title: The role of protein dynamics in increasing binding affinity for an engineered protein-protein interaction established by H/D exchange mass spectrometry.
abstract: It is generally accepted that protein and solvation dynamics play fundamental roles in the mechanisms of protein-protein binding; however, assessing their contribution meaningfully has not been straightforward. Here, hydrogen/deuterium exchange mass spectrometry (H/D-Ex) was employed to assess the role of dynamics for a high-affinity human growth hormone variant (hGHv) and the wild-type growth hormone (wt-hGH) each binding to the extracellular domain of their receptor (hGHbp). Comparative analysis of the transient fluctuations in the bound and unbound states revealed that helix-1 of hGHv undergoes significant transient unfolding in its unbound state, a characteristic that was not found in wt-hGH or apparent in the temperature factor data from the X-ray analysis of the unbound hGHv structure. In addition, upon hormone binding, an overall increase in stability was observed for the beta-sheet structure of hGHbp which included sites distant from the binding interface. On the basis of the stability, binding kinetics, and thermodynamic data presented, the increase in the binding free energy of hGHv is primarily generated by factors that appear to increase the energy of the unbound state relative to the free energy of the bound complex. This implies that an alternate route to engineer new interactions aiming to increase protein-protein association energies may be achieved by introducing certain mutations that destabilize one of the interacting molecules without destabilizing the resulting bound complex. Importantly, although the hGHv molecule is less stable than its wt-hGH counterpart, its resulting active ternary complex with two copies of hGHbp has comparable stability to the wt complex.
authors.affiliation: Department of Biochemistry and Molecular Biology, The University of Chicago, 929 East 57th Street, Chicago, Illinois 60637, USA.
authors.forename: James R
authors.initials: JR
authors.lastname: Horn
authors.forename: Brian
authors.initials: B
authors.lastname: Kraybill
authors.forename: Elizabeth J
authors.initials: EJ
authors.lastname: Petro
authors.forename: Stephen J
authors.initials: SJ
authors.lastname: Coales
authors.forename: Jeffrey A
authors.initials: JA
authors.lastname: Morrow
authors.forename: Yoshitomo
authors.initials: Y
authors.lastname: Hamuro
authors.forename: Anthony A
authors.initials: AA
authors.lastname: Kossiakoff
mesh_terms: D001665:Binding Sites; D002942:Circular Dichroism; D003903:Deuterium / Q000737:chemistry; D019382:Human Growth Hormone / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D006859:Hydrogen / Q000737:chemistry; D007700:Kinetics; D013058:Mass Spectrometry; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism; D015202:Protein Engineering; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary
chemical_list: D008565:Membrane Proteins; C405010:delta-hGHR; D019382:Human Growth Hormone; D006859:Hydrogen; D003903:Deuterium