title: Functional epitopes for site 1 of human prolactin.
abstract: Human prolactin (hPRL) binds two human prolactin receptor molecules, creating active heterotrimeric complexes. Receptors bind dissimilar hormone surfaces termed site 1 and site 2 in an obligate ordered process. We sought to map the functional epitopes in site 1 of hPRL. Extensive alanine mutagenesis (102 of the 199 residues) showed approximately 40% of these mutant hPRLs changed the ΔG for site 1 receptor binding. Six of these residues are within 3.5 Å of the receptor and form the site 1 functional epitopes. We identified a set of noncovalent interactions between these six residues and the receptor. We identified a second group of site 1 residues that are between 3.5 and 5 Å from the receptor where alanine mutations reduced the affinity. This second group has noncovalent interactions with other hormone residues and stabilized the topology of the functional epitopes by linking these to the body of the protein. Finally, we identified a third group of residues that are outside site 1 (>5 Å) and extend to site 2 and whose mutation to alanine significantly weakened receptor binding at site 1 of prolactin. These three groups of residues form a contiguous structural motif between sites 1 and 2 of human prolactin and may constitute structural features that functionally couple sites 1 and 2. This work identifies the residues that form the functional epitopes for site 1 of human prolactin and also identifies a set of residues that support the concept that sites 1 and 2 are functionally coupled by an allosteric mechanism.
authors.affiliation: Ohio State Biophysics Program, Ohio State University, 1925 Coffey Road, Columbus, OH 43210, USA.
authors.forename: Geeta Vittal
authors.initials: GV
authors.lastname: Rao
authors.forename: Charles L
authors.initials: CL
authors.lastname: Brooks
mesh_terms: D000409:Alanine; D001665:Binding Sites; D000939:Epitopes; D006801:Humans; D008958:Models, Molecular; D016296:Mutagenesis; D009154:Mutation; D011388:Prolactin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D020349:Surface Plasmon Resonance; D013816:Thermodynamics
chemical_list: D000939:Epitopes; D011388:Prolactin; D000409:Alanine