Thermodynamics of Nanobody Binding to Lactose Permease.
Camelid nanobodies (Nbs) raised against the outward-facing conformer of a double-Trp mutant of the lactose permease of Escherichia coli (LacY) stabilize the permease in outward-facing conformations. Isothermal titration calorimetry is applied herein to dissect the binding thermodynamics of two Nbs, one that markedly improves access to the sugar-binding site and another that dramatically increases the affinity for galactoside. The findings presented here show that both enthalpy and entropy contribute favorably to binding of the Nbs to wild-type (WT) LacY and that binding of Nb to double-Trp mutant G46W/G262W is driven by a greater enthalpy at an entropic penalty. Thermodynamic analyses support the interpretation that WT LacY is stabilized in outward-facing conformations like the double-Trp mutant with closure of the water-filled cytoplasmic cavity through conformational selection. The LacY conformational transition required for ligand binding is reflected by a favorable entropy increase. Molecular dynamics simulations further suggest that the entropy increase likely stems from release of immobilized water molecules primarily from the cytoplasmic cavity upon closure.
Department of Cell Physiology and Molecular Biophysics, Center for Membrane Protein Research, School of Medicine, Texas Tech University Health Sciences Center , Lubbock, Texas 79430, United States.
Parameswaran
P
Hariharan
Department of Theoretical Physics and Swedish e-Science Research Center, Science for Life Laboratory, KTH Royal Institute of Technology , SE-171 21 Solna, Sweden.
Magnus
M
Andersson
Department of Physiology, University of California , Los Angeles, California 90095, United States.
Xiaoxu
X
Jiang
VIB Center for Structural Biology Research, VIB , 1050 Brussel, Belgium.|Structural Biology Brussels, Vrije Universiteit Brussel , Pleinlaan 2, 1050 Brussel, Belgium.
Els
E
Pardon
VIB Center for Structural Biology Research, VIB , 1050 Brussel, Belgium.|Structural Biology Brussels, Vrije Universiteit Brussel , Pleinlaan 2, 1050 Brussel, Belgium.
Jan
J
Steyaert
Department of Physiology, University of California , Los Angeles, California 90095, United States.
H Ronald
HR
Kaback
Department of Cell Physiology and Molecular Biophysics, Center for Membrane Protein Research, School of Medicine, Texas Tech University Health Sciences Center , Lubbock, Texas 79430, United States.
Lan
L
Guan
D002151:Calorimetry; D004926:Escherichia coli / Q000201:enzymology; D026901:Membrane Transport Proteins / Q000378:metabolism*; D056004:Molecular Dynamics Simulation; D061905:Single-Domain Antibodies; D013379:Substrate Specificity; D013816:Thermodynamics
D026901:Membrane Transport Proteins; D061905:Single-Domain Antibodies; C021499:lactose permease
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