Characterization of the structural requirements for assembly and nucleotide binding of an ATP-binding cassette transporter. The maltose transport system of Escherichia coli.

The periplasmic maltose-binding protein-dependent, maltose transport system of Escherichia coli is a well studied member of the ATP-binding cassette family of transport ATPases. In addition to the water-soluble maltose-binding protein, the system comprises three membrane proteins, MalF, MalG, and MalK, which form a heterotetrameric complex (FGK2) in the cytoplasmic membrane. The purified complex exhibits transport-associated ATPase activity. To characterize the requirements for nucleotide binding and hydrolysis by the FGK2 complex, we used plasmids to express different combinations of the individual subunits as well as mutant forms of the MalK subunit. Prior to measuring nucleotide binding, we examined membrane preparations for the presence of each subunit from strains that contained all possible permutations of the three structural genes, malF, malG, and malK. We found that when all three genes were present or when malF and malK were present together, the corresponding antigens were detected easily on Western immunoblots and were soluble in the non-ionic detergent, Triton X-100. In contrast, all other permutations resulted in decreased amounts of antigen or antigen that was Triton X-100-insoluble. We relied on photocross-linking with 8-azido-[32P]ATP and ATP hydrolysis as indicators of the ability of the transport complex to interact with purine nucleotides. 8-Azido-[32P]ATP was photocross-linked to the MalK subunit. Photolabeling of MalK was inhibited by ATP, ADP, and GTP and not by other nucleotides. Photolabeling of MalK required the presence of MalF but not MalG. Mutations in malK that affect amino acid residues thought to be directly involved in nucleotide binding did indeed abolish labeling and resulted in loss of transport activity without affecting protein stability. In general, ATP hydrolysis correlated with the photocross-linking. A notable exception is the MalK941 mutant protein which retained the ability to be labeled by 8-azido-[32P]ATP but was unable to catalyze detectable levels of ATP hydrolysis. Some, but not all, of the malK mutations were dominant to wild type. To study the mechanism of dominance we devised a means of measuring the ability of different wild-type and mutant MalK proteins to interact with the MalF and MalG subunits. This assay relies on the fact that, when a bifunctional MalK-LacZ hybrid protein is associated with the MalF and MalG subunits, it is membrane-bound. Excess MalK competed with the MalK-LacZ hybrid protein for sites in the membrane and resulted in the hybrid fractionating as a soluble protein.(ABSTRACT TRUNCATED AT 400 WORDS)

The activities of the Escherichia coli MalK protein in maltose transport, regulation, and inducer exclusion can be separated by mutations.

The maltose regulon consists of several genes encoding proteins involved in the uptake and utilization of maltose and maltodextrins. Five proteins make up a periplasmic binding-protein-dependent active transport system. One of these proteins, MalK, contains an ATP-binding site and is thought to couple the hydrolysis of ATP to the accumulation of substrate. Beside its function in transport, MalK has two additional roles: (i) it negatively regulates mal regulon expression and (ii) it serves as the target for regulation of transport activity by enzyme IIIGlc of the phosphotransferase system. To determine whether the three functions of MalK are separable, we have isolated and characterized three classes of malK mutations. The first type (class I) exhibited constitutive mal gene expression but still allowed normal transport of maltose; the second type (class II) lacked the ability to transport maltose but retained the ability to repress the mal genes. Class I mutations were localized in the last third of the gene, at amino acids 267 (Trp to Gly) and 346 (Gly to Ser). Mutations of class II were found at the positions 137 (Gly to Ala), 140 (delta Gln Arg), and 158 (Asp to Asn). These mutations are near or within the region of MalK that exhibits extensive homology to the B site of an ATP-binding fold. In addition, site-directed mutagenesis was used to add or remove one amino acid in the A site of the ATP-binding fold. Plasmids carrying these mutations also behaved as class II mutants. The third class of malK mutations resulted in resistance to the enzyme IIIGlc-mediated inhibitory effects of alpha-methylglucoside. These mutations did not interfere with the regulatory function of MalK. One of these mutations (exchanging a serine at position 282 for leucine) is located in a short stretch of amino acids that exhibits homology to a sequence in the Escherichia coli Lac permease in which alpha-methylglucoside-resistant mutations have been found.