Thioredoxin (TRX)-fold proteins are ubiquitous in nature. This redox scaffold has evolved to enable a variety of functions, including redox regulation, protein folding, and oxidative stress defense. In bacteria, the TRX-like disulfide bond (Dsb) family mediates the oxidative folding of multiple proteins required for fitness and pathogenic potential. Conventionally, Dsb proteins have specific redox functions with monomeric and dimeric Dsbs exclusively catalyzing thiol oxidation and disulfide isomerization, respectively. This contrasts with the eukaryotic disulfide forming machinery where the modular TRX protein disulfide isomerase (PDI) mediates thiol oxidation and disulfide reshuffling. In this study, we identified and structurally and biochemically characterized a novel Dsb-like protein from Salmonella enterica termed bovine colonization factor protein H (BcfH) and defined its role in virulence.
Results:
In the conserved bovine colonization factor (bcf) fimbrial operon, the Dsb-like enzyme BcfH forms a trimeric structure, exceptionally uncommon among the large and evolutionary conserved TRX superfamily. This protein also displays very unusual catalytic redox centers, including an unwound α-helix holding the redox active site and a trans-proline instead of the conserved cis-proline active site loop. Remarkably, BcfH displays both thiol oxidase and disulfide isomerase activities contributing to Salmonella fimbrial biogenesis.
Innovation and Conclusion:
Typically, oligomerization of bacterial Dsb proteins modulates their redox function, with monomeric and dimeric Dsbs mediating thiol oxidation and disulfide isomerization, respectively. This study demonstrates a further structural and functional malleability in the TRX-fold protein family. BcfH trimeric architecture and unconventional catalytic sites permit multiple redox functions emulating in bacteria the eukaryotic PDI dual oxidoreductase activity. Antioxid. Redox Signal. 35, 21–39.
Get full access to this article
View all access options for this article.
References
1.
AdamsPD, Grosse-KunstleveRW, HungLW, IoergerTR, McCoyAJ, MoriartyNW, ReadRJ, SacchettiniJC, SauterNK, and TerwilligerTC. PHENIX: building new software for automated crystallographic structure determination. Acta Crystallogr D Biol Crystallogr, 58: 1948–1954, 2002.
2.
AtkinsonHJ and BabbittPC. An atlas of the thioredoxin fold class reveals the complexity of function-enabling adaptations. PLoS Comput Biol, 5: e1000541, 2009.
3.
BardwellJC, LeeJO, JanderG, MartinN, BelinD, and BeckwithJ. A pathway for disulfide bond formation in vivo. Proc Natl Acad Sci U S A, 90: 1038–1042, 1993.
4.
BiranS, GatY, and FassD. The Eps1p protein disulfide isomerase conserves classic thioredoxin superfamily amino acid motifs but not their functional geometries. PLoS One, 9: e113431, 2014.
5.
BouwmanCW, KohliM, KilloranA, TouchieGA, KadnerRJ, and MartinNL. Characterization of SrgA, a Salmonella enterica serovar Typhimurium virulence plasmid-encoded paralogue of the disulfide oxidoreductase DsbA, essential for biogenesis of plasmid-encoded fimbriae. J Bacteriol, 185: 991–1000, 2003.
6.
CowtanK. The Buccaneer software for automated model building. 1. Tracing protein chains. Acta Crystallogr D Biol Crystallogr, 62: 1002–1011, 2006.
7.
CowtanK. Recent developments in classical density modification. Acta Crystallogr D Biol Crystallogr, 66: 470–478, 2010.
8.
CrespoMD, PuorgerC, ScharerMA, EidamO, GrutterMG, CapitaniG, and GlockshuberR. Quality control of disulfide bond formation in pilus subunits by the chaperone FimC. Nat Chem Biol, 8: 707–713, 2012.
DenoncinK and ColletJF. Disulfide bond formation in the bacterial periplasm: major achievements and challenges ahead. Antioxid Redox Signal, 19: 63–71, 2013.
11.
Dos SantosAMP, FerrariRG, and Conte-JuniorCA. Virulence factors in Salmonella Typhimurium: The sagacity of a bacterium. Curr Microbiol, 76: 762–773, 2019.
12.
DufresneK, Saulnier-BellemareJ, and DaigleF. Functional analysis of the chaperone-usher fimbrial gene clusters of Salmonella enterica serovar Typhi. Front Cell Infect Microbiol, 8: 26, 2018.
13.
EdgarRC. MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res, 32: 1792–1797, 2004.
14.
EmsleyP and CowtanK. Coot: model-building tools for molecular graphics. Acta Crystallogr D Biol Crystallogr, 60: 2126–2132, 2004.
15.
EschenfeldtWH, LucyS, MillardCS, JoachimiakA, and MarkID. A family of LIC vectors for high-throughput cloning and purification of proteins. Methods Mol Biol, 498: 105–115, 2009.
16.
FucheFJ, SowO, SimonR, and TennantSM. Salmonella serogroup C: Current status of vaccines and why they are needed. Clin Vaccine Immunol, 23: 737–745, 2016.
17.
FurlongEJ, KurthF, PremkumarL, WhittenAE, and MartinJL. Engineered variants provide new insight into the structural properties important for activity of the highly dynamic, trimeric protein disulfide isomerase ScsC from Proteus mirabilis. Acta Crystallogr D Struct Biol, 75: 296–307, 2019.
18.
FurlongEJ, LoAW, KurthF, PremkumarL, TotsikaM, AchardMES, HaliliMA, HerasB, WhittenAE, ChoudhuryHG, SchembriMA, and MartinJL. A shape-shifting redox foldase contributes to Proteus mirabilis copper resistance. Nat Commun, 8: 16065, 2017.
19.
GruberCW, CemazarM, HerasB, MartinJL, and CraikDJ. Protein disulfide isomerase: the structure of oxidative folding. Trends Biochem Sci, 31: 455–464, 2006.
20.
GuptaSD, WuHC, and RickPD. A Salmonella typhimurium genetic locus which confers copper tolerance on copper-sensitive mutants of Escherichia coli. J Bacteriol, 179: 4977–4984, 1997.
21.
HatahetF and RuddockLW. Protein disulfide isomerase: a critical evaluation of its function in disulfide bond formation. Antioxid Redox Signal, 11: 2807–2850, 2009.
22.
HavelaarAH, KirkMD, TorgersonPR, GibbHJ, HaldT, LakeRJ, PraetN, BellingerDC, de SilvaNR, GargouriN, SpeybroeckN, CawthorneA, MathersC, SteinC, AnguloFJ, DevleesschauwerB, and World Health Organization Foodborne Disease Burden Epidemiology ReferenceGroup. World Health Organization global estimates and regional comparisons of the burden of foodborne disease in 2010. PLoS Med, 12: e1001923, 2015.
23.
HerasB, EdelingMA, ByrielKA, JonesA, RainaS, and MartinJL. Dehydration converts DsbG crystal diffraction from low to high resolution. Structure, 11: 139–145, 2003.
24.
HerasB, EdelingMA, SchirraHJ, RainaS, and MartinJL. Crystal structures of the DsbG disulfide isomerase reveal an unstable disulfide. Proc Natl Acad Sci U S A, 101: 8876–8881, 2004.
25.
HerasB, KurzM, ShouldiceSR, and MartinJL. The name's bond.…..disulfide bond. Curr Opin Struct Biol, 17: 691–698, 2007.
26.
HerasB, ShouldiceSR, TotsikaM, ScanlonMJ, SchembriMA, and MartinJL. DSB proteins and bacterial pathogenicity. Nat Rev Microbiol, 7: 215–225, 2009.
27.
HerasB, TotsikaM, JarrottR, ShouldiceSR, GuncarG, AchardME, WellsTJ, ArgenteMP, McEwanAG, and SchembriMA. Structural and functional characterization of three DsbA paralogues from Salmonella enterica serovar typhimurium. J Biol Chem, 285: 18423–18432, 2010.
28.
HolmgrenA. Thioredoxin catalyzes the reduction of insulin disulfides by dithiothreitol and dihydrolipoamide. J Biol Chem, 254: 9627–9632, 1979.
29.
HumphriesAD, RaffatelluM, WinterS, WeeningEH, KingsleyRA, DroleskeyR, ZhangS, FigueiredoJ, KhareS, NunesJ, AdamsLG, TsolisRM, and BaumlerAJ. The use of flow cytometry to detect expression of subunits encoded by 11 Salmonella enterica serotype Typhimurium fimbrial operons. Mol Microbiol, 48: 1357–1376, 2003.
30.
HutchinsonEG and ThorntonJM. PROMOTIF—a program to identify and analyze structural motifs in proteins. Protein Sci, 5: 212–220, 1996.
31.
InabaK and ItoK. Paradoxical redox properties of DsbB and DsbA in the protein disulfide-introducing reaction cascade. EMBO J, 21: 2646–2654, 2002.
32.
Jacob-DubuissonF, PinknerJ, XuZ, StrikerR, PadmanhabanA, and HultgrenSJ. PapD chaperone function in pilus biogenesis depends on oxidant and chaperone-like activities of DsbA. Proc Natl Acad Sci U S A, 91: 11552–11556, 1994.
33.
JarrottR, ShouldiceSR, GuncarG, TotsikaM, SchembriMA, and HerasB. Expression and crystallization of SeDsbA, SeDsbL and SeSrgA from Salmonella enterica serovar Typhimurium. Acta Crystallogr Sect F Struct Biol Cryst Commun, 66: 601–604, 2010.
34.
JiaoL, KimJS, SongWS, YoonBY, LeeK, and HaNC. Crystal structure of the periplasmic disulfide-bond isomerase DsbC from Salmonella enterica serovar Typhimurium and the mechanistic implications. J Struct Biol, 183: 1–10, 2013.
35.
KabschW. Integration, scaling, space-group assignment and post-refinement. Acta Crystallogr D Biol Crystallogr, 66: 133–144, 2010.
36.
KadokuraH, TianH, ZanderT, BardwellJC, and BeckwithJ. Snapshots of DsbA in action: detection of proteins in the process of oxidative folding. Science, 303: 534–537, 2004.
37.
KonarevPV, VolkovVV, SokolovaAV, KochMHJ, and SvergunDI. PRIMUS: a Windows PC-based system for small-angle scattering data analysis. J Appl Crystallogr, 36: 1277–1282, 2003.
38.
LandetaC, BoydD, and BeckwithJ. Disulfide bond formation in prokaryotes. Nat Microbiol, 3: 270–280, 2018.
39.
LaueTM, ShahBD, RidgewayTM, and PelletierSL. Computer-aided interpretation of analytical sedimentation data for proteins. In: Analytical Ultracentrifugation in Biochemistry and Polymer Science, edited by Harding SE, Rowe AJ, and Horton JC. Cambridge, United Kingdom: Royal Society of Chemistry, 1992, 90–125.
40.
LedeboerNA, FryeJG, McClellandM, and JonesBD. Salmonella enterica serovar Typhimurium requires the Lpf, Pef, and Tafi fimbriae for biofilm formation on HEp-2 tissue culture cells and chicken intestinal epithelium. Infect Immun, 74: 3156–3169, 2006.
41.
LinD, KimB, and SlauchJM. DsbL and DsbI contribute to periplasmic disulfide bond formation in Salmonella enterica serovar Typhimurium. Microbiology, 155: 4014–4024, 2009.
42.
McCoyAJ, StoroniLC, and ReadRJ. Simple algorithm for a maximum-likelihood SAD function. Acta Crystallogr D Biol Crystallogr, 60: 1220–1228, 2004.
43.
McGinnisS and MaddenTL. BLAST: at the core of a powerful and diverse set of sequence analysis tools. Nucleic Acids Res, 32: W20–W25, 2004.
44.
MurshudovGN, SkubakP, LebedevAA, PannuNS, SteinerRA, NichollsRA, WinnMD, LongF, and VaginAA. REFMAC5 for the refinement of macromolecular crystal structures. Acta Crystallogr D Biol Crystallogr, 67: 355–367, 2011.
45.
NuccioSP and BaumlerAJ. Evolution of the chaperone/usher assembly pathway: fimbrial classification goes Greek. Microbiol Mol Biol Rev, 71: 551–575, 2007.
46.
OtwinowskiZ and MinorW. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol, 276: 307–326, 1997.
47.
PanjikarS, ParthasarathyV, LamzinVS, WeissMS, and TuckerPA. Auto-rickshaw: an automated crystal structure determination platform as an efficient tool for the validation of an X-ray diffraction experiment. Acta Crystallogr D Biol Crystallogr, 61: 449–457, 2005.
48.
PanjikarS, ParthasarathyV, LamzinVS, WeissMS, and TuckerPA. On the combination of molecular replacement and single-wavelength anomalous diffraction phasing for automated structure determination. Acta Crystallogr D Biol Crystallogr, 65: 1089–1097, 2009.
49.
PannuNS and ReadRJ. The application of multivariate statistical techniques improves single-wavelength anomalous diffraction phasing. Acta Crystallogr D Biol Crystallogr, 60: 22–27, 2004.
50.
PetoukhovMV, FrankeD, ShkumatovAV, TriaG, KikhneyAG, GajdaM, GorbaC, MertensHDT, KonarevPV, and SvergunDI. New developments in the ATSAS program package for small-angle scattering data analysis. J Appl Crystallogr, 45: 342–350, 2012.
51.
PilipczukJ, Zalewska-PiatekB, BruzdziakP, CzubJ, WieczorM, OlszewskiM, WanarskaM, NowickiB, Augustin-NowackaD, and PiatekR. Role of the disulfide bond in stabilizing and folding of the fimbrial protein DraE from uropathogenic Escherichia coli. J Biol Chem, 292: 16136–16149, 2017.
52.
QuanS, SchneiderI, PanJ, Von HachtA, and BardwellJC. The CXXC motif is more than a redox rheostat. J Biol Chem, 282: 28823–28833, 2007.
53.
RenG, StephanD, XuZ, ZhengY, TangD, HarrisonRS, KurzM, JarrottR, ShouldiceSR, HinikerA, MartinJL, HerasB, and BardwellJC. Properties of the thioredoxin fold superfamily are modulated by a single amino acid residue. J Biol Chem, 284: 10150–10159, 2009.
54.
RietschA, BessetteP, GeorgiouG, and BeckwithJ. Reduction of the periplasmic disulfide bond isomerase, DsbC, occurs by passage of electrons from cytoplasmic thioredoxin. J Bacteriol, 179: 6602–6608, 1997.
55.
SchneiderTR and SheldrickGM. Substructure solution with SHELXD. Acta Crystallogr D Biol Crystallogr, 58: 1772–1779, 2002.
56.
SchuckP. Size-distribution analysis of macromolecules by sedimentation velocity ultracentrifugation and lamm equation modeling. Biophys J, 78: 1606–1619, 2000.
57.
ShepherdM, HerasB, AchardME, KingGJ, ArgenteMP, KurthF, TaylorSL, HowardMJ, KingNP, SchembriMA, and McEwanAG. Structural and functional characterization of ScsC, a periplasmic thioredoxin-like protein from Salmonella enterica serovar Typhimurium. Antioxid Redox Signal, 19: 1494–1506, 2013.
58.
ShouldiceSR, HerasB, WaldenPM, TotsikaM, SchembriMA, and MartinJL. Structure and function of DsbA, a key bacterial oxidative folding catalyst. Antioxid Redox Signal, 14: 1729–1760, 2011.
59.
SieversF, WilmA, DineenD, GibsonTJ, KarplusK, LiW, LopezR, McWilliamH, RemmertM, SodingJ, ThompsonJD, and HigginsDG. Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega. Mol Syst Biol, 7: 539, 2011.
60.
StudierFW. Protein production by auto-induction in high density shaking cultures. Protein Expr Purif, 41: 207–234, 2005.
61.
SubediP, PaxmanJJ, WangG, UkuwelaAA, XiaoZ, and HerasB. The Scs disulfide reductase system cooperates with the metallochaperone CueP in Salmonella copper resistance. J Biol Chem, 294: 15876–15888, 2019.
62.
SvergunD, BarberatoC, and KochMHJ. CRYSOL—a program to evaluate x-ray solution scattering of biological macromolecules from atomic coordinates. J Appl Crystallogr, 28: 768–773, 1995.
63.
SvergunDI. Determination of the regularization parameter in indirect-transform methods using perceptual criteria. J Appl Crystallogr, 25: 495–503, 1992.
64.
SvergunDI. Restoring low resolution structure of biological macromolecules from solution scattering using simulated annealing. Biophys J, 76: 2879–2886, 1999.
65.
TerwilligerTC. Maximum-likelihood density modification. Acta Crystallogr D Biol Crystallogr, 56: 965–972, 2000.
66.
TianG, XiangS, NoivaR, LennarzWJ, and SchindelinH. The crystal structure of yeast protein disulfide isomerase suggests cooperativity between its active sites. Cell, 124: 61–73, 2006.
67.
TotsikaM, VagenasD, PaxmanJJ, WangG, DhouibR, SharmaP, MartinJL, ScanlonMJ, and HerasB. Inhibition of diverse DsbA enzymes in multi-DsbA encoding pathogens. Antioxid Redox Signal, 29: 653–666, 2018.
68.
VerderosaAD, DhouibR, Fairfull-SmithKE, and TotsikaM. Nitroxide functionalized antibiotics are promising eradication agents against Staphylococcus aureus biofilms. Antimicrob Agents Chemother, 64: e01685-19, 2019.
69.
VivianJP, ScoullarJ, RimmerK, BushellSR, BeddoeT, WilceMC, ByresE, BoyleTP, DoakB, SimpsonJS, GrahamB, HerasB, KahlerCM, RossjohnJ, and ScanlonMJ. Structure and function of the oxidoreductase DsbA1 from Neisseria meningitidis. J Mol Biol, 394: 931–943, 2009.
70.
VivianJP, ScoullarJ, RobertsonAL, BottomleySP, HorneJ, ChinY, WielensJ, ThompsonPE, VelkovT, PiekS, ByresE, BeddoeT, WilceMC, KahlerCM, RossjohnJ, and ScanlonMJ. Structural and biochemical characterization of the oxidoreductase NmDsbA3 from Neisseria meningitidis. J Biol Chem, 283: 32452–32461, 2008.
71.
VolkovVV and SvergunDI. Uniqueness of ab initio shape determination in small-angle scattering. J Appl Crystallogr, 36: 860–864, 2003.
72.
WaldenPM, WhittenAE, PremkumarL, HaliliMA, HerasB, KingGJ, and MartinJL. The atypical thiol-disulfide exchange protein alpha-DsbA2 from Wolbachia pipientis is a homotrimeric disulfide isomerase. Acta Crystallogr D Struct Biol, 75: 283–295, 2019.
73.
WeeningEH, BarkerJD, LaarakkerMC, HumphriesAD, TsolisRM, and BaumlerAJ. The Salmonella enterica serotype Typhimurium lpf, bcf, stb, stc, std, and sth fimbrial operons are required for intestinal persistence in mice. Infect Immun, 73: 3358–3366, 2005.
74.
YehSM, KoonN, SquireC, and MetcalfP. Structures of the dimerization domains of the Escherichia coli disulfide-bond isomerase enzymes DsbC and DsbG. Acta Crystallogr D Biol Crystallogr, 63: 465–471, 2007.
75.
YueM, RankinSC, BlanchetRT, NultonJD, EdwardsRA, and SchifferliDM. Diversification of the Salmonella fimbriae: a model of macro- and microevolution. PLoS One, 7: e38596, 2012.
76.
ZapunA, MissiakasD, RainaS, and CreightonTE. Structural and functional characterization of DsbC, a protein involved in disulfide bond formation in Escherichia coli. Biochemistry, 34: 5075–5089, 1995.
Supplementary Material
Please find the following supplemental material available below.
For Open Access articles published under a Creative Commons License, all supplemental material carries the same license as the article it is associated with.
For non-Open Access articles published, all supplemental material carries a non-exclusive license, and permission requests for re-use of supplemental material or any part of supplemental material shall be sent directly to the copyright owner as specified in the copyright notice associated with the article.
