiently promote FctA polymerization as compared to FctB incorporation by the FctA polymer. Furthermore, formation of the heterodimer complex of FctB with FctA or Cpa, which is mediated by SipA, is demonstrated.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStrains and plasmids\u003c/h2\u003e \u003cp\u003eA \u003cem\u003eStreptococcus pyogenes\u003c/em\u003e serotype M49 strain 591 harboring an FCT type 3 region and isogenic mutant strains were used for immunoblot analyses. The \u003cem\u003eEscherichia coli\u003c/em\u003e strain XL10-gold (Stratagene) served as a host for plasmids and \u003cem\u003eE. coli\u003c/em\u003e strain NiCo21 (DE3) (New England Biolabs) transformed with pREP4 (Qiagen) was used for expression of recombinant FctB3. All \u003cem\u003eE. coli\u003c/em\u003e strains were cultured in Luria-Bertani (LB) medium (Nacalai Tesque) at 37\u0026deg;C with constant agitation. The \u003cem\u003eS. pyogenes\u003c/em\u003e strains were cultured in Todd-Hewitt broth (Becton Dickinson) supplemented with 0.2% yeast extract (Becton Dickinson) (THY medium) at 25\u0026deg;C in an ambient atmosphere (Nakata et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). When appropriate, antibiotics were added to the media at the following concentrations: ampicillin, 100 \u0026micro;g/ml for \u003cem\u003eE. coli\u003c/em\u003e; spectinomycin (FUJIFILM Wako Pure Chemical), 100 \u0026micro;g/ml for \u003cem\u003eE. coli\u003c/em\u003e and 100 \u0026micro;g/ml for \u003cem\u003eS. pyogenes\u003c/em\u003e; erythromycin (Sigma Aldrich), 150 \u0026micro;g/ml for \u003cem\u003eE. coli\u003c/em\u003e and 1 \u0026micro;g/ml for \u003cem\u003eS. pyogenes\u003c/em\u003e. All strains used in this study are listed in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eConstruction of S. pyogenes mutant strains and alignment of FctB sequence\u003c/h2\u003e \u003cp\u003eGenomic DNA preparations from \u003cem\u003eS. pyogenes\u003c/em\u003e were conducted using a DNA extraction kit (Takara). Plasmid DNA was prepared from \u003cem\u003eE. coli\u003c/em\u003e overnight cultures with a plasmid purification kit (Macherey-Nagel). Transformation of \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eS. pyogenes\u003c/em\u003e was performed, as previously described (Nakata et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). All primers listed in Table S2 were designed using the reported genome sequences of strains SSI-1 (NC004606, Nakagawa et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2003\u003c/span\u003e) and 591 (NZ_CP077685.1, Patenge et al \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eConstruction of in-frame markerless deletion mutants was conducted using the temperature-sensitive shuttle vector pSET4s, as previously reported (Takamatsu et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2001\u003c/span\u003e, Nakata et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Mutated \u003cem\u003efctB\u003c/em\u003e was created using an overlapping PCR with primers listed in Table S2. The fragment was cloned into the shuttle vector pAT18-P\u003cem\u003egyrA\u003c/em\u003e (Nakata et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Introduction of the mutation was confirmed by DNA sequencing. Amino acid sequences of FctB from serotypes M1, M3, M5, M18, and M49 were aligned using ClsutalW software (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://clustalw.ddbj.nig.ac.jp\u003c/span\u003e\u003cspan address=\"http://clustalw.ddbj.nig.ac.jp\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and visualized with CLC Main workbench software (CLC bio).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eCell fractionization and immunoblot analysis\u003c/h2\u003e \u003cp\u003eExtraction of cell wall and culture supernatant fractions of \u003cem\u003eS. pyogenes\u003c/em\u003e strains was conducted, as previously reported (Nakata et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). For preparation of cell wall fractions, \u003cem\u003eS. pyogenes\u003c/em\u003e cells grown to the late exponential phase were washed with PBS and suspended in protoplast buffer (0.1 M KPO\u003csub\u003e4\u003c/sub\u003e, pH 6.2, 40% sucrose, 10 mM MgCl\u003csub\u003e2\u003c/sub\u003e) containing complete EDTA-free protease inhibitors (Roche) and 250 \u0026micro;g/ml of \u003cem\u003eN\u003c/em\u003e-acetylmuramidase (Seikagaku Biobusiness). The suspension was incubated at 37˚C for 3 h, followed by centrifugation at 4˚C and 16,000 \u0026times; \u003cem\u003eg\u003c/em\u003e for 10 min, with the resultant supernatant used as a cell wall fraction. For preparation of culture supernatant fractions, the \u003cem\u003eS. pyogenes\u003c/em\u003e overnight culture was centrifuged at 4˚C and 16,000 \u0026times; \u003cem\u003eg\u003c/em\u003e for 20 min and proteins in the supernatant were precipitated overnight at 4˚C with 10% trichloroacetic acid. After centrifugation at 20,000 \u0026times; \u003cem\u003eg\u003c/em\u003e and 4˚C for 10 min, the protein pellet was washed three times with cold ethanol, resuspended in 1 M Tris buffer (pH 8.0), and used as the culture supernatant fraction.\u003c/p\u003e \u003cp\u003eUsing a 5\u0026ndash;12% acrylamide gel (FUJIFILM Wako Pure Chemical), proteins in those fractions were separated by sodium dodecyl sulfatepoly-acrylamide gel electrophoresis, then blotted onto a polyvinylidene difluoride membrane. The membrane was blocked overnight at 4˚C with a solution containing a casein-based blocking reagent (Megmilk Snow Brand) and then incubated at room temperature (RT) for 2 h with mouse anti-pilin antiserum diluted 1:2000 in Tris-buffered saline (TBS) containing 0.2% Tween 20 (TBST). Following washing steps, the membrane was incubated at RT for 2 h with a horseradish peroxidase-conjugated anti-mouse IgG antibody (Cell Signaling Technology). Finally, the membrane was washed three times with TBST and developed with Pierce western blotting substrate (Thermo Scientific). Signals were detected using X-ray film (FUJIFILM).\u003c/p\u003e \u003cp\u003eMouse antiserum was prepared by immunizing five-week-old female BALB/c mice (Japan SLC) with recombinant pilus proteins and TiterMax Gold adjuvant (CytRx), as previously described (Kubota et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003ePurification of recombinant FctB3\u003c/h2\u003e \u003cp\u003eThe DNA fragment encoding FctB without the putative signal peptide and cell-wall sorting signal was PCR-amplified using genomic DNA of the serotype M3 strain SSI-1 and cloned into pQE30 (Qiagen). N-terminal His-tagged FctB was expressed in NiCo21 (DE3) transformed with pREP4 and purified by affinity chromatography using Ni-NTA agarose (QIAGEN). The eluted protein was further purified by gel filtration with Superdex 200 Increase 10/300GL (Cytiva) equilibrated with Tris-buffered saline (50 mM Tris, 150 mM NaCl, pH7.6). The protein concentration was measured based on 280 nm absorbance using Nanodrop 2000 (Thermo Fisher Scientific).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eCrystallization of FctB and X-ray diffraction data collection\u003c/h2\u003e \u003cp\u003eInitial screening was conducted with screening kits, including Crystal Screen HT, Index HT (Hampton Research), and Wizard Classic 1 \u0026amp; 2 HT96 (Rigaku) using a sitting-drop vapor diffusion method at 20˚C. Crystals were obtained within 1 week from solutions containing 0.1 M sodium acetate trihydrate (pH 4.5) and 2.0 M ammonium sulfate. Further optimization of the conditions was performed using hanging- and sitting-drop vapor diffusion methods with a mixture of 1 \u0026micro;l of protein solution (45.12 mg ml⁻\u0026sup1;) and 1 \u0026micro;l reservoir solution. Ranges of ammonium sulfate concentration (1.8\u0026ndash;2.4 M) and pH conditions (4.2\u0026ndash;4.6) were tested. The final crystallization condition is summarized in Table S3.\u003c/p\u003e \u003cp\u003eThe crystals were cryoprotected with reservoir solution containing 25% (vol/vol) glycerol and flash-cooled with liquid nitrogen. First, X-ray diffraction experiments were conducted in BL44XU (SPring-8, Sayo-cho, Hyogo Pref.) and the condition was confirmed. The final data were obtained in BL-1A (Photon Factory, Tsukuba city, Ibaraki Pref.), with a wavelength of 1.1 \u0026Aring; and 1˚ oscillation. The diffraction datasets were integrated using the XDS Package (Kabsch \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2010\u003c/span\u003e) and scaled using CCP4 suite (v. 6.4.0) (Winn et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2011\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eStructure solution and refinement\u003c/h2\u003e \u003cp\u003eThe structure of FctB3 was determined by molecular replacement using MOLREP (Vagin and Teplyakov \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). A previously reported FctB structure from a serotype T9 strain harboring FCT type 7 region (Linke et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2010\u003c/span\u003e, PDB ID:3klq, sequence identity 97.9%) was used as a model molecule. Structural refinement was carried out using REFMAC5 (Murshudov et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2011\u003c/span\u003e) and PHENIX package (Adams et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2010\u003c/span\u003e), while manual model building was performed using Coot (Emsley et al. 2004). The final model was deposited in wwPDB (PDB ID: 8K6T).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eEthics statement\u003c/h2\u003e \u003cp\u003eExperiments of mouse immunization for antiserum preparation were conducted according to a protocol approved by the Animal Care and Use Committee of Osaka University Graduate School of Dentistry (authorization number 29-014-0).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results and Discussion","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eCell wall anchoring of FCT region type 3 pili is mediated by FctB\u003c/h2\u003e \u003cp\u003eTo confirm the reported role of FctB in a strain possessing FCT region type 3, in-frame deletion mutants of each gene in the \u003cem\u003ecpa\u003c/em\u003e operon (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA) and a deletion mutant of the house-keeping transpeptidase gene \u003cem\u003esrtA\u003c/em\u003e were constructed in the background of a serotype M49 strain harboring the FCT type 3 region, then the effects of the mutation on pilus assembly and cell wall anchoring were examined by immunoblot analyses (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). In a wild-type cell wall fraction (CWF), high molecular weight ladder bands (HLBs) reflecting FctA polymerization were detected using antisera against the pilus proteins FctA, Cpa, and FctB (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB-D). In contrast, HLBs were not detected in the CWFs of all of the mutant strains, except for ∆\u003cem\u003efctB\u003c/em\u003e and ∆\u003cem\u003esrtA\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB-D). In concentrated supernatant fractions (SF), HLBs detected with anti-FctA were more prominent in ∆\u003cem\u003efctB\u003c/em\u003e and ∆\u003cem\u003esrtA\u003c/em\u003e, as compared with the wild type, which indicated that greater amounts of assembled pili were released into culture supernatants of ∆\u003cem\u003efctB\u003c/em\u003e and ∆\u003cem\u003esrtA\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). Therefore, in a strain harboring the FCT type 3 genomic region, FctB and SrtA also participate in cell wall anchoring of pili.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn the background of ∆\u003cem\u003ecpa\u003c/em\u003e, HLBs disappeared, while a band representing a monomeric form of FctA and a band sized\u0026thinsp;~\u0026thinsp;47 kDa were detected with anti-FctA (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). The ~\u0026thinsp;47 kDa band was also detected with anti-FctB (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD), indicating an FctA and FctB complex. Also, in the fraction of ∆\u003cem\u003efctA\u003c/em\u003e, in addition to a band representing a monomeric form of Cpa, a band sized approximately\u0026thinsp;~\u0026thinsp;100 kDa was detected with anti-Cpa (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC), while the same sized band was also detected with anti-FctB (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD), indicating formation of a Cpa and FctB complex. The level of detection of bands potentially representing FctA/FctB and Cpa/FctB complexes were decreased with \u003cem\u003esipA\u003c/em\u003e deletion. These findings suggest that FctB possesses an ability to bind to FctA and Cpa, with that binding mediated by SipA (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB-D).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eOverall structure of FctB3\u003c/h2\u003e \u003cp\u003eTo gain insight into the underlying mechanism of FCT type 3 pilus assembly and pilin interactions, the crystal structure of recombinant FctB from a serotype M3/T3 strain (hereafter referred to as FctB3) was determined and used as a representative of FCT type 3 pili. The structure is summarized in Table\u0026nbsp;1. The crystal belonged to the space group \u003cem\u003eP\u003c/em\u003e4\u003csub\u003e3\u003c/sub\u003e22 and the unit-cell parameters were as follows; a\u0026thinsp;=\u0026thinsp;b\u0026thinsp;=\u0026thinsp;44.47 \u0026Aring;, c\u0026thinsp;=\u0026thinsp;181.37 \u0026Aring;, and α, β and γ\u0026thinsp;=\u0026thinsp;90˚. Matthews coefficient (VM) was calculated to be 3.17 \u0026Aring;\u003csup\u003e3\u003c/sup\u003eDa\u003csup\u003e\u0026minus;1\u003c/sup\u003e and the solvent content was 61.14%, which indicated that a monomer molecule was contained in the asymmetric unit cell. The FctB3 crystal structure was solved at 2.8 \u0026Aring; resolution (R\u003csub\u003efactor\u003c/sub\u003e = 0.209, R\u003csub\u003efree\u003c/sub\u003e = 0.260). FctB3 was composed of 11 β-sheets folded into an immunoglobulin (Ig)-like fold and a proline-rich tail that formed a poly proline Ⅱ helix (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). The Ig-like domain was characterized by β-sandwiches, which were composed of five-stranded (β3, β4, β7, β8, β9) and four-stranded (β1, β2, β5, β6) β-sheets. In addition, another specific structure was an omega (Ω) loop, composed of the region from Arg 104 to Thr 114 in the β9-β10 loop. The inner surface of the molecules (i.e., region not exposed to external milieu) was mainly composed of hydrophobic and aromatic residues with the exception of Asn 13 and Gln 67 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB-D). For example, Phe 50 on the β56 loop formed a CH-π interaction with Leu 28 and Ile 40 on the β3 and β4 strands, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC), while Val 84, Val 86, and Val 88, a group of valine residues on the β8 strand, formed hydrophobic interactions with Ile 9, Val 100, and Phe 55 on the β1-β2 loop, β9 strand, and β6 strand, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). On the tail side of the Ig-like domain, Tyr 75 formed a π-π interaction with the C-terminal Trp 119 and Trp 119 formed a CH-π interaction with Val 121 on the C-terminal flexible linker, as discussed below. Thus, hydrophobic interactions extended from the inner surface to the proline-rich tail region. This strong hydrophobic network stabilizes the FctB structure by functioning like adhesive tape to hold β-sandwiches together.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eUnlike in FctA, there was no isopeptide bond in FctB3, as reported in a study of the structure of FctB from a serotype T9 strain (FctB9) (PDB ID: 3klq) (Linke et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2010\u003c/span\u003e), which also showed that the FctB9 structure was similar to the structure of the FctA N-terminal domain. In the present study, structures of FctB3 and FctA3 (PDB ID: 6BBW) were found to be superposed, based on the backbone Cα atoms. FctA3 Lys 15 and Asn 152, responsible for isopeptide bond formation, correspond to Asn 13 and Pro 17 of FctB3, respectively, whereas Glu 101, a catalytic residue of FctA3 for the formation, was found to be merged with Gln 67 of FctB3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA and B). FctB3 Pro 17 formed CH-π interactions with side chains of Leu 15, Phe 24, and Asp 78. Furthermore, a hydrogen bond between Asn 13 and Gln 67 was noted. These interactions together with above-mentioned hydrophobic interactions likely compensate for the absence of an isopeptide bond to stabilize FctB structure. Furthermore, findings showing that Asn 13 and Gln 67 are the only hydrophilic residues on the inner surface of FctB3 and replacement of Pro 17 of FctB3 by a glutamine residue in FctB1 (Fig. S2) suggest a relic of an isopeptide bond in FctB.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eComparison of FctB3 and FctB9 structures\u003c/h2\u003e \u003cp\u003ePreviously, FctB9 structure was solved at 1.9 \u0026Aring; (Linke et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2010\u003c/span\u003e) (Fig.\u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). The sequence identity of FctB3 and FctB9 is 97.9%, and a structural comparison of FctA and FctB9 showed root-mean-square deviation (RMSD) values less than 1.5\u0026Aring;. Fitting of the main chain atoms revealed that the direction of the proline-rich tail was quite different, and RMSD value for Cα fit between FctB3 and FctB9 was 0.397 \u0026Aring;. The RMSDs of the Ig-like domain and the proline-rich tail region were 0.500\u0026Aring; and 0.460\u0026Aring;, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). The intramolecular hydrophobic interaction network observed in FctB3 was also present in FctB9 and hydrophobic residues were conserved in FctB9 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB-E).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFctB9 Lys 110 links to the Thr residue in the FctA LPXTG-like motif via isopeptide bond formation, and Lys 110 is positioned in the omega loop (Linke et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Therefore, the omega loop structure has been noted as important for recognition of the LPXTG-like motif of FctA (Linke et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). A comparison of the omega loop conformation between FctB3 and FctB9 showed differences in the main chain from Arg 104 to Glu 109, though the location of Cα of Lys 110 was well overlapped (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA-D). This is because the nitrogen atom and carbonyl group of the Ser 111 main chain form hydrogen bonds to the carbonyl group of the Ser 102 main chain and the hydroxyl group of the Ser 102 side chain, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA-C). Furthermore, Ser 102 and Ser 111 were conserved among FctB of various serotype strains (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD, Fig. S2). The hydrogen bond network in the omega loop, including the hydrogen bond between the nitrogen atom in the Arg 104 main chain and the oxygen atom in Val 81 main chain, was also noted to be conserved. Arg 104 was located on the N-terminus of the omega loop and this hydrogen bond may stabilize the loop structure by fixing the omega loop to the Ig-like domain. Another conserved hydrogen bond was formed between the hydroxyl group of the Ser 111 side chain, oxygen atom of the Ala 112 main chain, and nitrogen atom of the Thr 114 main chain. This hydrogen bond network likely stabilizes the C terminus of the omega loop in conjunction with the hydrogen bond between Ser 102 and Ser 111. Most of conserved hydrogen bonds in the omega loop were formed between main chains, thus this network is considered to be less susceptible to the surrounding environment.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAlthough the amino acid sequences of the omega loop of FctB3 and FctB9 are identical, the structure was found to be changed. This is likely due to the hydrogen bond network formed by the Arg 103 and Arg 104 side chains. The side chain of FctB3 Arg 104 was found to be interacting with Asp 107 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA), whereas that of FctB9 Arg 104 was interacting with Ser 111 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Furthermore, the hydrogen bond interaction of the Arg 103 side chain with Glu 108 noted in FctB9 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eBC) was not observed in FctB3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Based on the high proportions of acidic and basic amino acid residues in the constituent of the omega loop, large amounts of residues are capable of forming hydrogen bonds between side chains, and switching of the hydrogen bond network by Arg 103 and Arg 104 may occur. The switching mode of the network without changing the Lys 110 position might be advantageous for linkage to FctA and subsequent anchoring of assembled pili to the cell wall.\u003c/p\u003e \u003cp\u003eOn the other hand, the proline-rich tails of FctB3 and FctB9 showed protrusions in different directions, caused by distinct hydrogen bond networks formed by Trp 119 (FctB3) and Arg 119 (FctB9) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA-C). Trp 119 was conserved in FctB1, whereas Arg 119 was not conserved in FctB from other serotypes, including M5, M9, M12, M18, and M49. Trp 119 of FctB3 formed a CH-π interaction with Arg 17, Tyr 75, and Val 121, but no hydrogen bond with the main chains was formed (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). Nevertheless, the side chain of Arg 119 in FctB9 formed hydrogen bonds with the main chains of Val 121 and Lys 122 in the linker region, as well as the CH-π interaction with Tyr 75 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). Val 121 and Lys 122 of FctB9 were found adjacent to the proline-rich tail. Hence, it is likely that either Arg 119 or Trp 119 is a determinant for the direction of the proline-rich tail. In support of this, another structure in the asymmetric unit of FctB9 showed that a disorder of the Arg 119 side chain caused a conformational change in the proline-rich tail, and Val 121 and Lys 122 were disordered (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eFormation of FctB/FctA and FctB/Cpa heterodimer complex involves SipA\u003c/h2\u003e \u003cp\u003eFctB3 and the FctA1 N-terminal domain also showed high homology, as previously noted for FctB9 (Linke et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). In the crystal packing of FctA1, a state mimicking the linkage has been reported (Kang et al, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2007\u003c/span\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). The C-terminus sequence EPT, of which Thr is linked to the neighboring pilin, was not found contained in the reported structure, whereas Lys 161 responsible for interaction with other subunits, and the groove for binding to LPXTG-like motifs composed of β7, β8, and β9, were suggested to be important for the linkage (Kang et al, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2007\u003c/span\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). We modeled the FctA/FctB complex by superposition based on this structure (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). Comparisons of FctA1/ FctA1 and FctA1/ FctB3 models showed structural differences in the contact area, including the groove. When the FctA1 N-terminal domain and FctB3 structures were superposed, the positions of FctB3 Lys 110 and FctA Lys 161 were shown to be overlapped (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAn \u003cem\u003ein vivo\u003c/em\u003e experiment was conducted to determine whether Lys 110 of FctB mediates formation of an isopeptide bond to growing FCT type 3 pili. Culture supernatants obtained from ∆\u003cem\u003efctB\u003c/em\u003e expressing wild-type or mutant FctB were subjected to immunoblot analyses with anti-FctA and anti-FctB (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD, E). In addition to Lys 110, the conserved lysine residues Lys 47, Lys 116, Lys 118, and Lys 122 were chosen to be replaced with alanine residues. In the culture supernatant obtained from ∆\u003cem\u003efctB\u003c/em\u003e expressing K110A mutant FctB, prominent HLBs were detected with anti-FctA (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD), and HLBs detected with anti-FctB completely disappeared (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE), confirming the linkage of FctB to growing FCT type 3 pili via Lys 110, as previously reported for FctB9 (Linke et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2010\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe β67 loop, as well as β8 and β9 of FctB are most likely responsible for recognition of FctA, since those regions were found to be well overlapped with the FctA1 β56 loop, β8, and β9 (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC). The FctA1 β56 loop, β8, and β9, together with the β23 loop constitute the N-terminal tip of the Ig-like domain responsible for recognition of Cpa and FctA. Structural differences were observed mainly between the FctB β34 loop and corresponding FctA β23 loop, indicating that a difference in loop structure affects binding to other subunits. The shorter loop of FctB was considered extremely unfavorable for binding to other pilus subunits, as compared with the corresponding region of FctA, since the contact area for the binding is smaller and the groove of the binding pocket for LPXTG-like motifs is shallower. Therefore, as compared with FctA, FctB is not favorable in regard to association with other pilus subunits and, as in the case of FctA, it is suggested that the association may require auxiliary proteins, such as the putative chaperone SipA.\u003c/p\u003e \u003cp\u003eSuperposition of the C-terminal domains of FctA and Cpa showed a high homology (RMSD: 1.395\u0026Aring;). Although the C terminus sequence of VVPT in Cpa, in which Thr is linked to FctA, was also not contained in the reported Cpa structure, the binding mode was predicted based on the position of Lys161 in FctA, and the location of the C terminus of Cpa, and groove in FctA domain 1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA). In addition, the structural similarity between the C-terminal domains of Cpa and FctA allowed modeling of the Cpa/FctB complex (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB), which was performed using an FctA/FctB complex model. With this complex model, Lys 110 of FctB was shown to be close to the Cpa C-terminus sequence VVPT and that region could be located in the groove on the FctB surface, suggesting that FctB can be linked to Cpa (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB). In fact, a band potentially representing the Cpa-FctB complex was detected in immunoblot data (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo clarify whether Lys 110 is linked to Cpa and FctA, wild type FctB, K47A, or a K110A mutant was expressed in either the double-deletion mutant of \u003cem\u003ecpa\u003c/em\u003e and \u003cem\u003efctB\u003c/em\u003e (∆\u003cem\u003ecpa\u003c/em\u003e∆\u003cem\u003efctB\u003c/em\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eC) or that of \u003cem\u003efctA\u003c/em\u003e and \u003cem\u003efctB\u003c/em\u003e (∆\u003cem\u003efctA\u003c/em\u003e∆\u003cem\u003efctB\u003c/em\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eD). In the CWF of ∆\u003cem\u003ecpa\u003c/em\u003e∆\u003cem\u003efctB\u003c/em\u003e expressing wild-type \u003cem\u003efctB\u003c/em\u003e (Comp) or K47A \u003cem\u003efctB\u003c/em\u003e, a\u0026thinsp;~\u0026thinsp;46 kDa band corresponding to the FctA/FctB complex was detected, whereas it was not detectable in empty vector-transformed ∆\u003cem\u003ecpa\u003c/em\u003e∆\u003cem\u003efctB\u003c/em\u003e (Mock) and ∆\u003cem\u003ecpa\u003c/em\u003e∆\u003cem\u003efctB\u003c/em\u003e expressing K110A (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eC). Similarly, a\u0026thinsp;~\u0026thinsp;100 kDa band reflecting the complex of Cpa and FctB was detected in the CWF of ∆\u003cem\u003efctA\u003c/em\u003e∆\u003cem\u003efctB\u003c/em\u003e expressing either wild-type \u003cem\u003efctB\u003c/em\u003e (Comp) or K47A \u003cem\u003efctB\u003c/em\u003e, but not in that of ∆\u003cem\u003efctA\u003c/em\u003e∆\u003cem\u003efctB\u003c/em\u003e expressing K136A \u003cem\u003efctB\u003c/em\u003e or empty vector-transformed control (Mock) (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eD). When the cell wall sorting signal mutant of either \u003cem\u003ecpa\u003c/em\u003e or \u003cem\u003efctA\u003c/em\u003e was expressed in the background of ∆\u003cem\u003ecpa\u003c/em\u003e∆\u003cem\u003efctA\u003c/em\u003e, no band of the complex was detected (data not shown). Therefore, Lys 110 of FctB is suggested to be linked to both FctA and Cpa.\u003c/p\u003e \u003cp\u003eFinally, to examine the requirement of SipA in formation of heterodimers, i.e., FctA/FctB and Cpa/FctB complexes, an \u003cem\u003esipA\u003c/em\u003e mutation was introduced into both ∆\u003cem\u003ecpa\u003c/em\u003e∆\u003cem\u003efctB\u003c/em\u003e and ∆\u003cem\u003efctA\u003c/em\u003e∆\u003cem\u003efctB\u003c/em\u003e, and then complemented with wild-type \u003cem\u003efctB\u003c/em\u003e (∆\u003cem\u003ecpa\u003c/em\u003e∆\u003cem\u003efctB\u003c/em\u003e∆\u003cem\u003esipA::fctB\u003c/em\u003e and ∆\u003cem\u003efctA\u003c/em\u003e∆\u003cem\u003efctB\u003c/em\u003e∆\u003cem\u003esipA::fctB\u003c/em\u003e). As a control, revertant strains (∆\u003cem\u003ecpa\u003c/em\u003e∆\u003cem\u003efctB\u003c/em\u003eWr\u003cem\u003esipA::fctB\u003c/em\u003e and ∆\u003cem\u003efctA\u003c/em\u003e∆\u003cem\u003efctB\u003c/em\u003eWr\u003cem\u003esipA::fctB\u003c/em\u003e), which arose during the mutant construction were also utilized. In the background of ∆\u003cem\u003ecpa\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eE, lane 1, Wr\u003cem\u003esipA\u003c/em\u003e) and ∆\u003cem\u003efctA\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eF, lane 1, Wr\u003cem\u003esipA\u003c/em\u003e), FctA-FctB and Cpa-FctB complexes, respectively, were detected. Notably, those complexes were not clearly detected following the \u003cem\u003esipA\u003c/em\u003e deletion (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eE and F, lane 2, ∆\u003cem\u003esipA\u003c/em\u003e). These findings indicate that SipA is required for the linkage between Cpa and FctB, as well as between FctA and FctB via FctB Lys 110.\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn the present study, the FctB3 structure was determined. Comparisons with the previously reported FctB9 structure showed that the main differences are the omega loop structure and direction of the proline-rich tail. The difference in the omega loop structure is likely caused by the distinct hydrogen bond network, which also indicates that network switching without changing the position of the lysine residue responsible for the linkage of pilin via an isopeptide bond may be advantageous for FctA polymerization and subsequent anchoring of pili to the cell wall. The direction of the proline-rich tail is potentially caused by a single residue located at the root of the tail. Furthermore, it is notable that the FctB structure is stabilized by intramolecular large hydrophobic interactions instead of an isopeptide bond. It is proposed that structural features that allow polymerization of FctA to occur more efficiently than incorporation of FctB to halt FctA polymerization are present. In addition, the findings show that formation of the heterodimer complex of FctB with FctA or Cpa is mediated by SipA. Thus, the present study provides an alternative structure for FctB as well as insight into the interactions between pilins of strains possessing FCT genomic region type 3.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAcknowledgments\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank T. Sekizaki (Kyoto University) and D. Takamatsu (National Institute of Animal Health) for providing the pSET4s plasmid. pAT18 was kindly provided by P. Trieu-Cuot (Institut Pasteur).\u0026nbsp;This work was performed using a synchrotron beamline BL44XU at SPring-8 under the Collaborative Research Program of Institute for Protein Research, Osaka University. Preliminary diffraction data were collected at the Osaka University beamline BL44XU at SPring-8 (Harima, Japan) (Proposal No. 2017A6732, 2017B6732, and 2018A6832). Also, this work was performed under the approval of the Photon Factory Program Advisory Committee (Proposal No. 2016G609, 2017G169).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eFunding\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by JSPS KAKENHI Grants‐in‐Aid for Scientific Research (Grant Nos. 19K22715, 19H03825, 22H03262, 22H03263).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAuthor Contribution\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eKT and MN contributed to the study conception and design. Material preparation, data collection, and analysis were performed by KT, MS, TS, and MN. The first draft of the manuscript was written by KT and MN, and all authors contributed to revision of the manuscript. All authors approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eData Availability\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe protein structure data are available in the wwPDB repository under accession number 8K6T.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eDeclarations\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAbbot EL, Smith WD, Siou GP, Chiriboga C, Smith RJ, Wilson JA, Hirst BH, Kehoe MA (2007) Pili mediate specific adhesion of \u003cem\u003eStreptococcus pyogenes\u003c/em\u003e to human tonsil and skin. 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Infect Immun 87:e00205-19. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1128/IAI.00205-19\u003c/span\u003e\u003cspan address=\"10.1128/IAI.00205-19\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZ\u0026auml;hner D, Scott JR (2008) SipA is required for pilus formation in \u003cem\u003eStreptococcus pyogenes\u003c/em\u003e serotype M3. J Bacteriol 190:527\u0026ndash;535. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1128/JB.01520-07\u003c/span\u003e\u003cspan address=\"10.1128/JB.01520-07\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTable 1 is available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"archives-of-microbiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"aomi","sideBox":"Learn more about [Archives of Microbiology](https://www.springer.com/journal/203)","snPcode":"203","submissionUrl":"https://submission.nature.com/new-submission/203/3","title":"Archives of Microbiology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Streptococcus pyogenes, pilus, FctB, crystal structure, SipA","lastPublishedDoi":"10.21203/rs.3.rs-3233333/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3233333/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e \u003cem\u003eStreptococcus pyogenes\u003c/em\u003e harboring an FCT type 3 genomic region display pili composed of three types of pilins. In this study, the structure of the base pilin FctB from a serotype M3 strain (FctB3) was determined at 2.8 \u0026Aring; resolution. In accordance with the previously reported structure of FctB from a serotype T9 strain (FctB9), FctB3 was found to consist of an immunoglobulin-like domain and proline-rich tail region. Data obtained from structure comparison reveled main differences in the omega loop structure and the proline-rich tail direction. In the omega loop structure, a differential hydrogen bond network was observed, while the lysine residue responsible for linkage to growing pili was located at the same position in both structures, which indicated that switching of the hydrogen bond network in the omega loop without changing the lysine position is advantageous for linkage to the backbone pilin FctA. The difference in direction of the proline-rich tail is potentially caused by a single residue located at the root of the proline-rich tail. Also, the FctB3 structure was found to be stabilized by intramolecular large hydrophobic interactions instead of an isopeptide bond. Comparisons of the FctB3 and FctA structures indicated that the FctA structure is more favorable for linkage to FctA. Additionally, the heterodimer formation of FctB with Cpa or FctA was shown to be mediated by the putative chaperone SipA. Together, these findings provide an alternative FctB structure as well as insight into the interactions between pilin proteins.\u003c/p\u003e","manuscriptTitle":"Analysis of FctB3 crystal structure and insight into its structural stabilization and pilin linkage mechanisms","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-08-10 15:08:08","doi":"10.21203/rs.3.rs-3233333/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revision","date":"2023-08-17T09:11:26+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2023-08-15T12:49:13+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"ef6e8097-b770-4635-9d63-ddab1662237a","date":"2023-08-06T11:32:43+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"4da18f3f-fb3c-4e32-9f2c-ab4486d8e30b","date":"2023-08-06T08:08:09+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2023-08-06T07:14:10+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2023-08-06T07:05:06+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2023-08-05T02:20:49+00:00","index":"","fulltext":""},{"type":"submitted","content":"Archives of Microbiology","date":"2023-08-04T05:18:25+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"archives-of-microbiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"aomi","sideBox":"Learn more about [Archives of Microbiology](https://www.springer.com/journal/203)","snPcode":"203","submissionUrl":"https://submission.nature.com/new-submission/203/3","title":"Archives of Microbiology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"fbdbd39d-1b11-43d4-b4b0-15fcecb846fd","owner":[],"postedDate":"August 10th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2023-11-27T15:09:19+00:00","versionOfRecord":{"articleIdentity":"rs-3233333","link":"https://doi.org/10.1007/s00203-023-03727-1","journal":{"identity":"archives-of-microbiology","isVorOnly":false,"title":"Archives of Microbiology"},"publishedOn":"2023-11-23 15:01:48","publishedOnDateReadable":"November 23rd, 2023"},"versionCreatedAt":"2023-08-10 15:08:08","video":"","vorDoi":"10.1007/s00203-023-03727-1","vorDoiUrl":"https://doi.org/10.1007/s00203-023-03727-1","workflowStages":[]},"version":"v1","identity":"rs-3233333","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3233333","identity":"rs-3233333","version":["v1"]},"buildId":"7rjqhiLT3MXkJMwkYKINL","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}