Disruption of LptA oligomerization and affinity of the LptA-LptC interaction. Protein Sci 2013 Nov;22(11):1639-45
Date
10/15/2013Pubmed ID
24123237Pubmed Central ID
PMC3831678DOI
10.1002/pro.2369Scopus ID
2-s2.0-84892738005 (requires institutional sign-in at Scopus site) 29 CitationsAbstract
The lipopolysaccharide (LPS)-rich outer membrane (OM) is a unique feature of Gram-negative bacteria, and LPS transport across the inner membrane (IM) and through the periplasm is essential to the biogenesis and maintenance of the OM. LPS is transported across the periplasm to the outer leaflet of the OM by the LPS transport (Lpt) system, which in Escherichia coli is comprised of seven recently identified proteins, including LptA, LptC, LptDE, and LptFGB2 . Structures of the periplasmic protein LptA and the soluble portion of the membrane-associated protein LptC have been solved and show these two proteins to be highly structurally homologous with unique folds. LptA has been shown to form concentration dependent oligomers that stack end-to-end. LptA and LptC have been shown to associate in vivo and are expected to form a similar protein-protein interface to that found in the LptA dimer. In these studies, we disrupted LptA oligomerization by introducing two point mutations that removed a lysine and glutamine side chain from the C-terminal β-strand of LptA. This loss of oligomerization was characterized using EPR spectroscopy techniques and the affinity of the interaction between the mutant LptA protein and WT LptC was determined using EPR spectroscopy (Kd = 15 µM) and isothermal titration calorimetry (Kd = 14 µM). Kd values were also measured by EPR spectroscopy for the interaction between LptC and WT LptA (4 µM) and for WT LptA oligomerization (29 µM). These data suggest that the affinity between LptA and LptC is stronger than the affinity for LptA oligomerization.
Author List
Schultz KM, Feix JB, Klug CSAuthors
Jimmy B. Feix PhD Professor in the Biophysics department at Medical College of WisconsinCandice S. Klug PhD Professor in the Biophysics department at Medical College of Wisconsin
Kathryn 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
Amino Acid SequenceBinding Sites
Calorimetry
Carrier Proteins
Circular Dichroism
Electron Spin Resonance Spectroscopy
Escherichia coli
Escherichia coli Proteins
Membrane Proteins
Molecular Sequence Data
Point Mutation
Protein Interaction Domains and Motifs
Protein Multimerization
Protein Structure, Quaternary
Protein Structure, Secondary
Recombinant Proteins
Sequence Homology