NUCLEOTIDE-INDUCED CONFORMATIONAL-CHANGES OF MALK, A BACTERIAL ATP BINDING CASSETTE TRANSPORTER PROTEIN

Autor(en): SCHNEIDER, E 
WILKEN, S
SCHMID, R
Stichwörter: ADENYLATE KINASE; Biochemistry & Molecular Biology; CYSTIC-FIBROSIS; ESCHERICHIA-COLI; MALTOSE TRANSPORT; MECHANISM; MULTIDRUG-RESISTANCE; PERIPLASMIC PERMEASES; SALMONELLA-TYPHIMURIUM; STRUCTURAL MODEL; SUGAR PHOSPHOTRANSFERASE SYSTEM
Erscheinungsdatum: 1994
Herausgeber: AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
Journal: JOURNAL OF BIOLOGICAL CHEMISTRY
Volumen: 269
Ausgabe: 32
Startseite: 20456
Seitenende: 20461
Zusammenfassung: 
Nucleotide-induced structural rearrangements of MalK, the ATP-hydrolyzing component of the ATP binding cassette transporter for maltose from Salmonella typhimurium were investigated by means of analysis of intrinsic tryptophan fluorescence and limited proteolysis. ATP was found to decrease the tryptophan fluorescence of purified MalK by 37 /- 1%. ADP or adenosine 5'-O-(3-(thio)triphosphate) (ATP gamma S) caused similar quenching while AMP was rather ineffective. Mg2+ ions were not required. Exposure of MalK to increasing concentrations of trypsin and subsequent analysis by SDS polyacrylamide gel electrophoresis and immunoblotting revealed the formation of three major transiently stable peptide fragments of 24 (T2), 23 (T3), and 20 kDa (T4), respectively. In addition, a minor rapidly degraded fragment of 33 kDa (T1) was observed. However, in the presence of MgATP, fragment T1 as well as a substantial fraction of native MalK were strongly protected against proteolytic attack. Similar protection against the protease was observed in the presence of MgGTP or, to a lesser extent, MgCTP. In contrast, MgADP, ATP in the presence of EDTA, CaATP or nonhydrolyzable nucleotides such as MgATP gamma S or MgAMP-PNP (beta,gamma-imidoadenosine-5'-triphosphate) failed to significantly affect the susceptibility of MalK to the protease. MgATP similarly affected the tryptic digestion pattern of a mutant protein (MalKK42R) that exhibits only a much reduced ATPase activity but has retained the capability to bind nucleotides. N-terminal protein sequence analysis of the peptides revealed cleavage by trypsin at Arg(66) (T1), Arg(146) (T2), Arg(153) (T3), and Arg(185) (T4), respectively. These results indicate that (i) nucleotide binding to MalK is accompanied by a global conformational change of the protein; (ii) a very specific interaction occurs with substrates of the MalK-ATPase, resulting in structural changes that involve the helical domain from Arg(66) to Arg(146); and (iii) the C-terminal half of MalK is rather resistant to proteolysis.
ISSN: 00219258

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