Open AccessDiversity in kinetics correlated with structure in nano body-stabilized LacY
Author(s) -
Atul Kumar Johri,
J. Finer-Moore,
И. Н. Смирнова,
Vladimir N. Kasho,
Els Pardon,
Jan Steyaert,
H. Ronald Kaback,
Robert M. Stroud
Publication year - 2020
Publication title -
plos one
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.99
H-Index - 332
ISSN - 1932-6203
DOI - 10.1371/journal.pone.0232846
Subject(s) - lactose permease , periplasmic space , chemistry , symporter , mutant , receptor–ligand kinetics , kinetics , binding site , protein structure , wild type , crystallography , stereochemistry , biophysics , permease , biochemistry , biology , escherichia coli , receptor , transporter , quantum mechanics , gene , physics
The structure of lactose permease, stabilized in a periplasmic open conformation by two Gly to Trp replacements (LacYww) and complexed with a nanobody directed against this conformation, provides the highest resolution structure of the symporter. The nanobody binds in a different manner than two other nanobodies made against the same mutant, which also bind to the same general region on the periplasmic side. This region of the protein may represent an immune hotspot. The CDR3 loop of the nanobody is held by hydrogen bonds in a conformation that partially blocks access to the substrate-binding site. As a result, k on and k off for galactoside binding to either LacY or the double mutant complexed with the nanobody are lower than for the other two LacY/nanobody complexes though the K d values are similar, reflecting the fact that the nanobodies rigidify structures along the pathway. While the wild-type LacY/nanobody complex clearly stabilizes a similar ‘extracellular open’ conformation in solution, judged by binding kinetics, the complex with wild-type LacY did not yet crystallize, suggesting the nanobody does not bind strongly enough to shift the equilibrium to stabilize a periplasmic side-open conformation suitable for crystallization. However, the similarity of the galactoside binding kinetics for the nanobody-bound complexes with wild type LacY and with LacY WW indicates that they have similar structures, showing that the reported co-structures reliably show nanobody interactions with LacY.