Effects of the L511P and D512G mutations on the Escherichia coli ABC transporter MsbA. Biochemistry 2011 Apr 05;50(13):2594-602
Date
02/25/2011Pubmed ID
21344946Pubmed Central ID
PMC3110719DOI
10.1021/bi1018418Scopus ID
2-s2.0-79953175250 (requires institutional sign-in at Scopus site) 16 CitationsAbstract
MsbA is a member of the ABC transporter superfamily and is homologous to ABC transporters linked to multidrug resistance. The nucleotide binding domains (NBDs) of these proteins include conserved motifs that are involved in ATP binding, including conserved SALD residues (D-loop) that are diagnostic in identifying ABC transporters but whose roles have not been identified. Within the D-loop, single point mutations L511P and D512G were discovered by random mutational analysis of MsbA to disrupt protein function in the cell [Polissi, A., and Georgopoulos, C. (1996) Mol. Microbiol. 20, 1221-1233] but have not been further studied in MsbA or in detail in any other ABC transporter. In these studies, we show that both L511P and D512G mutants of MsbA are able to bind ATP at near-wild-type levels but are unable to maintain cell viability in an in vivo growth assay, verifying the theory that they are dysfunctional at some point after ATP binding. An ATPase assay further suggests that the L511P mutation prevents effective ATP hydrolysis, and an ATP detection assay reveals that only small amounts of ATP are hydrolyzed; D512G is able to hydrolyze ATP at a rate 3-fold faster than that of the wild type. EPR spectroscopy studies using reporter sites within the NBDs also indicate that at least some hydrolysis occurs in L511P or D512G MsbA but show fewer spectral changes than observed for the same reporters in the wild-type background. These studies indicate that L511 is necessary for efficient ATP hydrolysis and D512 is essential for conformational rearrangements required for flipping lipid A.
Author List
Schultz KM, Merten JA, Klug CSAuthors
Candice S. Klug PhD Professor in the Biophysics department at Medical College of WisconsinKathryn M. Schultz Research Scientist I in the Biophysics department at Medical College of Wisconsin
MESH terms used to index this publication - Major topics in bold
ATP-Binding Cassette TransportersAdenosine Triphosphate
Amino Acid Substitution
Bacterial Proteins
Biocatalysis
Conserved Sequence
Electron Spin Resonance Spectroscopy
Escherichia coli
Escherichia coli Proteins
Kinetics
Microbial Viability
Mutagenesis, Site-Directed
Mutant Proteins
Point Mutation
Protein Interaction Domains and Motifs
Recombinant Proteins