seminars in CELL & DEVELOPMENTAL BIOLOGY, Vol 11, 2000: pp. 27]34doi:10.1006rscdb.1999.0348, available online at http:rrwww.idealibrary.com on

PapD-like chaperones and pilus biogenesis

i

Frederic G. Sauer a, Stefan D. Knight c, GabrScott J. Hultgrena,U

The assembly of adhesive pili from individual subunits byperiplasmic PapD-like chaperones in Gram-negative bacteriaoffers insight into the complex process of organelle biogenesis.PapD-like chaperones bind, stabilize, and cap interactivesurfaces of subunits until they are assembled into the pilus.Subunits lack the seventh b-strand necessary to completetheir immunoglobulin-like folds; the chaperone supplies thismissing strand. Indeed, the chaperone may act as a template,providing steric information to facilitate subunit folding. Inthe mature pilus, each subunit is thought to supply themissing strand to complete the fold of its neighbor. Thus,one general function of chaperones in organelle biogenesismay be to cap highly interactive surfaces of subunits untilthey reach the proper assembly site.

Key words: chaperone r pilus r organelle biogenesis rprotein folding

Q2000 Academic Press

Introduction

A FUNDAMENTAL QUESTION in molecular biology ishow proteins fold into domains that can serve asassembly modules for building up larger macro-molecular structures. The biogenesis of large fibrousorganelles, called pili, on the surface of Gram-nega-tive bacteria requires the orchestration of a complexprocess that includes protein synthesis, folding, secre-

From the aDepartments of Molecular Microbiology, bBiochemistryand Molecular Biophysics, Washington University School ofMedicine, St. Louis, MO 63110, USA, and cDepartment of

Molecular Biology, Uppsala Biomedical Center, Swedish Universityof Agricultural Sciences, Uppsala, Sweden. UCorresponding au -thor.

Q2000 Academic Press1084-9521r00r010027q08 $35.00r0

27

el Waksmanb and

tion, and assembly.1 The PapD-like superfamily ofperiplasmic chaperones directs the assembly of anarray of adhesive surface organelles that mediateattachment to host tissues, a critical early step in the

1,2 Ž .development of disease, Table 1 . The elucidationof the function of PapD-like chaperones has revealedmolecular details of a mechanism that has con-tributed to our understanding both of general princi-ples of organelle development and of the pathogene-sis of a variety of bacterial diseases.

The more than 30 known PapD-like chaperonesfacilitate the assembly of both pilus and non-pilus

2 Ž .organelles Table 1 . Much of our knowledge of thefunction of these molecules comes from the study ofPapD and FimC, which assemble P and type 1 pili,

3 ] 5 Ž .respectively, in E. coli Figure 1 . Genes importantŽ . Ž .in P papA]papK and type 1 fimA]fimH pilus

biogenesis are organized in the pap and fim operons,respectively.1 P pili consist of six structural proteinsthat interact to form a composite fiber with twosubassemblies: a 7-nm-thick pilus rod comprised ofPapA subunits arranged in a right-handed helicalcylinder, and a thin fibrillum comprised primarily ofPapE subunits arranged in an open helical configu-ration.6,7 The PapK adaptor protein links the twosubassemblies.8 The PapG adhesin, a virulence factorin uropathogenic E. coli, is joined to the distal end ofthe tip fibrillum by the PapF adaptor protein.8 ] 10

Type 1 pili are also composite fibers consisting of ashort tip fibrillum containing the FimH adhesin,joined to the distal end of a pilus rod.11

During pilus biogenesis, the chaperone interactswith each nascently translocated pilus subunit in the

Ž .periplasm Figure 1 via a mechanism termed donorŽ .strand complementation see below . This interaction

facilitates the release of the subunit from the cyto-

plasmic membrane in a process that my be driven byits folding directly on the chaperone template. Thechaperone remains bound to the folded subunit,stabilizing it and capping its interactive surfaces and

F. G. Sauer et al.

Table 1. Bacterial surface organelles assembled by PapD-like chaperones

Ž .Organelle reference Organism Chaperone Usher Disease

FibersP pilus E. coli PapD PapC Pyelonephritis, cystitisPrs pilus E. coli PrsD PrsC Cystitis?Type 1 pilus E. coli, Salmonella ssp., FimC FimD Cystitis

Klebsiella pneumoniaeF1C pilus E. coli FocC FocD Cystits?S pilus E. coli SfaE SfaF UTI, NBMHif pilus Hemophilus influenzae HifB HifC Otitis media, meningitisHaf pilus H. influenzae biogroup HafB HafC Brazilian purpuric fever

aegyptiusŽ . Ž .Type 2 and 3 pili Bordetella pertussis FimB FhaD FimC FhaA Whopping cough

Pef pilus S. typhimurium PefD PefC Gastroenteritis, samonellosisLpf pilus S. typhimurium LpfB LpfC Gastroenteritis?, samonellosis?MRrP pilus Proteus mirabilis MrpD MrpC Nosocomial UTIPMF pilus P. mirabilis PmfD PmfC Nosocomial UTIAft pilus P. mirabilis AftB AftC UTI

38AFrR1 pilus E. coli AfrC AfrB Diarrhea in rabbitsK99 pilus E. coli FanE FanD Neonatal diarrhea in calves,

lambs, pigletsK88 pilus E. coli FaeE FaeD Neonatal diarrhea in piglets987P pilus E. coli FasB FasD Diarrhea in pigletsF17 pilus ETEC F17D F17papC Diarrhea in piglets

Ž .MRrK Type 3 pilus K. pneumoniae MrkB MrkC Pneumonia

Non-fimbrial AdhesinsNFA1-6 family E. coli NfaE NfaC UTI, NBMAfa-1 E. coli AfaB AfaC PyelonephritisDrrAfa-111 E. coli DraE DraD UTI, diarrheaM E. coli BmaB BmaC Pyelonephritis

Atypical structuresCS3 ETEC Cs3-1 Cs3-2 Traveler’s diarrheaCS31A pilus ETEC ClpE ClpD DiarrheaCS6 pilus ETEC CssC CssD? DiarrheaAAFr1 ETEC AggD AggC DiarrheaSef S. enteritidis SefB SefC Gastroenteritis, salmonellosisF1 antigen Yersinia pestis Caf1M Caf1A PlaguePH6 antigen Y. pestis, PsaB PsaC Plague

Y. pseudotuberculosisMyf Y. enteritidis MyfB MyfC EntercolitisUnknown structures

E

? REPEC RalE

Notes. Adapted from Table 1 in Thanassi et al.17 See refs 2NBM, newborn meningitis; ETEC, enterotoxigenic E. coli; R

thus preventing premature aggregation in theperiplasm.12 ] 16 Each PapD-like chaperone functionsin concert with a corresponding outer membraneusher protein.17 Chaperone]subunit complexes arespecifically targeted to the usher, which forms a 2]3-

nm diameter donut-shaped channel, large enough toallow the passage of pilus subunits.18,19 In the fim andpap systems, it has been shown that the chaperone]adhesin complex binds most rapidly and tightly tothe usher in a process that is thought to initiate pilus

28

RalD Diarrhea in rabbits

and 17 for references not listed. UTI, urinary tract infection;PEC, rabbit enteropathogenic E. coli.

assembly. Formation of a chaperone]adhesin]usherternary complex induces a conformational change inthe usher to an assembly-competent form that ismaintained throughout pilus assembly.20 The usherfacilitates chaperone uncapping to expose the inter-

active surfaces on the subunits that drive their assem-bly into the pilus. The pilus is thought to growthrough the usher as a linear fiber that packages intoits final quaternary structure upon reaching the bac-terial surface.19

Chaperones and pilus biogenesis

Figure 1. Schematic of P pilus biogenesis. P pili are members of a large family of surfaceorganelles assembled by PapD-like chaperones and their corresponding ushers. Electron micro-

Ž .graphs of a P pilus and a type 1 pilus from E. coli are shown 1 . Pilus subunits enter theperiplasm through the Sec transport system. In the absence of the PapD periplasmic chaperone,

Ž .subunits aggregate and are degraded 2 , inducing signal transduction in the bacterium. In theŽ .presence of PapD, subunits form stable chaperone]subunit complexes 3 through a mechanism

Ž .termed donor strand complementation the PapD]PapK chaperone]subunit complex is shownŽ . Ž4 , in which the chaperone donates its G b-strand to complete the Ig fold of the subunit Figure1.2 . This interaction permits subunit folding and prevents both subunit aggregation and prema-

ture subunit polymerization. The chaperone]subunit complexes are then targeted to an outerŽ .membrane protein pore termed the usher PapC , where the subunits assemble into a pilus. The

Ž .chaperone]adhesin complex PapD]PapG binds most rapidly and tightly to the usher, initiatingŽ . Ž .pilus assembly 5 . Assembly of subsequent subunits 6 occurs through a mechanism termed

Ž .donor strand exchange 7 , in which the disordered N-terminus of an incoming subunit displacesthe G strand of the chaperone in the most recently assembled chaperone]subunit complex1

tsos

Ž .Figure 3 . Thus, in the mature pilus, the Ig fostrand of the adjacent subunit. The rod subuni

Ž .outside the cell 8 , a process that is thought telectron micrograph of an unwound pilus rod i

The Cpx pathway

In the absence of the chaperone, subunits are un-stable and misfold, aggregate, and are proteolyticallydegraded. OFF-pathway subunits have been shown toactivate a two-component signal transduction system

29

ld of a subunit is completed by the N-terminalŽ .PapA wind into their final helical form oncefacilitate the outward growth of the pilus. An

Ž .shown 9 .

known as the Cpx pathway.12,21 The Cpx pathwayresponds to periplasmic stress by inducing the activa-

tion of a variety of genes encoding periplasmic pro-

Ž .tein folding factors DsbA and prolyl isomerases andŽ . 21 ] 23periplasmic proteases DegP . DegP degrades

misfolded pilins, preventing their toxic buildup in

sis, the N-terminal extension of one subunit displaces

F. G. Sauer et al.

the periplasm. DsbA catalyzes disulfide bond forma-tion in proteins required for the assembly of P pili.24

Thus, Cpx is part of a pilus biogenesis sensorrcontrolŽ .circuit Figure 1 that ensures that toxic OFF-pathway

products do not accumulate.12

Chaperone–subunit complexes: donor strandcomplementation

The crystal structure of PapD and the solution struc-ture of FimC, and more recently, the crystal struc-tures of the FimC]FimH chaperone]adhesin com-plex and the PapD]PapK chaperone]pilin complexhave all been solved.25 ] 28 The chaperone consists of

Ž .two immunoglobulin-like Ig domains oriented in anL-shape to form a cleft between them. The apo andcomplexed forms of the chaperone are virtually su-perimposable, with the exception of the F ]G loop,1 1as discussed below. The FimH adhesin consists of twodomains: a pilin domain and a receptor-binding do-main. The PapK pilin and the pilin domain of FimHhave Ig folds; however, they lack the seventh and

Ž .C-terminal b-strand strand G present in canonicalŽ .Ig folds Figure 2 . The absence of this strand pro-

duces a deep groove along the surface of the pilindomain and exposes its hydrophobic core}hencethe instability of pilins when expressed without thechaperone. In the chaperone]subunit complex, theG strand of the chaperone and a portion of the1F ]G loop, which extends to lengthen the G strand,1 1 1complete the Ig fold of the subunit by occupying thegroove. This donor strand complementation interac-tion stabilizes the subunit by shielding its hy-drophobic core. Indeed, alternating hydrophobicresidues in the G strand become an integral part of1the hydrophobic core of the subunit. The G strand1provides the missing strand of the subunit Ig domain

Ž .by interacting with strands A2 also called A0 and Flocated on either side of the groove, while the C-terminal carboxyl group of the subunit, at the end ofthe F strand, is anchored between the conserved Argand Lys residues29 in the cleft of the chaperoneŽ .Figure 2 . The Ig fold that is produced is atypicalsince the G strand of the chaperone runs parallel,1rather than anti-parallel, to the F strand, as it does ina canonical Ig fold.27,28,30 While stabilizing the sub-unit and completing its fold, the chaperone simulta-

neously caps one of its interactive surfaces. Mutatio-nal and biochemical studies indicate that the grooveof the pilus subunit that participates in donor strandcomplementation is the same surface that interacts

30

Figure 2. Donor strand complementation interactions inthe FimC]FimH chaperone]adhesin complex. FimC isshown in black and FimH is in gray. The G b-strand1completes the Ig fold of the FimH pilin domain between

Ž .the A0 i.e. A2 and F strands. The G b-strand runs1parallel to the F strand. The alternating hydrophobic

Žresidues of the G b-strand Leu103, Leu105, Ile107 in1.FimC form part of the hydrophobic core of the pilin

domain and may facilitate the collapse of the hydrophobiccore during subunit folding. The C-terminal carboxyl groupŽ .COOH is anchored in the cleft between the two domainsof FimC by the conserved Arg8 and Lys112 residues.

with other subunits in the pilus.14,16 Thus, the G1strand of the chaperone, by occupying the groove,prevents premature pilus formation in the periplasm.

Donor strand exchange and pilus biogenesis

ŽPilus subunits have an N-terminal extension residues.1]13 in PapK, for example with a highly conserved

alternating hydrophobic motif that has been shownto participate in subunit]subunit interactions.16 Thismotif is similar to the alternating hydrophobic motifpresent in the G b-strand of the chaperone that1participates in donor strand complementation. TheN-terminal extension does not contribute to the Igfold of the subunit, but rather projects away from therest of the pilin domain where it would be free tointeract with another subunit.28 Based on theseobservations, we propose that during pilus biogene-

the chaperone G strand from its neighboring sub-1unit in a mechanism termed donor strand

27,28 Ž .exchange Figure 3 . The pilin domain of theadhesin lacks this N-terminal extension, consistent

Figure 3. Proposed model of subunit]subunit interactionsin a pilus. The N-terminal extension of one subunit re-places the chaperone G strand and completes the Ig fold1of the preceding subunit, running anti-parallel to the Fstrand.

with its location at the tip of the pilus.27 The subse-quent order of subunits in the pilus may at least inpart be determined by the stereochemical comple-mentarity between the N-terminal extension of thesubunit and the groove of its neighbor.27,28 Theknown geometry of pilus rods indicates that the N-terminal strand would insert anti-parallel to the Fstrand of the neighboring subunit, unlike the chaper-one G strand, which inserts in a parallel fashion.27,28

1The mature pilus would thus consist of an array ofperfectly canonical Ig domains, each of which con-tributes a strand to the fold of the preceding subunitto produce the organelle.

Protein folding with a novel twist

Donor strand complementation simultaneouslystabilizes the pilus subunit and caps the interactivegroove. In addition, donor strand complementationpermits subunit folding. In the absence of the chap-erone, subunits aggregate in the periplasm and aredegraded by the DegP protease.12 These non-produc-tive interactions are thought to interfere with the

proper folding pathway. For example, PapG ex-pressed in the absence of the chaperone in a degPy

strain does not fold into a native receptor bindingconformation.12 Therefore, by providing the seventhG strand to complete the subunit Ig fold, the chaper-

31

Chaperones and pilus biogenesis

one prevents non-productive interactions, therebykeeping it ON pathway. However, the PapD-likechaperones may play a more active role by providinga structural context for subunit folding. Donor strandcomplementation might occur either early or late inthe folding pathway, but once the subunit is folded,the chaperone remains bound, capping the criticalsubunit groove. In other words, the chaperone maycouple the folding of the subunit with the capping ofthe groove. Then, during donor strand exchange, theN-terminal extension of the neighboring subunitwould replace the G strand of the chaperone in the1groove, thus preventing the exposure of the interac-tive groove at any time during the entire folding andassembly pathway.

b-sheet formation is thought to be determined in alarge part by tertiary context, even at solvent-accessi-ble sites, and not by intrinsic secondary structuralpreferences.31 Donation of the G strand of the chap-1erone may provide the necessary tertiary context forthe formation of the G , F, C1 b-sheet,16 which forms1half of the immunoglobulin fold of the pilindomain.27,28 Indeed, PapD is known to induce theformation of b-strands in peptides corresponding tothe C-terminal F strands of subunits.15,16,32 Duringchaperone]subunit complex formation, the interac-tion between the C-terminal carboxyl group at theend of the subunit F strand and the conserved Argand Lys residues in the chaperone cleft would posi-tion the F strand correctly in relation to the chaper-

Ž .one G strand during subunit folding Figure 2 . At1the same time, this interaction would position thehydrophobic side chains of strand F to allow them tomake the appropriate contacts with the alternatinghydrophobic residues in the chaperone G strand.1Thus, by providing the correct context for beta sheetformation, the G strand of the chaperone may facili-1tate the proper collapse of the hydrophobic core of

Ž .the subunit Figure 2 .

Cytoplasmic and periplasmic chaperones

The PapD-like chaperones differ in several respectsfrom the DnaJrDnaKrGrpE and GroELrGroES cy-toplasmic chaperone systems reviewed in this issue

Ž .see preceding paper by Agashe and Hartl as well asin ref 33. DnaJ and DnaK bind to semi-unfolded polypeptides and maintain them infolding-competent conformations by preventing themfrom engaging in non-productive interactions. DnaJ

F. G. Sauer et al.

is thought to be released from the ternary complex,leaving DnaK tightly bound to the polypeptide. GrpEis then thought to facilitate the release of thepolypeptide from DnaK, allowing it to fold in solu-tion. In the GroELrGroES system, semi-unfolded ormisfolded polypeptides interact with the hydrophobicwalls of the GroEL chamber. This is thought toprevent non-productive interactions andror to facili-tate the folding of misfolded proteins.33,34 Release ofthe folding-competent polypeptide from the hy-drophobic walls allows folding to occur in solution.In contrast, in the PapD-like chaperone system, pilussubunits may fold directly on the chaperone templateand remain bound to the chaperone in their fullyfolded states until they are assembled into a pilus atthe usher. Also, the DnaJrDnaKrGrpE andGroELrGroES systems require ATP hydrolysis, whilethe PapD-like chaperones function in the absence ofknown cellular energy sources.35 Finally, the cyto-plasmic and periplasmic chaperones differ in howthey facilitate folding. Both the DnaJrDnaKrGrpEand GroELrGroES systems appear to prevent orovercome the misfolding of proteins, rather thancontribute steric information during substrate fold-ing.33 PapD-like chaperones, on the other hand, maycontribute steric information during the folding of

their substrates by providing an integral part of theirIg fold. In this model, the complete informationrequired to produce a correctly folded Ig domainwould reside not in a single polypeptide chain butrather in two distinct polypeptides.27

Table 2. Partial list of chaperones involved in organelle bioge

Organelle Representative Ochaperone

Pilus PapD Pilus subunitCS1 pilus family CooB CooA, CooD

ŽCooC outerCurli CsgG? CsgA,CsgB

ŽFlagellum FlgN FlgK, FlgL hŽFlagellum FliT? FliD filamen

ŽFlagellum FliJ FlgD, FlgE hŽType II secretion PulS PulD outer

systemType III secretion SycD YopB, YopD

systemType III secretion YscB YopN

systemŽType III secretion VirG? YscC outer m

system

Notes. Only representative chaperones from homologous fafinal assembled organelle.

32

A model for organelle biogenesis?

The various surface organelles assembled by Gram-negative bacteria, including pili, flagella, and othermacromolecular structures, have to withstand theconsiderable forces that they experience in carryingout their functions. A flagellum that breaks apartdaring the first stroke is of little use to the cell. Apilus that breaks upon attachment loses its adhesive-ness. The proposed pilus structure explains how sucha sturdy organelle can be built. The contribution of aportion of the fold from one molecule to another, asin the pilus model, produces a very tight interaction,one that can be repeated over many molecules toproduce large structures. Protein polymerization bydomain swapping could also lead to the formation ofrobust macromolecular assemblies.36 Indeed, any rel -atively strong interaction between two molecules thatcan be repeated over a network of molecules in oneor more dimensions could be used to build an or-ganelle. Many variations of such interactions could beimagined to produce different structures. Such inter-actions might play a role in the formation of detri-mental structures as well, for example the amyloidfibers that characterize a variety of human diseases.

The coordinated assembly of such organelles, how-

ever, requires that the cell allow the subunits tointeract only at the site of assembly, since interactionselsewhere might be non-productive. In pilus biogene-sis, the PapD-like chaperone assures that subunitsinteract with each other only at the usher. The FlgN

nesis

rganelle subunit Reference

s 2,17, Table 1 hereinŽ .pilus subunits Reviewed in ref 39

.membrane protein40

.ook associated proteins 37.t cap protein 37.ook proteins , FliE? 41

.membrane protein 42

43,44

45

.embrane channel protein 46

milies are listed. Chaperones may or may not be part of the

and FliT chaperones have been proposed to functionanalogously in flagellar biogenesis.37 Thus, one com-mon function of the growing number of chaperones

Žknown to be involved in organelle biogenesis Table.2 may be to regulate subunit assembly. By capping

the interactive surfaces of subunits until they reachthe appropriate assembly site, chaperones wouldguarantee that organelle assembly proceeds in anorderly fashion.

Acknowledgements

SJH is supported by NIH grants RO1DK51406 andRO1AI29549, GW by NIH grant RO1GM54033, and SDKby grants from the Swedish Research Council NFR and the

ŽSwedish Foundation for Strategic Research Structural Bi-.ology Network . FGS was supported by an NSF Predoctoral

Fellowship.

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