Functional characterization of the maltose ATP-binding-cassette transporter of Salmonella typhimurium by means of monoclonal antibodies directed against the MalK subunit.
The maltose ATP-binding cassette transporter of Salmonella typhimurium is composed of a membrane-associated complex (MalFGK2) and a periplasmic receptor (MalE). In addition to its role in transport, the complex acts as a repressor of maltose-regulated gene expression and is subject to inhibition in the process of inducer exclusion. These activities are thought to be mediated by interactions of the ATPase subunit, MalK, with the transcriptional activator, MalT, and nonphosphorylated enzyme IIA of the glucose phosphotransferase system, respectively. To gain further insight in protein regions that are critical for these functions, we have generated nine MalK-specific monoclonal antibodies. These bind to four nonoverlapping linear epitopes: 60-LFig-63 (5B5), 113-RVNQVAEVLQL-123 (represented by 4H12), 309-GHETQI-314 (2F9) and 352-LFREDGSACR-361 (represented by 4B3). All mAbs recognize their epitopes in soluble MalK and in the MalFGK2 complex with Kd values ranging from 10-6 to 10-8 m. ATP reduced the affinity of the mAbs for soluble MalK, indicating a conformational change that renders the epitopes less accessible. 4H12 and 5B5 inhibit the ATPase activity of MalK and the MalE/maltose-stimulated ATPase activity of proteoliposomes, while their Fab fragments displayed no significant effect. The results suggest a similar solvent-exposed position of helix 3 in the MalK dimer and in the intact complex and might argue against a direct role in the catalytic process. 4B3 and 2F9 exhibit reduced binding to the MalFGK2 complex in the presence of MalT and enzyme IIAGlc, respectively, thereby providing the first direct evidence for the C-terminal domain of MalK being the site of interaction with the regulatory proteins.
Humboldt Universität zu Berlin, Institut für Biologie, Berlin, Germany.
Anke
A
Stein
Martina
M
Seifert
Rudolf
R
Volkmer-Engert
Jörg
J
Siepelmeyer
Knut
K
Jahreis
Erwin
E
Schneider
D018528:ATP-Binding Cassette Transporters / Q000276:immunology / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D000907:Antibodies, Bacterial / Q000276:immunology* / Q000302:isolation & purification; D000911:Antibodies, Monoclonal / Q000276:immunology* / Q000302:isolation & purification; D000918:Antibody Specificity; D000942:Antigens, Bacterial / Q000276:immunology; D001426:Bacterial Proteins / Q000276:immunology / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism*; D004268:DNA-Binding Proteins; D019281:Dimerization; D000939:Epitopes / Q000276:immunology; D029968:Escherichia coli Proteins; D005260:Female; D058619:Maltose-Binding Proteins; D051379:Mice; D008807:Mice, Inbred BALB C; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System / Q000378:metabolism; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011510:Proteolipids / Q000378:metabolism; D012486:Salmonella typhimurium / Q000276:immunology / Q000378:metabolism*; D014157:Transcription Factors
D018528:ATP-Binding Cassette Transporters; D000907:Antibodies, Bacterial; D000911:Antibodies, Monoclonal; D000942:Antigens, Bacterial; D001426:Bacterial Proteins; D002352:Carrier Proteins; D004268:DNA-Binding Proteins; D000939:Epitopes; D029968:Escherichia coli Proteins; C068021:MalK protein, Bacteria; C489398:MalK protein, E coli; C489399:MalK protein, Salmonella typhimurium; C058976:MalT protein, E coli; D058619:Maltose-Binding Proteins; D011510:Proteolipids; D014157:Transcription Factors; C020485:proteoliposomes; D000255:Adenosine Triphosphate; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System; C031629:phosphoenolpyruvate-glucose phosphotransferase