Ligand-induced protein transition state stabilization switches the binding pathway from conformational selection to induced fit
Stenström, Olof LU ; Diehl, Carl LU ; Modig, Kristofer LU
and Akke, Mikael
LU
(2024)
In Proceedings of the National Academy of Sciences of the United States of America
121(14).
p.2317747121-2317747121
- Abstract
Protein-ligand complex formation is fundamental to biological function. A central question is whether proteins spontaneously adopt binding-competent conformations to which ligands bind conformational selection (CS) or whether ligands induce the binding-competent conformation induced fit (IF). Here, we resolve the CS and IF binding pathways by characterizing protein conformational dynamics over a wide range of ligand concentrations using NMR relaxation dispersion. We determined the relative flux through the two pathways using a four-state binding model that includes both CS and IF. Experiments conducted without ligand show that galectin-3 exchanges between the ground-state conformation and a high-energy conformation similar to the... (More)
Protein-ligand complex formation is fundamental to biological function. A central question is whether proteins spontaneously adopt binding-competent conformations to which ligands bind conformational selection (CS) or whether ligands induce the binding-competent conformation induced fit (IF). Here, we resolve the CS and IF binding pathways by characterizing protein conformational dynamics over a wide range of ligand concentrations using NMR relaxation dispersion. We determined the relative flux through the two pathways using a four-state binding model that includes both CS and IF. Experiments conducted without ligand show that galectin-3 exchanges between the ground-state conformation and a high-energy conformation similar to the ligand-bound conformation, demonstrating that CS is a plausible pathway. Near-identical crystal structures of the apo and ligand-bound states suggest that the high-energy conformation in solution corresponds to the apo crystal structure. Stepwise additions of the ligand lactose induce progressive changes in the relaxation dispersions that we fit collectively to the four-state model, yielding all microscopic rate constants and binding affinities. The ligand affinity is higher for the bound-like conformation than for the ground state, as expected for CS. Nonetheless, the IF pathway contributes greater than 70% of the total flux even at low ligand concentrations. The higher flux through the IF pathway is explained by considerably higher rates of exchange between the two protein conformations in the ligand-associated state. Thus, the ligand acts to decrease the activation barrier between protein conformations in a manner reciprocal to enzymatic transition-state stabilization of reactions involving ligand transformation.
(Less)
- author
- Stenström, Olof
LU
; Diehl, Carl
LU
; Modig, Kristofer
LU
and Akke, Mikael
LU
- organization
- publishing date
- 2024-04-02
- type
- Contribution to journal
- publication status
- published
- subject
- keywords
- ligand-binding kinetics, molecular recognition, protein dynamics
- in
- Proceedings of the National Academy of Sciences of the United States of America
- volume
- 121
- issue
- 14
- pages
- 2317747121 - 2317747121
- publisher
- National Academy of Sciences
- external identifiers
-
- pmid:38527204
- scopus:85189060003
- ISSN
- 1091-6490
- DOI
- 10.1073/pnas.2317747121
- language
- English
- LU publication?
- yes
- id
- 868fcaa1-7c8c-4213-8423-ca6609aea382
- date added to LUP
- 2024-04-19 08:43:41
- date last changed
- 2024-11-03 03:39:38
@article{868fcaa1-7c8c-4213-8423-ca6609aea382,
abstract = {{<p>Protein-ligand complex formation is fundamental to biological function. A central question is whether proteins spontaneously adopt binding-competent conformations to which ligands bind conformational selection (CS) or whether ligands induce the binding-competent conformation induced fit (IF). Here, we resolve the CS and IF binding pathways by characterizing protein conformational dynamics over a wide range of ligand concentrations using NMR relaxation dispersion. We determined the relative flux through the two pathways using a four-state binding model that includes both CS and IF. Experiments conducted without ligand show that galectin-3 exchanges between the ground-state conformation and a high-energy conformation similar to the ligand-bound conformation, demonstrating that CS is a plausible pathway. Near-identical crystal structures of the apo and ligand-bound states suggest that the high-energy conformation in solution corresponds to the apo crystal structure. Stepwise additions of the ligand lactose induce progressive changes in the relaxation dispersions that we fit collectively to the four-state model, yielding all microscopic rate constants and binding affinities. The ligand affinity is higher for the bound-like conformation than for the ground state, as expected for CS. Nonetheless, the IF pathway contributes greater than 70% of the total flux even at low ligand concentrations. The higher flux through the IF pathway is explained by considerably higher rates of exchange between the two protein conformations in the ligand-associated state. Thus, the ligand acts to decrease the activation barrier between protein conformations in a manner reciprocal to enzymatic transition-state stabilization of reactions involving ligand transformation.</p>}},
author = {{Stenström, Olof and Diehl, Carl and Modig, Kristofer and Akke, Mikael}},
issn = {{1091-6490}},
keywords = {{ligand-binding kinetics; molecular recognition; protein dynamics}},
language = {{eng}},
month = {{04}},
number = {{14}},
pages = {{2317747121--2317747121}},
publisher = {{National Academy of Sciences}},
series = {{Proceedings of the National Academy of Sciences of the United States of America}},
title = {{Ligand-induced protein transition state stabilization switches the binding pathway from conformational selection to induced fit}},
url = {{http://dx.doi.org/10.1073/pnas.2317747121}},
doi = {{10.1073/pnas.2317747121}},
volume = {{121}},
year = {{2024}},
}