Functional and Structural Characterization of Polysaccharide Co

Functional and Structural Characterization of PolysaccharideCo-polymerase Proteins Required for Polymer Export inATP-binding Cassette Transporter-dependent CapsuleBiosynthesis Pathways*

Received for publication, February 4, 2011, and in revised form, March 17, 2011 Published, JBC Papers in Press, March 18, 2011, DOI 10.1074/jbc.M111.228221

Kane Larue‡, Robert C. Ford§, Lisa M. Willis‡, and Chris Whitfield‡1

From the ‡Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario, Canada N1G 2W1 and the§Faculty of Life Science, University of Manchester, Manchester M60 1QD, United Kingdom

Neisseria meningitidis serogroup B and Escherichia coli K1bacteria produce a capsular polysaccharide (CPS) that is com-posed of �2,8-linked polysialic acid (PSA). Biosynthesis of PSAin these bacteria occurs via an ABC (ATP-binding cassette)transporter-dependent pathway. In N. meningitidis, export ofPSA to the surface of the bacterium requires two proteins thatform an ABC transporter (CtrC and CtrD) and two additionalproteins, CtrA and CtrB, that are proposed to form a cell enve-lope-spanning export complex. CtrA is a member of the outermembrane polysaccharide export (OPX) family of proteins,which are proposed to form a pore to mediate export of CPSsacross the outer membrane. CtrB is an inner membrane proteinbelonging to the polysaccharide co-polymerase (PCP) family.PCP proteins involved in other bacterial polysaccharide assem-bly systems form structures that extend into the periplasm fromthe inner membrane. There is currently no structural infor-mation available for PCP or OPX proteins involved in an ABCtransporter-dependent CPS biosynthesis pathway to supporttheir proposed roles in polysaccharide export. Here, we reportcryo-EM images of purified CtrB reconstituted into lipid bilay-ers. These images contained molecular top and side views ofCtrB and showed that it formed a conical oligomer thatextended �125 A from the membrane. This structure is consis-tentwithCtrB functioning as a component of an envelope-span-ning complex. Cross-complementation of CtrA and CtrB inE. coli mutants with defects in genes encoding the correspond-ing PCP and OPX proteins show that PCP-OPX pairs requireinteractions with their cognate partners to export polysaccha-ride. These experiments add further support for themodel of anABC transporter-PCP-OPX multiprotein complex that func-tions to export CPS across the cell envelope.

Capsular polysaccharides (CPSs)2 are produced by manybacteria and form a layer (the capsule) that coats the surface of

the cell. The capsule helps prevent bacteria from desiccationand is important in infection because it provides protectionfrom the host immune response by interfering with both com-plement-mediated killing (1–3) and the adhesion of monocytesand neutrophils to the bacteria (4, 5). Consequently, capsulesare an important virulence factor for numerous pathogenicbacteria including those that cause disease in livestock, likePas-teurella multocida, and bacteria such as Escherichia coli andNeisseria meningitidis, which cause bacterial meningitis andsepsis in humans (6).CPSs vary significantly in carbohydrate composition and gly-

cosidic linkages. Despite the diversity of structures, the major-ity of CPSs produced by Gram-negative bacteria are assembledvia either an ATP-binding cassette (ABC) transporter-depen-dent biosynthesis pathway or a Wzy-dependent biosynthesispathway (for review, see Refs. 7, 8). These pathways are funda-mentally different in the topology of the polysaccharide polym-erization reaction at the inner membrane. In Wzy-dependentpathways, polymer repeat units are assembled at the cytoplas-mic face of the inner membrane on undecaprenol diphosphateand flipped to the periplasmic face of the inner membrane in aprocess that requires a putative flippase protein, Wzx. Polym-erization occurs on the periplasmic face of the innermembranein a process that is dependent on the polymerase Wzy. In con-trast, CPSs assembled via ABC transporter-dependent path-ways are polymerized entirely at the cytoplasmic face of theinner membrane. The complete polysaccharide is exportedacross the inner membrane in a process that requires an ABCtransporter that is composed of two nucleotide binding domainsubunits and two transmembrane domain subunits.Translocation of full-length CPS to the surface of the cell

requires a polysaccharide co-polymerase (PCP) protein andan outer membrane polysaccharide export (OPX) protein inboth the Wzy-dependent pathway and the ABC transporter-dependent pathway (for review, see Refs. 7, 8). PCP proteinsshare a conserved membrane topology that consists of N- andC-terminal membrane-spanning regions (or a C-terminalmembrane-associated region) that flank a predominantly�-helical periplasmic domain (for review, see 7, 9, 10). Most

* This work was supported by operating funding from the Canadian Institutesof Health Research (to C. W.).

1 Recipient of a Canada Research Chair. To whom correspondence should beaddressed. E-mail: [email protected].

2 The abbreviations used are: CPS, capsular polysaccharide; ABC, ATP-bindingcassette; DM, decyl �-D-maltopyranoside; DMPC, 1,2-dimyristoyl-sn-glycero-3-phosphocholine; DOPC, 1,2-dioleoyl-sn-glycero-3-phosphocho-line; DOPE, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine; DOPS, 1,2-dioleoyl-sn-glycero-3-phospho-L-serine; MTSET, methanethiosulfonate

ethyltrimethylammonium; OGM, Oregon Green 488 maleimide carboxylicacid; OPX, outer membrane polysaccharide export; PCP, polysaccharideco-polymerase; PSA, polysialic acid; SB3-14, dimethylmyristylammoniopropanesulfonate.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 286, NO. 19, pp. 16658 –16668, May 13, 2011© 2011 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

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PCP proteins possess a proline-containing consensus motif(PX2PX4SPKX11GGMXGAG) that is located at the interfacebetween the periplasmic domain and the C-terminal mem-brane-spanning region, as well as amino acid sequences that arepredicted to form periplasmic coiled-coils (7, 9–11). ThreePCP subfamilies have been established based on their roles indifferent polysaccharide biosynthesis pathways (9), and theirphylogenetic relationships have been investigated (7). PCP-1proteins (Wzz homologs) determine LPS O antigen chainlength in aWzy-dependent biosynthesis pathway and representthe best characterized family members. PCP-2a proteins areinvolved in Wzy-dependent CPS biosynthesis and possess aC-terminal cytoplasmic domain with tyrosine kinase activity;this domain is absent in PCP-1 and PCP-3 proteins. CertainPCP-2 proteins (including proteins from prominent Gram-positive bacteria) lack the C-terminal cytoplasmic domain butfunction in conjunction with a separate cognate kinase poly-peptide. PCP-3 proteins are confined to the ABC transporter-dependent CPS biosynthesis. Structural information is avail-able for PCP-1 (12, 13) and PCP-2a (14) proteins and shows thatthey form conical oligomers that extend�100Å from the innermembrane into the periplasm.OPX proteins have limited sequence similarity and are

identified by a polysaccharide export sequence motif (7).Wza from E. coliK30 is the prototype OPX protein and oper-ates in aWzy-dependent assembly system. The crystal struc-ture of Wza shows that it is an octamer that forms a large(140 � 105 Å) barrel with eight amphipathic �-helices thatinsert into the outer membrane to form a channel (15). Themajority of Wza is located in the periplasm, where it encloses acentral water-filled cavity. A cryo-EM structure of a complex ofWza and the PCP-2a protein Wzc shows that PCP-OPX pro-teins are capable of assembling into cell envelope-spanningstructures that are consistent with their proposed roles in poly-saccharide export (16). PCP-3 proteins and their partner OPXproteins are thought to form a similar polysaccharide exportcomplex in ABC transporter-dependent assembly systems, butthere are currently no structural data available supporting thishypothesis.E. coliK1 andN.meningitidis group B bothmake ABC trans-

porter-dependent capsules that are polymers of �2,8-linkedpolysialic acid (PSA) (17). The biosynthesis of PSA in E. coliserves as an influential model for ABC transporter-dependentCPS assembly and export (reviewed in (18, 19). The E. coli PSAexport apparatus consists of two proteins providing the trans-membrane domains and nucleotide binding domains of theABC transporter (KpsM and KpsT, respectively), the PCP-3protein (KpsE) and the OPX protein (KpsD). Homologous pro-teins in other bacteria are proposed to function in similar CPSexporters. As expected, given the shared mode of assembly,conserved components are encoded by the capsule loci fromE. coli and N. meningitidis (7, 18, 20). PSA export in N. menin-gitidis requires two proteins that form an ABC transporter(CtrC and CtrD), a PCP-3 protein (CtrB), and an OPX protein(CtrA). In this study, we provide evidence that the PCP-3-OPXpairs from E. coli and N. meningitidis are functionally inter-changeable, but they require cognate interactions betweenpartner proteins for CPS export. In addition, cryo-EM is used to

examine the PCP-3 protein CtrB from N. meningitidis recon-stituted in lipid bilayers and shows that it forms a structureresembling PCP-1 and PCP-2 familymembers. These data sup-port the model for a cell envelope-spanning CPS export systemin ABC transporter-dependent biosynthesis pathways.

EXPERIMENTAL PROCEDURES

Strains, Plasmids, and Growth Conditions—The bacterialstrains and plasmids used in this study are listed in Table 1.Bacteria were grown at 37 °C in LBmedium supplementedwithampicillin (100 �g/ml), chloramphenicol (34 �g/ml), anhydro-tetracycline (10 ng/ml), and arabinose (0.1% w/v) as required.K1F phage (21) was propagated using E. coli EV36 and stored ata titer of 1012 pfu/ml in LB medium containing chloroform.DNA Methods—Genes were amplified by PCR using Pwo

polymerase (Roche Applied Science) with custom oligonucleo-tides (Sigma) designed to introduce appropriate restrictionsites and N-terminal polyhistidine tags when required. PCRproducts were purified by gel extraction using the PureLinkGelExtraction kit (Invitrogen). Plasmid DNA was purified usingthe PureLink Plasmid Miniprep kit (Invitrogen). Purified PCRproducts and plasmid DNA were digested using restrictionendonucleases and ligated according to individual manufactur-er’s specifications. pWQ557, pWQ565, pWQ566, pWQ567,and pWQ568 were generated by site-directed mutagenesisaccording to the Stratagene QuikChange Site-directed Mu-tagenesis procedure using PfuUltra polymerase (Stratagene)and DpnI restriction endonuclease. Gene constructs weretransformed into E. coli TOP10 by electroporation or by heatshock and verified by sequencing (University of Guelph Labo-ratory Services).Phage Sensitivity Assays—10 �l of overnight culture from

appropriate E. coli strains was cross-streaked on LB agar platesprepared with 50 �l of K1F bacteriophage (1011 pfu/ml) spreadover half of the surface of the media. Plates were photographedafter an incubation period of 4–6 h at 37 °C.Site-directed Fluorescence Labeling—CtrB proteins with

N-terminal decahistidine tags were expressed from pWQ555,pWQ556, pWQ557, and pWQ558 in E. coli BL21. Labeling ofmembrane-embedded proteins with Oregon Green 488maleimide carboxylic acid (OGM) was based on the protocoldescribed by Culham et al. (22). Cells expressing each CtrBvariant were grown in 200 ml LB of broth inoculated with anovernight culture (1% v/v) and incubated at 37 °C with shakingfor 2 h. Cells were harvested by centrifugation at 5000� g for 10min at 4 °C, resuspended in 10 ml of buffer A (50 mM Tris-Cl,pH 8.0, containing 100 mM NaCl), and divided into three ali-quots. OGM (40 �M) was added to aliquot 1, labeling Cys resi-dues accessible to the periplasm. The sample was incubated for15 min at room temperature and quenched with 1 mM �-mer-captoethanol. In aliquot 2, periplasmic Cys residues wereblocked with methanethiosulfonate ethyltrimethylammonium(MTSET) (2 mM) for 15 min at room temperature. Aliquots 1and 2 were washed twice by centrifugation using buffer A toresuspend the cell pellets. All three aliquots were pelleted bycentrifugation and resuspended in 2ml of buffer A containing 5mMEDTA, 100�g/ml lysozyme, and 20% (w/v) sucrose. After a15-min incubation at room temperature, 18 ml of cold water

Analysis of Bacterial Polysaccharide Co-polymerase Proteins

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was added to each aliquot to disrupt the cells. OGM (10 �M)was then added to aliquots 2 and 3, and all three aliquots weresubjected to 30 s of sonication (10 s on, 10 s off), followed by a15-min incubation at room temperature. This labeled any avail-able and unblocked cytoplasmic Cys residues in aliquot 2 andlabeled all of the accessible Cys residues in aliquot 3. The label-ing reactionwas quenched in aliquots 2 and 3with 1mM �-mer-captoethanol, and the unbroken cells and cell debris were col-lected from all three aliquots by centrifugation at 15,000� g for15 min at 4 °C. Membranes were collected from the cell-freelysates by centrifugation at 100,000 � g for 30 min at 4 °C andsolubilized overnight at 4 °C in 1 ml of buffer A containing 1%(w/v) dimethylmyristylammonio propanesulfonate (SB3-14)(Sigma-Aldrich). Insoluble material was removed by centrifu-gation at 16,000 � g for 10 min at room temperature, and thesupernatant wasmixed with 100 �l of nickel affinity resin (Qia-gen) for 1 h at room temperature. The solubilized membrane-resin mixture was then loaded into 1-ml minicolumns (Bio-Rad) andwashed twice with 1.5ml of buffer A containing 0.05%(w/v) SB3-14. Proteins were eluted with 200 �l of buffer A con-taining 1% (w/v) SDS and 500 mM imidazole into 200 �l ofSDS-PAGE loading buffer. 15-�l samples were analyzed bySDS-PAGE using 12% (w/v) gels, and fluorescence was re-cordedwith exposure of the gels toUV light using aBio-RadGelDoc equippedwith a CCD camera. Gels were stainedwith Coo-massie Blue following UV exposure to visualize protein bands.The experiment was conducted in duplicate and produced thesame results.To confirm the interpretation of SDS-PAGE results from the

OGM-labeling experiments described above, purified CtrB

variants were labeled with OGM in an SDS solution and ana-lyzed by SDS-PAGE followed by Western blotting. The CtrBvariants were expressed from their respective plasmids in200-ml cultures and purified according to the proceduredescribed above but omitting OGM labeling and MTSETblocking. The solubilizedmembrane-resinmixturewaswashedwith 2ml of buffer A containing 0.05% (w/v) SB3-14 and 20mM

imidazole followed by 0.5ml of buffer A containing 0.05% (w/v)SB3-14 and 50 mM imidazole. Proteins were eluted with 300 �lof buffer A containing 1% (w/v) SDS and 500 mM imidazole. A50-�l aliquot from each CtrB sample was incubated with OGM(40�M) for 15min at room temperature and quenchedwith�1mM �-mercaptoethanol. Untreated and OGM-treated sampleswere analyzed by SDS-PAGE using 12% (w/v) gels and trans-ferred to nitrocellulose membranes. The nitrocellulose mem-branes were exposed to UV light, and fluorescence wasrecorded as described above. After exposure to UV light, thenitrocellulosemembranes were probed with anti-His5 antibod-ies (Qiagen) using an alkaline phosphatase-conjugated second-ary antibody (Jackson ImmunoResearch Laboratories). Sepa-rate gels were also stained with Coomassie Blue to visualizeprotein bands (data not shown).Purification of CtrB for EM Studies—His10-CtrB was overex-

pressed from pWQ555 in E. coli TOP10. One liter of LB con-taining ampicillin was inoculated with 10 ml of overnight cul-ture and grown with shaking at 37 °C. When an A600 nm of 0.6was reached, expression of CtrB was induced by adding 0.1%(w/v) arabinose, and growth was continued for an additional2 h. Cells were harvested by centrifugation at 5000 � g for 10min at 4 °C. Cell pellets from 1-liter cultures were resuspended

TABLE 1Bacterial strains and plasmids

Strain/plasmid Description or genotype Ref. or source

E. coli TOP10 F� mcrA �(mrr-hsdRMSmcrBC) f80�lacZM15 �lacX74 deoR recA1 araD139 �(ara-leu)7697galU galK rpsL (StrR) endA1 nupG

Novagen

E. coli BL21 F� ompT hsdSB(rB� mB�) gal dcm Novagen

E. coli EV36 K-12/K1 hybrid; kps� (26, 59)E. coli RS3175 EV36 �kpsED; kps� R. SilverE. coli EV36 ÄkpsE kpsEmutant strain generated with � Red recombinase system; kps� E. Vimr and S.

SteenbergenE. coli EV36 ÄkpsD KpsDmutant strain generated with � Red recombinase system; kps� E. Vimr and S.

SteenbergenE. coli PA360 F� thr-1 leuB6(Am) fhuA2 lacY1 glnV44(AS) gal-6 �� hisG1(Fs) rfbC1 serA1 galP63 malT1(�R)

xyl-7 mtlA2 �argH1 rplL9(L?) thi-1; kps-null strainCGSC288a (59)

pBAD24 Protein expression vector with multiple cloning site under control of Para; AmpR (60)pWQ284 Protein expression vector, pBAD24 derivative; CmR (61)pACYC184 Protein expression vector; CmR, TcR. (62)pWQ572 Protein expression vector with Ptet promoter, pBAD24 multiple cloning site and p15A origin of

replication; CmR, TcRJ. King

pWQ555 pBAD24 derivative containing an EcoRI/SphI fragment encoding His10-CtrB This studypWQ556 pBAD24 derivative containing an EcoRI/PstI fragment encoding His10-CtrB-N-Cys This studypWQ557 pWQ555 derivative encoding His10-CtrB-R192C This studypWQ558 pBAD24 derivative containing an EcoRI/PstI fragment encoding His10-CtrB-C-Cys This studypWQ559 pBAD24 derivative containing an EcoRI/SalI fragment encoding CtrAB This studypWQ560 pBAD24 derivative containing an EcoRI/HindIII fragment encoding CtrA This studypWQ561 pBAD24 derivative containing an EcoRI/PstI fragment encoding CtrB This studypWQ562 pWQ284 derivative containing an NheI/HindIII fragment excised from pWQ571 encoding KpsED This studypWQ563 pBAD24 derivative containing a KpnI/PstI fragment encoding KpsE This studypWQ564 pBAD24 derivative containing a KpnI/PstI fragment encoding KpsD This studypWQ565 pWQ563 derivative encoding KpsE-P341A This studypWQ566 pWQ563 derivative encoding KpsE-P344A This studypWQ567 pWQ563 derivative encoding KpsE-P350A This studypWQ568 pWQ563 derivative encoding KpsE-P314A-P344A-P350A This studypWQ569 pACYC derivative containing a BamHI/SphI fragment excised form pWQ561 encoding CtrB; CmR This studypWQ570 pWQ572 derivative containing an EcoRI/HindIII fragment excised from pWQ560 encoding CtrA This studypWQ571 pBAD24 derivative containing an EcoRI/SphI fragment encoding KpsED This study

a Coli Genetic Stock Center number, Yale University.




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in 25 ml of 50 mM sodium phosphate, pH 7.0, containing 300mMNaCl and 5 mM EDTA. Cells were disrupted by sonication,and the cell-free lysate was collected by centrifugation at15,000 � g for 20 min at 4 °C. Membranes were collected fromthe cell-free lysate by centrifugation at 100,000 � g at 4 °C. Themembrane pellet was solubilized overnight in 10 ml of 50 mM

sodium phosphate, pH 7.0, containing 300 mM NaCl and 1%(w/v) decyl �-D-maltopyranoside (DM) (Fluka). Insolublematerial was removed by centrifugation at 100,000� g for 1 h at4 °C, and 2ml of His-Select Nickel Affinity Gel (Sigma-Aldrich)was added to the supernatant. The mixture of resin and solubi-lizedmembranes was incubated for 1 h at 4 °Cwith rocking andloaded into a 20-ml Bio-Rad chromatography column. Theresin was washed with 20 ml of 20 mM sodium phosphate, pH7.0, containing 300mMNaCl, 0.1% (w/v) DM, and 20mM imid-azole, followed by 5 ml of the same buffer containing 75 mM

imidazole. His10-CtrB was eluted with a 5-ml fraction of 20mM

sodiumphosphate, pH7.0, containing 300mMNaCl, 0.1% (w/v)DM, and 300mM imidazole, followed by 5ml of the same buffercontaining 500 mM imidazole. Fractions collected throughoutthe purification were analyzed by SDS-PAGE. The fractioncontaining purified CtrB was diluted to 50 ml using 20 mM

sodium phosphate buffer, pH 7.0, containing 150mMNaCl and0.1% (w/v) DM and concentrated by centrifugation using a100,000-Da cutoff filter to 1/100 of the original volume. Proteinconcentrations were determined using the Bio-Rad detergent-compatible protein assay kit with BSA as a standard.Reconstitution of CtrB in Proteoliposomes—Reconstitution

experiments were performed using 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phos-pho-L-serine (DOPS), 1,2-dioleoyl-sn-glycero-3-phosphocho-line (DOPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine(DOPE), DMPC-DOPS mixtures (3:1, 1:1, and 1:3 (w:w)), andDOPC-DOPE mixtures (3:1, 1:1, and 1:3 (w:w)) (Avanti PolarLipids). Lipids and lipid mixtures were prepared at a concen-tration of 2.5 mg/ml and solubilized with 1% (w/v) octyl �-D-1-thioglucopyranoside (Sigma-Aldrich) in a 20 mM sodiumphosphate buffer, pH 7.0, containing 150 mM NaCl. 50-�lreconstitution reactions were prepared at lipid-to-proteinratios of 0.25:1, 0.5:1, and 0.75:1 (w:w) at a protein concentra-tion of 0.5 mg/ml. Reconstitution was initiated by the additionof � 10 hydrophobic beads (Bio-Rad) that had been equili-brated in water. The beads were replaced at�12-h intervals for2–4 days. The formation of proteoliposomes and membranesheets was screened by transmission electron microscopy ofnegatively stained specimens, as described previously (12).Cryo-EM and Image Processing—3-�l samples from recon-

stitution experiments were applied undiluted to Quantifoil R2/2 holey carbon-coated EM grids and plunged frozen in liquidethane using a Vitrobot plunge freezing system (FEI, Hillsboro,OR) using 2 � 1-s blots and an offset setting of �1 mm.Cryo-EM was performed using a Tecnai F20 200kV EM oper-ating in low dose mode at 200 kV at the University of GuelphMicroscopy Imaging Facility.Micrographswere recordedusinga Gatan 4k CCD at defocus values of 2–8 �mwith a calibratedmagnification corresponding to 3.88 Å/pixel. Images wererecorded with a 1-s exposure time corresponding to an overallelectron dose of �2500 electrons/nm2. Images were manipu-

lated using the EMAN software package (23). Molecular sideviews and small membrane patches containing top views ofCtrBwere selected using BOXER. The degree of underfocus foreach micrograph was determined, and the CTF correction wasperformed using the CTFIT program within the EMAN pack-age. Classification of the particles (after CTF correction) andaveraging of translationally and rotationally aligned particleswithin each class was also carried out using the EMANpackage.The contour-delineated CtrB projection map shown in Fig. 5Ewas generated using the SPIDER software contouring function(24).Analysis of PSA—To examine total cellular PSA, E. coli

strains were grown overnight on LB agar at 37 °C andwhole celllysates were prepared for SDS-PAGE and Western blotting.Cells were harvested from the surface of the LB agar andresuspended in water. The equivalent of 1 ml of cells with anA600 nm � 3.0 was collected by centrifugation and resuspendedin 150 �l of 70% (v/v) ethanol. Samples were incubated for 15min at room temperature, harvested by centrifugation, resus-pended in 150 �l of 100% ethanol, and incubated for 15 min atroom temperature. Cells were collected again by centrifuga-tion, and the supernatant from each sample was discarded.After allowing the cell pellet to dry completely, it was resus-pended in a 50-�l solution containing 10 �g/ml DNase I, 10�g/ml RNaseA, 100 �g/ml lysozyme, and 10 mM MgCl2. Thesamples were incubated for 90 min at room temperature andmixed with 50 �l of 2� sample loading buffer (100 mM MOPS,100 mM Tris, 2% (w/v) SDS, 20% (v/v) glycerol, 0.01% (w/v)bromphenol blue, and 250�g/ml proteinaseK, pH7.7). The celllysates were incubated at 37 °C for 2 h, and 15-�l samples wereloaded into 5% (w/v) gels prepared with a 3% (w/v) stackinglayer. The gel buffer and running buffer contained 50 mM

MOPS, 100mMTris, 0.1% (w/v) SDS, and 1mMEDTA. Sampleswere separated by electrophoresis for 45 min at 200 V. AfterSDS-PAGE, samples were transferred to nitrocellulose mem-branes in a buffer containing 50 mM MOPS, 50 mM Tris, 1 mM

EDTA, and 20% (v/v) methanol at 200 mA for 1 h. Westernblots were probed with a monoclonal anti-PSA (mAb 2.2.B,obtained from Dr. Michael Apicella, University of Iowa), usingan alkaline phosphatase-conjugated secondary antibody tovisualize the PSA.

RESULTS

Functional Exchangeability of CtrAB and KpsDE—KpsE andCtrB, the PCP-3 proteins from E. coli K1 and N. meningitidisserogroup B, are phylogenetically related (7). They share 28%identity (50% similarity), and their predicted secondary struc-tures are almost identical (Fig. 1). In contrast, the correspond-ing OPX proteins are significantly different in both size andsequence, particularly in the C-terminal portion of the protein(Fig. 1), and they are well separated in a dendrogram of OPXhomologs (7). To examine the specificity of PCP-OPX pairsrequired for surface expression of PSA, we performed cross-complementation experiments with CtrA and CtrB in EV36-derivative strains carrying mutations in kpsD and kpsE (Fig. 2).Complementation was assessed using the K1F phage sensitivityassay; K1F is a lytic phage that requires capsular PSA for infec-tion in E. coli and is used to detect assembly and export of the




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polymer (21, 25–27). Expression in trans of the ctrAB genes in a�kpsED strain restored K1F phage sensitivity, confirming thatthe PCP-OPX pairs from E. coli and N. meningitidis are func-tionally equivalent. However, when expressed individually, ctrBdid not complement the PSA export defect in EV36 �kpsE. Toverify that CtrB was functional when expressed from pBAD24,a compatible plasmid expressing ctrA (pWQ570) was trans-formed into EV36�kpsE harboring pWQ561 (ctrB). The co-ex-pression of ctrA and ctrB from separate plasmids restoredphage sensitivity. The same results were obtained from recip-rocal experiments performed in EV36�kpsD transformed withplasmids carrying ctrA and ctrB, where ctrA could not restore

polymer export when expressed alone. These experiments sug-gest that cognate interactions between PCP and OPX pairs arerequired for polymers export but that either PCP protein canform a productive interface with the PSA ABC transporter inE. coli EV36.Membrane Topology and Orientation of CtrB—Studies with

KpsE have predicted that this PCP-3 prototype contains anN-terminal transmembrane �-helix and a membrane-associ-ated C-terminal amphipathic helix (28, 29). This differs fromthe canonical PCP structure, which contains two transmem-brane �-helices (7, 9, 10). The membrane topology was there-fore reinvestigated using a cysteine-labeling strategy with CtrB

FIGURE 1. Sequence alignments and predicted secondary structures of PCP-3 and OPX proteins from N. meningitidis serogroup B and E. coli K1.Sequences were aligned using ClustalW (54). Identical residues are highlighted in black, and similar residues are highlighted in dark gray. Predicted �-helices(gray bars) and �-structures (black arrows) were determined using Jpred (55). For CtrB and KpsE, predicted transmembrane-spanning helices are identified bydashed lines and were determined using HMMTOP (56), SOSUI (57), and TMpred (58). The PCP proline motif is boxed in black.




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as the model because this protein lacks natural Cys residues.Three single-Cys His10-CtrB variants were constructed,expressed in E. coli, and subjected to site-directed fluorescencelabeling experiments using the membrane-impermeant re-agents OGM andMTSET. This approach has been validated toestablish the membrane topology and orientation of severalpolytopic inner membrane proteins (22, 30). The location ofeach introduced Cys residue in the three single-Cys variantswas selected to test the general orientation and topology ofCtrB. His10-CtrB provided the negative control in labelingexperiments. CtrB-N-Cys has a Cys residue added immediatelyfollowing the His10-tag and is predicted to be on the cytoplas-mic side of TM1.TheCys residue inCtrB-R192C is predicted tobe periplasmic, whereas CtrB-C-Cys has a C-terminal cysteinethat is predicted to reside on the cytoplasmic end of TM2. Toconfirm that each variant was functionally active, His10-CtrB,CtrB-N-Cys, CtrB-C-Cys, and CtrB-R192C were co-expressed

with CtrA in E. coli RS3175 (�kpsDE) and tested for K1F phagesensitivity. The genes encoding all four CtrB variants restoredK1F sensitivity in RS3175 when co-expressed with ctrA. Toconfirm their reactivity with the dye, the CtrB variants werepurified from membrane preparations and incubated withOGM in an SDS solution and analyzed by SDS-PAGE followedby immunoblotting (Fig. 3A). Exposure of the nitrocellulosemembrane to UV light confirmed that the three His10-taggedCtrB Cys variants fluoresced after incubation with OGM,whereas His10-CtrB remained unlabeled. Immunoblottingshowed that CtrB-N-Cys, CtrB-R192C, and His10-CtrBmigrated as a doublet on SDS-PAGE, and the same was true ina Coomassie-stained gel (data not shown). The band withdecreased mobility corresponding to CtrB-N-Cys fluorescedafter labeling with OGM, and the band with increased mobilitycorresponding to CtrB-R192C fluoresced after labeling withOGM.To distinguish between Cys residues with a periplasmic ver-

sus cytoplasmic localization, intact cells expressing the CtrBproteins were either labeled with membrane-impermeableOGM (targeting periplasmic cysteine residues) or blocked withMTSET and subsequently lysed by sonication and then labeledwith OGM (targeting unblocked cytoplasmic cysteine resi-dues). Fig. 3B shows the fluorescence analysis of an SDS-poly-acrylamide gel containing the four CtrB variants, purified fol-lowing whole cell OGM-MTSET labeling experiments. Thesedata show that the N andC termini of CtrB are exposed and theabsence of OGM labeling in intact cell is indicative of a cyto-plasmic location.As expected, the central portion of the proteinis periplasmic. As with the labeling of purified CtrB variantswith OGM, CtrB-N-Cys migrated on SDS-PAGE as a doubletwith only the upper band fluorescing upon exposure to UVlight. Likewise, only the lower band of the CtrB-R192C doubletfluoresced upon exposure to UV light.Purification of His10-CtrB from E. coli TOP10—Fig. 4 shows

the SDS-PAGE analysis of fractions from a representative CtrB

FIGURE 2. Complementation of PCP-3 and OPX proteins from N. meningi-tidis serogroup B and E. coli K1. E. coli EV36 is a K-12/K1 hybrid strain thatharbors the K1 CPS biosynthesis gene locus (26, 59). Infection and lysis of theidentified E. coli strains by the K1F phage are dependent on the surfaceexpression of PSA.

FIGURE 3. Site-directed fluorescence labeling of CtrB. A, SDS-PAGE andWestern immunoblotting of SDS-solubilized CtrB variants labeled with OGM.The top panel shows a nitrocellulose membrane after exposure to UV lightand was subsequently probed with anti-His antibodies (bottom panel). B, puri-fied CtrB analyzed by SDS-PAGE after OGM and MTSET blocking experimentsin E. coli. Cytoplasmic (C), periplasmic (P), and the control periplasmic � cyto-plasmic (P/C) Cys residues were probed as described under “ExperimentalProcedures.” The top panel shows fluorescence, and the bottom panel showsthe same gel stained with Coomassie Blue.




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purification using DM-solubilized E. colimembranes obtainedfrom 2 liters of culture. The 300 mM imidazole fraction con-tained a protein that is consistent with the predicted molecularmass of His10-CtrB (44.7 kDa). After exchanging the sampleinto a 20 mM sodium phosphate buffer, pH 7.0, containing 150mM NaCl, it was concentrated to 2.75 mg/ml protein. Purifica-tions consistently yielded �0.75 mg of protein/liter of culture.

EM of CtrB—EM of negatively stained samples showed thatCtrB reconstitution reactions produced small proteoliposomesthat were densely packed with protein particles (data notshown). Occasionally, samples produced larger proteolipo-somes andmembrane sheets that contained visible protein par-ticles. Of the conditions investigated, reconstitution reactionsat a lipid-to-protein ratio of 0.25:1 using DOPC or a 1:1 DOPC-DOPE mixture produced the largest proteoliposomes andmembrane sheets. Samples generated under these conditionsare optimal for structural studies and were selected for cryo-EM. Fig. 5A illustrates a frozen-hydrated DOPC membranesheet containing CtrB particles, and Fig. 5B shows the edges ofthree frozen hydrated �0.2–0.3-�m diameter DOPC-DOPECtrB proteoliposomes. The membrane sheet shown in Fig. 5Acontains protein particles that appeared to form a two-dimen-sional crystal. However, crystallographic image analysisshowed that this area contained a mosaic crystalline array withonly short range ordered patches thatwas unsuitable for furtherprocessing using standard crystallographic image processing(31). To obtain low resolution structural data, we thereforeemployed an alternative patch-averaging approachwhere smallpatches from the mosaic crystal are selected and averaged aftertranslational and rotational orientation. 252 patches of 96 � 96pixels (corresponding to 372.48� 372.48Å) that were centeredon an individual CtrB particle were selected using BOXER.Classification of the patches revealed no major differences inthe patches, and therefore an averaged top view was generatedwith 212 of the patches using EMAN. Fig. 5D shows the aver-aged top view containing six ring-shaped CtrB particles packedaround the central CtrB. The approximate diameter of a singleCtrB particle is 100 Å.Liposomes with sizes of �0.2–0.5-�m diameter produced

distinct bands of density corresponding to the lipid bilayer atthe edge of the flattened vesicles. 93 particles extending fromthe folded edge of proteoliposomes were selected using

FIGURE 4. Purification of His10-CtrB. CtrB was purified from solubilized E. colimembranes as described under “Experimental Procedures.” The DM-solubi-lized protein fraction was applied to nickel-nitrilotriacetic acid resin andeluted with an imidazole step gradient. Samples were analyzed by SDS-PAGEand visualized by staining with Coomassie Blue.

FIGURE 5. Cryo-EM of CtrB reconstituted in lipid bilayers and frozen in vitreous buffer. A, membrane sheet containing CtrB particles. B, edges of threeflattened vesicles with side views of CtrB extending distally from the membrane (arrows). C, magnified view of the CtrB particles from A. D, averaged top viewcontaining seven CtrB molecules. The corresponding projection map shown in E. F, averaged side view of CtrB, with the lipid bilayer and the proposedperiplasmic region emerging from the membrane highlighted in G.




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BOXER, and an averaged side view of CtrB was generated usingEMAN (Fig. 5F). The dimensions of the averaged side view (Fig.5G) are consistent with the top view showing that CtrB forms aring-shape structure with a diameter of �100 Å and extends�125 Å from the lipid bilayer.Mutational Analysis of the Proline Consensus Motif in KpsE—

PCP proteins lack sequence identity between subfamiliesexcept for a Pro-containing motif that overlaps the C terminusof the periplasmic domain and TM2 (Fig. 1) (32). Mutationalanalysis in PCP-1 (Wzz) and PCP-2a (ExoP) proteins indicatesthat the second and third Pro residues in the motif are impor-tant for function (33, 34). PCP-3 proteins possess the least con-served Pro-containing motif in the family; KpsE has only thethree conserved proline residues from themotif (Fig. 6A). Con-structs encoding KpsE with single Pro to Ala substitutions(KpsE-P341A, KpsE-P344A, and KpsE-P350A) and a triplePro3Ala variant (KpsE-AAA) were generated to investigatewhether these residues are required for PSA polymerizationand export. When expressed in trans in EV36 �kpsE, all four ofthe KpsE variants restored K1F phage sensitivity, showing thatthe Pro-containing motif is not essential for PSA export (datanot shown). To investigate any effect of these Pro substitutions

on PSA polymerization, whole cell lysates were analyzed bySDS-PAGE. PSA was visualized by Western blotting probedwith amonoclonal anti-PSA antibody (Fig. 6B). PSA fromEV36produced a large smeared signal that represents polymers witha broad range of lengths. Compared with EV36, RS3175 andEV36 �kpsE produced polymers with a more limited size rangeconfined to very highmolecularmasses.When the kpsE knock-out strain was complemented with the kpsE gene expressed intrans, the resulting PSA exhibited a size distribution compara-ble with a combination of the PAGE profiles from the wild-typeand the kpsE mutant, presumably reflecting incomplete com-plementation.When EV36 �kpsEwas complemented with sin-gle Pro to Ala KpsE variants or KpsE-AAA, the PSA was dis-cernible from the polymer produced by the kpsE knock-outstrain complemented with wild-type KpsE. These results sug-gest that the KpsE Pro-containingmotif does not play an essen-tial role in PSA export or chain length regulation.

DISCUSSION

PCP proteins are involved in several fundamentally differentbacterial polysaccharide assembly pathways, and they play rolesin polymer chain-length regulation and/or export, depending

FIGURE 6. Analysis of the PCP-proline rich consensus motif. A, alignment of the PCP proline consensus motif from representative PCP-1, PCP-2, and PCP-3proteins: SeLT2, S. enterica serovar Typhimurium LT2 Wzz; EcO157, E. coli O157 Wzz; PaPAO1, Pseudomonas aeruginosa PAO1 Wzz; EcK30, E. coli K30 Wzc; Kp,Klebsiella pneumoniae Wzc; Cc, Caulobacter crescentus PCP2a-like protein; EcK1, E. coli K1 KpsE; Nm, N. meningitidis CtrB; Hi, Haemophilus influenzae BexC.Identical residues are highlighted in black, and similar residues are highlighted in gray. Sequences were aligned using ClustalW (54). B, Western immunoblot ofPSA from E. coli whole cell lysates, separated by SDS-PAGE, and probed with an anti-PSA monoclonal antibody.




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on the system (for review, see Ref. 7). PCP-1 (Wzz) proteinswere the first family members identified (35–37). PCP-1 pro-teins determine the chain length of O antigens in the Wzy-de-pendent LPS assembly pathway. In their absence, cells can onlysynthesize short undecaprenol-linked oligosaccharides con-taining a limited number of O-repeat units. It is not understoodhow PCP-1 proteins determine polymer chain length, but aninteraction between Wzz and polymerase protein Wzy ishypothesized (11, 13, 35, 38). PCP-1 proteins are not essentialfor either the polymerization of O antigens or for the export ofLPS to the surface the cell. There is currently no evidence thatPCP-1 proteins interact with an outer membrane component.PCP-2a proteins (like Wzc from E. coli K30) participate in theregulation of high level polymerization (39) of CPS assembledvia the Wzy-dependent pathway, and in this respect they par-allel the role of PCP-1 proteins. However, the interactionbetween the PCP-2a protein and its cognate OPX representa-tive (16, 40) strongly suggests that these proteins also form partof the trans-envelope export machinery. The size difference inthe periplasmic domains of the PCP-1 and PCP-2 proteins isproposed to reflect the interaction of PCP-2a proteins withOPX partners (7). The PCP-2a protein (Wzc) forms an oligo-meric structure whose periplasmic domain is superficially sim-ilar to those observed for PCP-1 proteins (12–14), althoughthere is somedebate about the precise oligomeric states. Crystalstructures for several PCP-1 proteins suggest varied numbers ofmonomers in the oligomer (13), but a consistent hexamericstructure was found when those proteins were examined bycryo-EMof proteoliposomes (12). Single-particle EM studies ofdetergent-solubilizedWzc showed that it is a tetramer (14, 16).However, members of this subfamily carry an extended cyto-plasmic kinase domain (not found in other PCP proteins) that,in isolated form, is an octamer in crystal structures (41, 42). It isunclear whether the difference in oligomeric states reflects theabsence of constraining transmembrane and periplasmicdomains in the crystal structure.In contrast to the relatively well studied PCP-1 and PCP-2a

proteins, PCP-3 proteins participate in ABC transporter-de-pendent pathways. In these systems, polymerization is com-pleted by cytoplasmic or peripheral membrane proteins priorto export mediated by the ABC transporter (8, 19). The PCP-3proteins therefore cannot play a role similar to that of theirPCP-1 and PCP-2 counterparts in polymerization. In compara-ble ABC transporter-dependent LPS O antigen biosynthesis,polymer chain-length regulation is achieved either by the addi-tion of unusual nonreducing terminal residues (43, 44) (whichare not evident in PSA) or by coordinating the activities of theABC transporter and biosynthesis enzymes (45). The lattermaybe a candidate for ABC transporter-dependent CPSs (7, 19). Inthe context of the ABC transporter-dependent systems, thePCP “polysaccharide co-polymerase” name may thereforeprove to be a misnomer. However, the PCP-3 (KpsE) proteinfrom E. coli is required for export of the polymer from theperiplasm to the cell surface (46, 47). KpsE co-localizes with itsOPX partner (KpsD) and forms part of a protein complex iden-tified by chemical cross-linking (48). It is therefore proposedthat PCP-3 proteins act in export, via interactionswith theOPXproteins, in a manner analogous to their PCP-2a counterparts.

The cross-complementation data presented here providefurther supporting evidence for a functional interactionbetween PCP and OPX proteins by showing that only cognateprotein pairs are active. The export machineries from CPS sys-tems have no specificity for a particular repeat-unit structure;that feature is established by the biosynthesis machinery beforethe product is exported. E. coli provides the best example,where the genes encoding the ABC transporter, PCP, and OPXproteins are identical in serotypes producing radically differentCPS structures (47, 49). Functional hybrid CPS exporterscomposed of the E. coli ABC transporter proteins paired withActinobacillus pleuropneumoniae PCP-3-OPX proteins(CpxC-CpxD) and the reciprocal exporter composed of A.pleuropneumoniae ABC transporter proteins (CpxBA) pairedwith E. coli PCP-3-OPX proteins have been described in areview, but the experimental data are not available (50). It hasalso been shown that the ABC transporter proteins encoded bytheMannheimia hemolyticaCPSbiosynthesis locus can restorePSA export in an E. coli�kpsMTmutant strain (51). The abilityto transfer the OPX-PCP-3 pairs between E. coli andN. menin-gitidis supports the concept of a generic polysaccharide exportconduit. Although no structural data are available forOPXpro-teins from ABC transporter-dependent CPS systems, second-ary structure analysis predicts that they share general featuresof the family (including the defining polysaccharide exportsequence motif) (7). Interestingly, KpsD and CtrA are signifi-cantly different in size and possess different export signalsequences (KpsD: 58.2 kDa, signal peptidase I cleavage site;CtrA: 38.4 kDa, signal peptidase II cleavage site) and cluster inseparate phylogenetic groups (7). The PCP-3 proteins fromE. coli (KpsE) and N. meningitidis B (CtrB) share nearly identi-cal predicted secondary structures, are the same size (KpsE,43.1 kDa; CtrB, 43.4 kDa), and cluster in the same group inphylogenetic analyses (7) (Fig. 1). This may explain the require-ment for a cognate interaction between PCP-3 and OPX pro-teins, whereas PCP-3 proteins are suited to form a genericinterface with the ABC transporter proteins. These observa-tions reinforce the analogies made between the structure andfunctions of PCP-2a and PCP-3 proteins and the adapter pro-teins that link outer membrane channels to inner membraneexporters in tripartite efflux pumps (7).This work provides the first evidence that a PCP-3 protein

forms an oligomeric periplasmic structure with a central cavity,similar to PCP-1 and PCP-2a proteins. Cryo-EMof CtrB recon-stituted into lipid bilayers provided top and side views of a par-ticle that were used to generate averaged images. These lowresolution images of CtrB are roughly similar in shape and sizeto those that were observed for the PCP-1 protein from Salmo-nella enterica serovar Typhimurium (12). Cryo-EM structuresof PCP-1 proteins in proteoliposomes revealed hexamericstructure and the current data suggest that this is themost likelyorganization of the CtrB oligomer. However, other oligomericpossibilities cannot be ruled out unequivocally without a higherresolution dataset. This is dependent on the protein formingsignificant areas of better ordered two-dimensional arrays.Although such arrays have been obtained for the PCP-1 pro-teins, this has not been possible with CtrB, despite the applica-tion of an extensive range of conditions. The averaged top view




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of CtrB was generated using small membrane patches that con-tained several particles that were organized comparably to thetwo-dimensional crystals obtained with PCP-1 hexamers. TheEM images of CtrB are consistent with the structures of PCP-2aproteins and suggest that PCP-3 proteins may also assembleinto structures that are capable of forming the interfacebetween the cytoplasmic membrane and the periplasm in a cellenvelope-spanning complex required for CPS export.A defining characteristic of the PCP family of proteins is a

conserved predictedmembrane topology (7, 9, 10). Hydropathyanalysis yields nearly identical results for representative pro-teins from all three PCP subfamilies, where transmembrane-spanning regions are predicted at each end of the polypeptide.Experimental data for the membrane topology and orientationof PCP-2a (Wzc) and PCP-1 (Wzz) proteins confirm the pres-ence of N- and C-terminal transmembrane helices that flank aperiplasmic region (11, 52). To investigate whether this is aconserved theme among PCP-3 proteins, site-directed fluores-cence labeling experiments were used to analyze themembranetopology of CtrB and showed that the N and C termini arelocated in the cytoplasm, whereas the central potion of the pro-tein is located in the periplasm. These results show that CtrB isa bitopic membrane protein, and the shared functions of CtrBand KpsE, as well as the conservation of predicted secondarysequences, suggest that other members of the PCP-3 familymay also be bitopic. Previous studies with KpsE predicted aC-terminal amphipathic helix that interacts with, rather thanspans, the membrane (28, 29). It is difficult to reconcile the twosets of data. They could reflect either the different methods ofanalysis or subtle differences between KpsE and CtrB that haveno significant impact on function.The PCPPro-containingmotif has been proposed tomediate

protein-protein interactions that play a role in determiningpolymer chain length. Site-directed mutagenesis of the PCP-1(Wzz) protein from Shigella flexneri suggested that the secondPro in the motif is critical for LPS O antigen chain-length reg-ulation (33). A similar analysis of the PCP-2a (ExoP) proteinfrom Rhizobium meliloti showed that the Pro in the conservedSPK region of the motif was required for a wild-type ratio ofhigh molecular mass to low molecular mass exopolysaccharide(34). KpsE from E. coli K1 has a minimally conserved Pro-con-taining consensus motif that is well suited to probe the role ofthese residues in a PCP-3 homolog. Complementation experi-ments were performed using KpsE variants with specific aminoacid substitutions at the three conserved Pro residues. Theseexperiments showed that none of the Pro residues was requiredfor polymer export, and all three residues could be mutatedwithout effect. PSA was also analyzed by SDS-PAGE to deter-mine whether the KpsE variants affected chain-length regula-tion. We were unable to detect any significant alteration in themolecular mass profile of PSA produced by these strains com-pared with the strain expressing wild-type KpsE. The �kpsEmutant does accumulate CPS chains with higher average chainlengths than the wild type, based on SDS-PAGE analysis. Thiseffect (increased size) is the opposite of the phenotype of PCP-1and PCP-2a mutants, and there are several potential explana-tions for this phenotype. The assembly process dictates thatCPS polymerization is established before export, but commu-

nication between the PCP-3 protein and the ABC transporter,or other assembly components on the cytoplasmic face of themembrane, could alter normal chain-length regulation. PAGEanalysis of PSA from an EV36 �kpsT mutant also showed theproduction of high molecular mass polymer (data not shown)that is consistent with the �kpsE mutant phenotype and sug-gests an interaction between the assembly and export systems.Communication between the export and biosynthesis machin-ery is evident in the Wzy-dependent CPS systems although, inthis case, the absence of the PCP-2a or OPX proteins also inac-tivates high level polymerization (53). Alternatively, the biosyn-thesis system might always produce PSA with a homogeneoussize range similar to the�kpsEmutant, but exposure of the PSAon the cell surface of thewild typemay lead to physical shearingand/or partial hydrolysis, resulting in awider range of sizes. Theapparent expendability of residues in the Pro-containing motifof PCP-3 proteins points to important functional differencesbetween PCP-1/2a and PCP-3 proteins. We conclude that thismotif is important for regulating polymerization (an activitymissing in the PCP-3 proteins) but not for export.There is a growing body of data from structural biology and

biochemical initiatives targeted toward proteins in the PCPfamily. Interestingly, a phylogenetic analysis of the PCP familyshowed that PCP-1 andPCP-3 proteins aremore closely relatedthan the PCP-2 family is to either subfamily 1 or 3 (7). Thedesignation of PCP is consistent with the data available forPCP-1 and PCP-2 familymembers, and there is a clear relation-ship in export between PCP-2 and PCP-3 families. Althoughthe general roles in regulating polymerization and export areknown, the next challenge is to define the structural featuresandmechanisms that are important for each of these processes.The functional link among all three PCP subfamilies will likelyonly be resolved by establishing how polysaccharides and othersecreted macromolecules interact with assembly and exportproteins at the inner membrane.

Acknowledgments—We acknowledge the gifts of bacterial strains andphage K1F from E. R. Vimr, S. Steenbergen, and R. P. Silver and PSA-specific antibody from M. Apicella. Plasmid pWQ572 was con-structed by J. King.

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Kane Larue, Robert C. Ford, Lisa M. Willis and Chris WhitfieldTransporter-dependent Capsule Biosynthesis Pathways

Proteins Required for Polymer Export in ATP-binding Cassette Functional and Structural Characterization of Polysaccharide Co-polymerase

doi: 10.1074/jbc.M111.228221 originally published online March 18, 20112011, 286:16658-16668.J. Biol. Chem.

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