The EMBO Journal vol.1 1 no.13 pp.4747-4756, 1992

Interactive surface in the PapD chaperone cleft is conserved in pilus chaperone superfamily and essential in subunit recognition and assembly Lynn N.Slonim, Jerome S.Pinkner, Carl-lvar Branden' and Scott J.Hultgren Department of Molecular Microbiology, Box 8230, Washington University School of Medicine, St Louis, MO 63110, USA, and 'ESRF, BP220, F-38043 Grenoble Cedex, France Communicated by S.Normark

The assembly of adhesive pili in Gram-negative bacteria is modulated by specialized periplasmic chaperone systems. PapD is the prototype member of the superfamily of periplasmic pilus chaperones. Previously, the alignment of chaperone sequences superimposed on the three dimensional structure of PapD revealed the presence of invariant, conserved and variable amino acids. Representative residues that protruded into the PapD cleft were targeted for site directed mutagenesis to investigate the pilus protein binding site of the chaperone. The ability of PapD to bind to fiber-forming pilus subunit proteins to prevent their participation in misassembly interactions depended on the invariant, solvent-exposed arginine-8 (R8) cleft residue. This residue was also essential for the interaction between PapD and a minor pilus adaptor protein. A mutation in the conserved methionine-172 (M172) cleft residue abolished PapD function when this mutant protein was expressed below a critical threshold level. In contrast, radical changes in the variable residue glutamic acid-167 (E167) had little or no effect on PapD function. These studies provide the first molecular details of how a periplasmic pilus chaperone binds to nascently translocated pilus subunits to guide their assembly into adhesive pili. Key words: adhesive pili/biogenesis/immunoglobulin fold/ pathogenesis/periplasmic chaperones

Introduction The folding and assembly of proteins in vivo involves sequential domain -domain interactions within and between proteins that must be biologically productive to ensure correct assembly (Crooke et al., 1988a,b; Lecker et al., 1989, 1990; Ellis and van der Vies, 1991). If correct domain-domain interactions are in competition with incorrect interactions, then the role of chaperones in vivo may be to block misassembly reactions by making non-productive interactions kinetically unfavorable (Ellis and van der Vies, 1991). Chaperones have been found to be essential in a variety of eukaryotic compartments including chloroplasts (Hemmingsen et al., 1988; Chen et al., 1990; Marshall et al., 1990), mitochondria (Ostermann et al., 1989; Kang et al., 1990; Manning-Krieg et al., 1991), the lumen of the endoplasmic reticulum ( Sadler et al., 1989; Flynn et al., 1991) and in the cytosol (Grimm et al., 1991). Most studies of chaperones in bacteria have been limited to those localized Oxford University Press

in the cytoplasm (Ellis and Hemmingsen, 1989; Goloubinoff et al., 1989; Morimoto et al., 1990; Randall et al., 1990; Skowyra et al., 1990). However, in Gram-negative bacteria, complicated heteropolymeric fibers called pili are assembled after the secretion of subunit proteins across the cytoplasmic membrane. The correct assembly of a single heteropolymeric pilus depends on transient interactions of each protein subunit type with a periplasmic chaperone. The function of periplasmic pilus chaperones seems to be to bind to and cap interactive subunits imported into the periplasmic space and target them to outer membrane uncapping and assembly sites (Kuehn et al., 1991). However, details of their mechanism of action are unknown. Microbial attachment to host epithelial surfaces is often an initial event in colonization and subsequent infection and results mainly from the binding of microbial adhesins to complementary receptors on host cells. P pili are surfaceassociated composite fibers and have been implicated as important virulence determinants in pyelonephritogenic strains of Escherichia coli (Lund et al., 1988; Kisielius et al., 1989; Pecha et al., 1989; Svanborg Eden et al., 1989). P pili mediate binding to a digalactoside component, Galci(I-4)Gal, of the globoseries of glycolipids present on uroepithelial cells (Lund et al., 1987; Lindberg et al., 1987). Eleven genes are necessary for the regulation and biogenesis of P pili and are localized in the pap gene cluster (Normark et al., 1986; Hultgren et al., 1991a, 1991b). The pilus is a composite fiber consisting of a helical rod and a thin tip fibrillum (Kuehn et al., 1992). The tip fibrillum is joined end to end to the pilus rod, and is composed mostly of repeating subunits of PapE arranged in an open helical configuration (Kuehn et al., 1992). PapG is the Galct(1-4)Gal binding adhesin which is located at the distal end of the tip fibrillum. The pilus rod is composed of repeating subunits of PapA arranged with a helical symmetry of 33 subunits in 10 turns with a 244.5 A axial repeat (Gong et al., 1992). PapF and PapK are minor components of the tip fibrillum and are thought to be required for the initiation of pilus tip fibrillum and rod formation respectively (Lindberg et al., 1986, 1987; Kuehn et al., 1992; F.JacobDubuisson, J.Heuser, K.Dodson, S.Normark and S.Hultgren, submitted). Pilus assembly depends on two proteins which are not components of the final pilus structure: PapD and PapC (Norgren et al., 1987; Lindberg et al., 1989; Hultgren et al., 1989; Kuehn et al., 1991). PapD is a periplasmic chaperone protein which binds to each pilus subunit forming assembly-competent complexes. PapC is thought to serve as an outer membrane assembly platform to which chaperone subunit complexes are targeted (K.Dodson, F.JacobDubuisson and S.Hultgren, submitted). The PapD three dimensional structure has been solved and recently refined to 2.0 A resolution (Holmgren and Branden, 1989). PapD consists of two globular domains oriented towards one another giving the molecule an overall boomerang shape with a cleft between the two domains. Each 4747

L.N.Slonim et al.

domain is a A-barrel structure formed by two antiparallel a pleated sheets giving this protein a topology similar to an immunoglobulin fold (Holmgren and Branden, 1989). It differs from the classical immunoglobulin fold by strand switching at the edges of the sheets. Similar modulations of the classical fold have been observed in CD4 (Ryu et al., 1990) and in the human growth hormone receptor (deVos et al., 1992). PapD is the prototype member of a family of periplasmic chaperone proteins in Gram-negative bacteria, all of which are predicted to have a similar immunoglobulinlike topology. Analysis of an alignment of the chaperone sequences superimposed on the three dimensional structure of PapD has revealed invariant, highly conserved and variable residues present in the chaperone superfamily (Holmgren et al., 1992, Figure 1). Most of the conserved residues were found concentrated in the 3 strands in the cleft region between the domains, while the variable residues were usually situated in loop regions between the ,B strands. In this study, an analysis of site directed mutations in solventexposed cleft residues revealed that the highly conserved cleft region between the two domains probably forms a pilus subunit binding pocket.

crystallized PapD which often corresponds to the location of a protein's active site thus making it an attractive residue to mutagenize. E167, the third amino acid targeted for mutagenesis, is part of a negatively charged cluster of variable amino acids in a loop of PapD which is situated at the rim of the cleft in the second domain. It was mutagenized to test the role of a variable loop structure in subunit binding. R8, M172 and E167 are representative of the three classes of non-structural amino acids identified in the chaperone superfamily of proteins: invariant, conserved and variable, respectively. Mutagenesis and analysis of the PapD chaperone The codons encoding R8, M172 and E167 of PapD were specifically altered by site directed mutagenesis to examine the effect of a point mutation at these positions on PapD activity. Each point mutant papD gene was subcloned downstream from the isopropyl ,B-D-thiogalactoside (IPTG) inducible Ptac promoter in pMMB91 resulting in the plasmids denoted in Table I and depicted in Figure 3 as pN # N'. The plasmid names indicate the nature of each site directed mutation (i.e. pR8G signifies that arginine at position 8 was changed to a glycine). Plasmid pLS101 carries the wild type papD gene on an isogenic construct (Figure 3). R8 was changed to a glycine (R8G), alanine (R8A) and a methionine (R8M). R8G is a radical mutation which was made to remove completely the conserved side chain and thus any interaction it may make. The R8A mutation was made for the same reasons but is a better substitution since it is less likely to result in structural complications. The R8M mutation is reasonably isosteric with the methionine side chain substituting for the hydrophobic aliphatic chain of arginine thereby maintaining side chain packing while at the same time removing the charged guanidinium group. This mutation would abolish the ability of this side chain to form hydrogen bonds or salt bridges with Pap subunits. M172 was changed to a lysine (M172K). The M172K mutation is largely isosteric and should not disturb the side chain packing for steric reasons. However, it introduces a new positively

Results Invariant, conserved and varable PapD cleft residues targeted for site directed mutagenesis Three surface exposed residues that have no apparent structural function, R8, M172 and E167, were targeted for site directed mutagenesis in order to investigate their role in the mechanism of action of PapD (Figure 2). R8 is an invariant cleft residue that is present in every member of the periplasmic chaperone family. It protrudes into the solvent in the empty cleft from domain 1. R8 is unique in that although it is a non-structural amino acid, it is invariant. The highly conserved M172 is also solvent exposed and protrudes into the cleft from domain 2. M172 was present in six of seven chaperones examined (Holmgren et al., 1992). This residue was the major heavy atom site of 1

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Fig. 1. PapD amino acid sequence and periplasmic chaperone superfamily consensus sequence. Amino acid sequence of the PapD chaperone is shown (Holmgren et al., 1992), using the conventional one-letter codes. Amino acids listed below, in boxes, are found at these positions throughout the entire chaperone superfamily and form part of a chaperone consensus sequence (Holmgren et al., 1992). Boxes that contain a line signify residues that are highly conserved but not invariant. Dots in the PapD sequence indicate regions where gaps were introduced in order to derive the consensus

sequence. Unboxed amino acids were variable throughout the chaperone family. The arrows below the sequences indicate the positions of ( strands of PapD.

4748

PapD subunit binding pocket

papDl lesion in HBO1I/pPAP37 (papDl) was complemented

Fig. 2. Alpha-carbon backbone photograph of PapD denoting point mutations. Alpha-carbon backbone of PapD (blue) containing residues which were mutagenized in this study. R8 and M172 (green) are invariant and highly conserved residues, respectively, located in the PapD cleft. E167 (red) is a variable residue located at the tip of the protein on the leading edge of the cleft. All of these residues are solvent exposed.

Table I. Summary of PapD point mutations Residue

Domain

Class

Mutation

Plasmid

R8

1

Invariant

G,A,M

pR8G pR8A pR8M

M172 E167

2 2

Conserved Variable

K

pM172K pE167D pE167H pE167T pE167G

D,H,T,G

in trans with the wild type papD gene on pLS101. Periplasmic extracts were obtained from identical late log phase cultures after induction of the Ptac promoter with increasing concentrations of IPTG ranging from 10-5 to 1.0 mM. The presence of PapD was analyzed after SDS -PAGE in immunoblots using anti-PapD antisera. The results demonstrated that induction of papD expression from pLS101 with increasing concentrations of IPTG yielded a corresponding increase in the concentration of PapD localized in the periplasmic space (Figure 4). In a similar analysis, all of the plasmids listed in Table I expressed mutant PapD proteins that were found to be stable and secreted into the periplasmic space in amounts that were also dependent on the IPTG concentration used for induction (data not shown). In addition, the amount of pili produced by HB101/pPAP37 (papDl) complemented in trans with wild type papD on pLS 101 was quantitated after growth in broth supplemented with IPTG concentrations ranging from 10-5 to 1.0 mM. Pili were purified from cultures adjusted to the same optical density (OD) at 540 nm and equal aliquots from each induction condition were analyzed by SDS-PAGE. Densitometric scanning of the Coomassie blue-stained PapA bands together with the PapD immunostained bands in Figure 4, revealed that the amount of isolatable pili produced by each culture was proportional to the amount of PapD present in the periplasm (Figure 5). In an analogous experiment, overexpression of papD from pLS101 in the presence of the pap operon in HB101/pPAP5 + pLS101 increased the amount of pili formed by 2-fold suggesting that PapD is limiting in pilus assembly (data not shown). Electron microscopy confirmed that an increase in the concentration of PapD resulted in an increase in the proportion of heavily piliated cells (data not shown), and that in the absence of PapD, the cells were essentially nonpiliated. This complementation system provided an on -off switch control of PapD expression and was used to synchronize pilus assembly in each strain examined.

PapD point mutations were constructed by using either PCR or oligodirected mutagenesis. The mutant papD genes were subcloned into vector pMMB91 under the IPTG-inducible Ptac promoter (Furste et al., 1989). The classification of each residue was derived from a consensus sequence compiled from sequence alignments of several Gram-negative pilus chaperones (Holmgren et al., 1992).

Role of R8, M172 and E167 in subunit binding The genetic system established above was used to examine the ability of each point mutant papD gene to complement in trans the papDl inactivated gene on pPAP37. Strain HB101/pPAP37 (papDI) was transformed with pLS101,

charged group into the cleft which may interfere with subunit binding directly or indirectly. E167 was changed to a glycine (E167G), a threonine (E167T), an aspartic acid (E167D) and a histidine (E167H). The E167G mutation resulted in the complete removal of the glutamic acid side chain and thus would abolish any interaction in which it is involved. The E167T mutation removes the negatively charged side chain, but leaves a polar group. The E167D mutation is a conservative change, leaving the negatively charged functional group, but shortening the side chain by one CH2 unit. This mutation examined the importance of the positioning of the charge. The E167H mutation replaced a negatively charged side chain with a potentially positively charged one which should have no structural effect on PapD. The ability to modulate the in vivo concentration of PapD expressed from each plasmid was used as a tool to study the effect of each point mutation on PapD activity. The

pE167D, pE167H, pE167T, pE167G, pM172K, pR8G, pR8A and pR8M. Pilus assembly was initiated in synchrony in each culture by growth on CFA agar supplemented with either 10-2 or 10-5 mM IPTG. Hemagglutination (HA) titers were determined for each of the complementation groups as one measurement of pilus assembly. The HA assay is essentially a measurement of the incorporation and presentation of the PapG adhesin in the pilus tip fibrillum, a process which requires PapD (Hultgren et al., 1989; Kuehn et al., 1992). If PapG is present in the tip fibrillum in a native-like confonnation, it will bind to Gala(14)Gal present in the globoseries of glycolipids on human type A erythrocytes and cause agglutination. HB101/pPAP37 (papDl) + pR8G was HA negative after growth on either concentration of IPTG (Figure 6). HB101/pPAP37 (papDl) + pR8A or pR8M were also HA negative. This result argued that the invariant R8 residue was critical for the ability of PapD to modulate the assembly of adhesive pili. In contrast, 4749

L.N.Slonim et al.

E

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1 Kb Fig. 3. Genetic constructs. Plasmid pPAP5 contains the entire pap gene cluster. Plasmid pPAP37 is isogenic to pPAP5 but contains an XhoI linker insertion in papD designated DI. * signifies pBR322. The general function of each gene product is indicated. pLSl01 contains the wild type papD gene downstream of the inducible Ptac promoter in plasmid pMMB91 (O). Plasmids isogenic to pLS1O1, except for the point mutation in papD (X), are designated as pN # N'. The first N indicates the wild type amino acid, # is the residue number and the N' is the mutagenic amino acid.

the E167D, E167H, E167T and E167G mutant PapD proteins were able to complement the papDl genetic lesion whether induced with high or low IPTG concentrations. Induction with 10-2 mM IPTG yielded HA titers that were virtually identical to HA titers observed when wild type papD was expressed in trans from pLS 101 (Figure 6). Induction with 10-5 mM IPTG yielded HA titers that were approximately 3.0, 2.5, 6.0 and 11-fold lower than wild type, respectively (Figure 6). These results demonstrated that the variable E167 residue was not critical for the ability of PapD to modulate the assembly of adhesive pili, but could play a minor role which was amplified under limiting concentrations of PapD. The mutation in the conserved M172 residue (M172K) reduced the HA titer by 2.3-fold in cultures grown on 10-2 mM IPTG, yet growth on 10-5 mM IPTG resulted in HA negative cultures (Figure 6) suggesting that a threshold concentration of M172K PapD was required to modulate the assembly of adhesive pili. The HA titers of each complementation group were correlated with the amount of pili produced by each culture. Pili were purified from each culture which had been adjusted to an identical ODMO after growth on CFA agar supplemented with either 10-2 or 10-5 mM IPTG. Equal aliquots of the pilus preparations were then examined by immunoblotting with anti-PapA antiserum (Figure 7A). Pili purified from cultures complemented with pLS101, pE167D, pE167H and pM172K grown on 10-2 mM IPTG all contained a 19.5 kDa band that reacted with anti-PapA antiserum (Figure 7A, lanes 1-4 respectively). The pR8G, pR8A or pR8M-complemented cells produced no detectable surface localized PapA (Figure 7A, lanes 5, 6 and 7 respectively).

4750

~~_

Fig. 4. Relationship between the concentration of IPTG used to induce papD expression and the subsequent amount of PapD localized in the periplasm. Anti-PapD antiserum was used in a Western blot of periplasmic extracts from cultures of strain HBl0l/pPAP37 (papDl) + pLS101 where the expression of papD was induced with the following concentrations of IPTG: 0.00001 (lane 1), 0.001 (lane 2), 0.01 (lane 3), 0.1 (lane 4) and 1.0 mM (lane 5). Note that increasing the IPTG concentration used for induction yielded corresponding increases in the concentration of PapD present in the periplasmic preparations.

Interestingly, after growth on 10-5 mM IPTG, pM172Kcomplemented cells became non-piliated as did the pR8G, pR8A and pR8M-complemented cells as determined by the lack of an immunoreactive PapA band (Figure 7B, lanes 4, 5, 6 and 7 respectively). In contrast, pLS101 (PapD), pE167D and pE167H-complemented cells grown on 10-5 mM IPTG produced isolatable pili that contained a 19.5 kDa PapA band as determined by Western blot analysis (Figure 7B, lanes 1, 2 and 3 respectively). These results were consistent with the HA titers reported in Figure 6, confirming that the invariant R8 residue was critical for PapD

PapD subunit binding pocket 100

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Fig. 5. Relationship between periplasmic PapD concentration and production of pili. Pili were isolated from strain HB1O1/pPAP37 (papDl) + pLSIOI after induction of papD expression for 1 h from pLSIO1 with five different IPTG concentrations ranging from 1.0 to i0-5 mM. Equal aliquots of each pilus preparation were analyzed by SDS-PAGE and each Coomassie blue-stained PapA band was scanned by a densitometer. The densitometric reading of the PapA band present after induction of PapD with 1.0 mM IPTG was normalized to 100%. All the other points are expressed as percentages of this value. The relative amount of PapD induced by the five different IPTG concentrations as shown in Figure 4 was also determined by densitometry. The densitometric reading of PapD induced by 1.0 mM IPTG was normalized to 100%. The relative amount of pili produced at five different IPTG concentrations was plotted against the known amount of PapD produced at the same concentrations. This graph shows that the amount of surface-localized pili is proportional to the in vivo concentration of periplasmic PapD

function. In addition, the M172 residue was required for PapD function when PapD was produced below a threshold concentration, in contrast to the variable E167 residue which was non-essential. The R8 mutation in PapD abolished interactions with fiber-forming pap subunits We examined the ability of R8G, R8A and R8M PapD to interact with PapA, PapE and PapG in order to investigate which event in the pilus biogenesis the R8 mutations had abolished. HBO1I strains carrying plasmids that expressed only the papA gene (pPAP43, Lund et al., 1985), only the papE gene (pPAP63, Lindberg et al., 1989) or the papG gene (pLS201, see Materials and methods) were complemented with plasmids containing the wild type papD gene (pLS 101) or the R8 mutant papD genes (pR8G, pR8A or pR8M). The ability of R8G, R8A and R8M PapD to bind to PapA and PapE was assayed by testing for the presence of these subunits in periplasmic extracts since it is known that subunits are degraded in the absence of an interaction with the chaperone (Hultgren et al., 1989; Lindberg et al., 1989; Kuehn et al., 1991). When either PapA or PapE was expressed in the absence of PapD they were proteolytically degraded as determined by immunoblotting with the corresponding antisera (Figure 8A and B, lane 1). However, expression of wild type papD, in trans, from plasmid pLS 101 stabilized PapA and PapE in the periplasm where they were detected by the appropriate antiserum (Figure 8A and B, lane 2). In contrast, expression of the R8G mutant PapD protein, in trans, did not block the proteolytic degradation of PapA, the major pilus subunit, or PapE, the major component of the tip fibrillum (Figure 8A and B, lane 3). The R8A and R8M PapD mutants slightly or

C], 0