title: Functional consequences of mutations in the conserved 'signature sequence' of the ATP-binding-cassette protein MalK.
abstract: The binding-protein-dependent maltose-transport system of enterobacteria, a member of the ATP-binding-cassette (ABC) transporter superfamily, is composed of two integral membrane proteins, MalF and MalG, and two copies of an ATPase subunit, MalK, which hydrolyze ATP, thus energizing the translocation process. Isolated MalK displays spontaneous ATPase activity, whereas in the assembled MalFGK2 complex, reconstituted in liposomes, ATP hydrolysis requires stimulation by the substrate-loaded extracellular maltose-binding protein, MalE. The ATPase domains of ABC transporters, including MalK, share a unique sequence motif ('LSGGQ', 'signature sequence' or 'linker peptide') with as yet unknown function. To elucidate its role in the transport process, we investigated the consequences of mutations affecting two highly conserved residues (G137, Q140) in the MalK-ATPase of Salmonella typhimurium, by biochemical means. Residues corresponding to Q140 in other ABC proteins have not yet been studied. All mutant alleles (G137--> A, V, T; Q140--> L, K, N) fail to restore a functional transport complex in vivo. In addition, the mutations increase the repressing activity of MalK on other maltose-regulated genes when compared with wild-type MalK. Purified variants of G137 have lost the ability to hydrolyze ATP but still display nucleotide-binding activity, albeit with reduced affinity. Binding of MgATP results in similar protection against trypsin, as observed with wild-type, indicating no major change in protein structure. In contrast, the variants of Q140 differ in their properties, depending on the chemical nature of the replacement residue. MalKQ140L fails to hydrolyze ATP and exhibits a strong intrinsic resistance to trypsin in the absence of MgATP, suggesting a drastically altered conformation. In contrast, the purified mutant proteins Q140K and Q140N display ATPase activities and MgATP-induced changes in the tryptic cleavage pattern similar to those of wild-type. However, mutant transport complexes containing the Q140K or Q140N variants, when studied in proteoliposomes, are severely impaired in MalE-maltose-stimulated ATPase activity. These results are discussed with respect to the crystal structure of the homologous HisP protein [Hung, L.-W., Wang, I.X., Nikaido, K., Liu, P.-Q., Ames, G.F.-L. & Kim, S.-H. (1998) Nature (London) 396, 703-707] and are interpreted in favor of a role of the signature sequence in activating the hydrolyzing activity of MalK upon substrate-initiated conformational changes in MalF/MalG.
authors.affiliation: Institut für Biologie/Bakterienphysiologie, Humboldt-Universität zu Berlin, Germany.
authors.forename: G
authors.initials: G
authors.lastname: Schmees
authors.forename: A
authors.initials: A
authors.lastname: Stein
authors.forename: S
authors.initials: S
authors.lastname: Hunke
authors.forename: H
authors.initials: H
authors.lastname: Landmesser
authors.forename: E
authors.initials: E
authors.lastname: Schneider
mesh_terms: D018528:ATP-Binding Cassette Transporters / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000483:Alleles; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002455:Cell Division; D003001:Cloning, Molecular; D006868:Hydrolysis; D008027:Light; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D012486:Salmonella typhimurium / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013997:Time Factors; D014357:Trypsin / Q000494:pharmacology
chemical_list: D018528:ATP-Binding Cassette Transporters; D001426:Bacterial Proteins; C068021:MalK protein, Bacteria; C489399:MalK protein, Salmonella typhimurium; D011506:Proteins; D011994:Recombinant Proteins; D000255:Adenosine Triphosphate; D014357:Trypsin; D000251:Adenosine Triphosphatases