title: Two independent histidines, one in human prolactin and one in its receptor, are critical for pH-dependent receptor recognition and activation.
abstract: Human prolactin (hPRL), a member of the family of hematopoietic cytokines, functions as both an endocrine hormone and autocrine/paracrine growth factor. We have previously demonstrated that recognition of the hPRL·receptor depends strongly on solution acidity over the physiologic range from pH 6 to pH 8. The hPRL·receptor binding interface contains four histidines whose protonation is hypothesized to regulate pH-dependent receptor recognition. Here, we systematically dissect its molecular origin by characterizing the consequences of His to Ala mutations on pH-dependent receptor binding kinetics, site-specific histidine protonation, and high resolution structures of the intermolecular interface. Thermodynamic modeling of the pH dependence to receptor binding affinity reveals large changes in site-specific protonation constants for a majority of interface histidines upon complexation. Removal of individual His imidazoles reduces these perturbations in protonation constants, which is most likely explained by the introduction of solvent-filled, buried cavities in the crystallographic structures without inducing significant conformational rearrangements.
authors.affiliation: Department of Laboratory Medicine, Yale School of Medicine, New Haven, Connecticut 06520, USA.
authors.forename: Mandar V
authors.initials: MV
authors.lastname: Kulkarni
authors.forename: M Cristina
authors.initials: MC
authors.lastname: Tettamanzi
authors.forename: James W
authors.initials: JW
authors.lastname: Murphy
authors.forename: Camille
authors.initials: C
authors.lastname: Keeler
authors.forename: David G
authors.initials: DG
authors.lastname: Myszka
authors.forename: Naomi E
authors.initials: NE
authors.lastname: Chayen
authors.forename: Elias J
authors.initials: EJ
authors.lastname: Lolis
authors.forename: Michael E
authors.initials: ME
authors.lastname: Hodsdon
mesh_terms: D045744:Cell Line, Tumor; D006639:Histidine / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008958:Models, Molecular; D011388:Prolactin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D011981:Receptors, Prolactin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013816:Thermodynamics
chemical_list: D011981:Receptors, Prolactin; D006639:Histidine; D011388:Prolactin