Specificity of Campylobacter jejuni Adhesin PEB3 for Phosphates and Structural Differences among Its Ligand Complexes † , ‡ Tongpil Min, § Masoud Vedadi, | David C. Watson, ⊥ Gregory A. Wasney, | Christine Munger, § Miroslaw Cygler, §,@ Allan Matte,* ,@ and N. Martin Young* ,⊥ Department of Biochemistry, McGill UniVersity, Montreal, QC, H3G 1Y6 Canada, Structural Genomics Consortium, UniVersity of Toronto, and Institute for Biological Sciences, National Research Council of Canada, Ottawa, ON, K1A 0R6 Canada, and Biotechnology Research Institute, National Research Council of Canada, Montreal, QC, H4P 2R2 Canada ReceiVed NoVember 28, 2008; ReVised Manuscript ReceiVed February 13, 2009 ABSTRACT: PEB3 is a glycoprotein adhesin from Campylobacter jejuni whose structure suggested a role in transport. We have investigated potential ligands for PEB3 and characterized their binding properties using biophysical methods in solution and by X-ray crystallography. A thermal aggregation assay of PEB3 with a library of physiological compounds identified three possible ligands [3-phosphoglycerate (3-PG), phosphoenolpyruvate (PEP), and aconitate], which stabilized wild-type PEB3 but did not stabilize either a PEB3 form containing two mutations at the ligand-binding site, T138A/S139A, or a second PEB3 mutant, K135E, at a site ∼14 Å away. Fluorescence titration experiments and cocrystal structures with various ligands were used to characterize the binding of 3-PG, PEP, and phosphate to PEB3. Further, a C. jejuni growth experiment in minimal medium supplemented with 3-PG showed that this molecule enhances the growth of wild-type C. jejuni, but not of the PEB3 mutants. Crystallographic analysis of PEB3 complexes revealed that the Ser171-Gln180 region in the presence of 3-PG or other phosphates is helical and similar to those of other transport proteins, but it is nonhelical when citrate is bound. The K135E mutation resulted in expression of a more highly glycosylated form of PEB3 in vivo, and its crystal structure showed the conformation of the first two residues of the glycan. On the basis of our findings, we suggest that PEB3 is a transport protein that may function in utilization of 3-PG or other phosphate-containing molecules from the host. Campylobacter jejuni adhesin PEB3 (Cj0289c) is one of more than 30 proteins that have been shown to be N- glycosylated in vivo by the pgl system (1, 2). The oligosac- charyltransferase PglB of this system attaches a heptasac- charide to Asn90 of PEB3, which is within the consensus sequon sequence, D/E-X-N-X-S/T (2). Crystallographic studies of PEB3 (3) showed that its sequon is located within a surface-exposed loop, suggesting that PglB could modify Asn90 when PEB3 is in its folded state. The structure also showed that PEB3 contains a centrally located ligand-binding cleft that was occupied by a citrate molecule from the crystallization medium. Citrate is unlikely to be PEB3′s natural ligand since another gene, cj0203, has been identified to encode a citrate transporter in the genome of C. jejuni (4). The overall structure of PEB3 is that of a dimer and is similar to those of other type II periplasmic transport proteins (3). The closest structural homologues of PEB3 are trans- porters for molybdate/tungstate, sulfate, and ferric iron, suggesting the natural ligand would be a di- or trianion or cation. Alternatively, the ligand-binding site might recognize a cell surface molecule on a host cell to provide adhesion. In the case of C. jejuni PEB1a, the protein has both adhesin and transport capabilities (5), so a similar dual function is also possible for PEB3. However, no experimental data have so far been reported in support of PEB3 functioning as a transporter, or of possible ligands that could originate from host cells. To identify potential ligands for PEB3 in its role as either an adhesin or a transporter, we applied two recently developed techniques that permitted the interrogation of relevant compound libraries. These were a glycan microarray for the adhesin role (6) and a protein stabilization (T agg ) assay with a library of physiologically relevant ligands (7, 8). The latter study reveals that PEB3 recognizes 3-phosphoglycerate and other molecules found in intermediary metabolism of cells, including those of eukaryotic hosts. Fluorescence spectroscopy was used to characterize the binding properties † This work was supported in part by CIHR Grant GSP-48370 (M.C. and A.M.). Use of the Advanced Photon Source was supported by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences, under Contract DE-AC02-06CH11357. Use of the SGX Collaborative Access Team (SGX-CAT) beamline facilities at Sector 31 of the Advanced Photon Source was provided by SGX Pharma- ceuticals, Inc., who constructed and operates the facility. ‡ Coordinates for the structures described in this paper have been deposited in the Protein Data Bank as entries 3FJG, 3FJ7, 3FJM, and 3FIR. * To whom correspondence should be addressed. N.M.Y.: phone, (613) 990-0855; fax, (613) 941-1327; e-mail, martin.young@nrc- cnrc.gc.ca. A.M.: phone, (514) 496-2557; fax, (514) 496-5143; e-mail, allan.matte@bri.nrc.ca. § McGill University. | University of Toronto. ⊥ Institute for Biological Sciences, National Research Council of Canada. @ Biotechnology Research Institute, National Research Council of Canada. Biochemistry 2009, 48, 3057–3067 3057 10.1021/bi802195d CCC: $40.75  2009 American Chemical Society Published on Web 02/23/2009