Two independent histidines, one in human prolactin and one in its receptor, are critical for pH-dependent receptor recognition and activation.
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.
Kulkarni
Mandar V
MV
Department of Laboratory Medicine, Yale School of Medicine, New Haven, Connecticut 06520, USA.
Tettamanzi
M Cristina
MC
Murphy
James W
JW
Keeler
Camille
C
Myszka
David G
DG
Chayen
Naomi E
NE
Lolis
Elias J
EJ
Hodsdon
Michael E
ME
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
D011981:Receptors, Prolactin; D006639:Histidine; D011388:Prolactin