RESUMEN
ATP-binding-cassette (ABC) subunit MalK of the binding protein-dependent transport system for maltose of Salmonella typhimurium and Escherichia coli is crucial to the transport process but also exhibits a repressing activity on other genes of the maltose regulon. The latter function has been attributed to a carboxy-terminal extension by which MalK differs in length from a prototype ABC protein. In order to define the boundaries of putative functional domains of MalK, we have analyzed pairs of N- and C-terminally truncated MalK proteins of S. typhimurium. Coexpressed half molecules of about equal lengths (MalKN1: residues 1 to 179; MalKC1: residues 179 to 369) restored the transport activity of a malK strain and displayed substantial regulatory activity. The same regulatory activity was obtained when malKC1 was expressed separately. These results indicate that a covalent linkage is not absolutely essential for function and that the protein might be composed of two structurally distinct entities. To elucidate further the minimal structural requirements for the regulatory function of MalK, we have studied chimeric proteins that have C-terminal portions of MalK fused to the corresponding amino-terminal fragments of its close homolog LacK. Functional analyses revealed that a fusion containing only the C-terminal extension of MalK (Q263 to V369) is sufficient to display half-maximal regulatory activity. This activity increased with the lengths of the MalK portions present in the chimeras. Furthermore, the failure of two chimeras to support maltose transport suggests a structurally critical region between residues 243 and 264. In the absence of a crystal structure, this work contributes to the understanding of the multiple functions of MalK.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Proteínas Bacterianas/metabolismo , Proteínas de Escherichia coli , Salmonella typhimurium/metabolismo , Transportadoras de Casetes de Unión a ATP/genética , Alelos , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Regulación Bacteriana de la Expresión Génica , Prueba de Complementación Genética , Maltosa/metabolismo , Datos de Secuencia Molecular , Fragmentos de Péptidos , Proteínas Recombinantes de Fusión/metabolismo , Salmonella typhimurium/genética , Eliminación de Secuencia , Homología de Secuencia de AminoácidoRESUMEN
The ATP-binding-cassette (ABC) protein LacK of Agrobacterium radiobacter displays high sequence similarity to the MalK subunit of the Salmonella typhimurium maltose-transport system (MalFGK2). We have used LacK as a tool to identify sites of interaction of MalK with the membrane-integral components MalF and MalG. Small amounts of LacK, resulting from the expression of the plasmid-borne lacK gene, proved to be sufficient for partial restoration of growth of a malK strain of S. typhimurium on maltose. LacK failed to substitute for MalK in regulating the expression of maltose-inducible genes but the hybrid complex MalFGLacK2 was sensitive to inducer exclusion. The lacK gene also complemented a ugpC mutant of Escherichia coli to growth on sn-glycerol-3-phosphate as the phosphate source. Partially purified LacK exhibited a spontaneous ATPase activity comparable to that of MalK. A MalK"-'LacK chimeric protein was isolated (by in vivo recombination) in which the N-terminal 140 amino acids of MalK are fused to residues 141-363 of LacK. The protein substituted for MalK in maltose transport considerably better than LacK. Furthermore, random mutagenesis of the plasmid-borne lacK gene yielded three clones that were superior to wild-type lacK in complementing a malK mutation. Single mutations (V114M or L123F) substantially improved the growth of a malK strain on maltose, whereas a double mutation (L123F, S295N) resulted in growth and transport rates that were indistinguishable from those obtained with MalK. In contrast, the introduction of the single change S295N into LacK had no effect but combination with the V114M mutation led to a further twofold increase in transport activity. These results indicate that a putative helical domain in MalK, encompassing residues 89-140, is crucial for a functional, high-affinity interaction with MalF and MalG.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Proteínas Bacterianas/metabolismo , Proteínas Portadoras/metabolismo , Proteínas de Escherichia coli , Proteínas de Transporte de Membrana , Proteínas de Transporte de Monosacáridos , Rhizobium/metabolismo , Salmonella typhimurium/metabolismo , Transportadoras de Casetes de Unión a ATP/genética , Adenosina Trifosfatasas/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Sitios de Unión , Transporte Biológico , Proteínas Portadoras/genética , Escherichia coli/metabolismo , Regulación Bacteriana de la Expresión Génica , Glicerofosfatos/metabolismo , Proteínas de Unión a Maltosa , Datos de Secuencia Molecular , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Salmonella typhimurium/genéticaRESUMEN
The ATP-binding protein, MalK, of the bacterial ABC (ATP-binding-cassette) transport complex MalFGK2 provides the energy for the translocation of maltose and maltodextrins across the cytoplasmic membrane. The MalK protein from Salmonella typhimurium was overexpressed in Escherichia coli and crystallized by the hanging-drop method using (NH4)2SO4as a precipitant. The crystals belong to space group P6x22 (most probably x = 1 or 5) with cell dimensions a = 181.8 and c = 182.5 A, corresponding to three or four molecules per asymmetric unit. They diffract to a resolution of about 3 A on a synchrotron X-ray source and are suitable for structure determination.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/química , Transportadoras de Casetes de Unión a ATP/aislamiento & purificación , Proteínas Bacterianas/química , Proteínas Bacterianas/aislamiento & purificación , Proteínas de Escherichia coli , Transportadoras de Casetes de Unión a ATP/genética , Proteínas Bacterianas/genética , Cristalización , Cristalografía por Rayos X , Escherichia coli/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Salmonella typhimurium/genéticaRESUMEN
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.