title: Cross-linking of trans reentrant loops in the Na(+)-citrate transporter CitS of Klebsiella pneumoniae.
abstract: The membrane topology model of the Na(+)-citrate transporter CitS of Klebsiella pneumoniae shows a core of two homologous domains with opposite orientation in the membrane and each containing a so-called reentrant loop. A split version of CitS was constructed to study domain interactions and proximity relationships of the putative reentrant loops. Split CitS retained 50% transport activity of the wild-type version in membrane vesicles. Unspecific cross-linking of the purified complex with glutaraldehyde revealed a tetrameric complex with two N and two C domains corresponding to dimeric CitS. The separately expressed domains were not detected in the membrane. Strong interaction between the two domains followed from successful purification of the whole complex by Ni(2+)-NTA chromatography when only one domain was His-tagged. The presence of citrate and/or the co-ion Na(+) during purification did not seem to affect the interaction significantly. Successful disulfide cross-linking was obtained between single cysteine residues introduced in the highly conserved GGNG sequence motif at the vertex of the reentrant loop in the N domain and either of two endogenous cysteine residues at the base of the reentrant loop in the C domain. The disulfide bond was formed within one subunit in the dimer. A model is proposed in which the reentrant loops in the N and C domains are overlapping at the domain interface in the 3D structure where they form (part of) the translocation pathway for substrate and co-ions.
authors.affiliation: Molecular Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Groningen, The Netherlands.
authors.forename: Adam
authors.initials: A
authors.lastname: Dobrowolski
authors.forename: Fabrizia
authors.initials: F
authors.lastname: Fusetti
authors.forename: Juke S
authors.initials: JS
authors.lastname: Lolkema
mesh_terms: D020816:Amino Acid Motifs / Q000235:genetics; D001798:Blood Proteins / Q000378:metabolism; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics; D019343:Citric Acid / Q000378:metabolism; D003545:Cysteine / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism; D007711:Klebsiella pneumoniae / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D009571:Nitrilotriacetic Acid / Q000031:analogs & derivatives / Q000737:chemistry; D009942:Organometallic Compounds / Q000737:chemistry; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D017434:Protein Structure, Tertiary / Q000235:genetics
chemical_list: D001798:Blood Proteins; D002352:Carrier Proteins; D047908:Intracellular Signaling Peptides and Proteins; D009942:Organometallic Compounds; C020723:citrate-binding transport protein; C016308:serum P-component; D019343:Citric Acid; C088321:nickel nitrilotriacetic acid; C115635:citron-kinase; D017346:Protein Serine-Threonine Kinases; D003545:Cysteine; D009571:Nitrilotriacetic Acid