Solution Structure of Human Prolactin

Abstract

We report the solution structure of human prolactin determined by NMR spectroscopy. Our result is a significant improvement over a previous structure in terms of number and distribution of distance restraints, regularity of secondary structure, and potential energy. More significantly, the structure is sufficiently different that it leads to different conclusions regarding the mechanism of receptor activation and initiation of signal transduction. Here, we compare the structure of unbound prolactin to structures of both the homologue ovine placental lactogen and growth hormone. The structures of unbound and receptor bound prolactin/ placental lactogen are similar and no noteworthy structural changes occur upon receptor binding. The observation of enhanced binding at the second receptor site when the first site is occupied has been widely interpreted to indicate conformational change induced by binding the first receptor. However, our results indicate that this enhanced binding at the second site could be due to receptor-receptor interactions or some other free energy sources rather than conformational change in the hormone. Titration of human prolactin with the extracellular domain of the human prolactin receptor was followed by NMR, gel filtration and electrophoresis. Both binary and ternary hormone-receptor complexes are clearly detectable by gel filtration and electrophoresis. The binary complex is not observable by NMR, possibly due to a dynamic equilibrium in intermediate exchange within the complex. The ternary complex of one hormone molecule bound to two receptor molecules is on the contrary readily detectable by NMR. This is in stark contrast to the widely held view that the ternary prolactinreceptor complex is only transiently formed. Thus, our results lead to improved understanding of the prolactin-prolactin receptor interaction.

Crystal Structure of an Affinity-matured Prolactin Complexed to Its Dimerized Receptor Reveals the Topology of Hormone Binding Site 2

“…Indeed, the first structural data reported for the PRL system involved PRLR-ECDs bound to non-PRL ligands: a 1:1 complex between hGH and the hPRLR (22), and a 1:2 complex between ovine PL (oPL) and the rat (r) PRLR (23). As for hPRL, its structure in solution was obtained by NMR, confirming its four antiparallel ␣-helix bundle fold (24). More recently, we reported the first set of x-ray coordinates for a PRL-core hormone and determined the structure of the pure antagonist Del1-9-G129R-hPRL (20), and the structure of this compound bound to the hPRLR-ECD (1:1 complex) was then determined (25).…”

“…After transformation, Escherichia coli BL21(DE3) colonies were analyzed for their DNA content; plasmids were sequenced to verify the presence of the expected mutations. Sequences encoding the ECD of human or rat PRLR were inserted into the pQE-70 expression plasmid (Qiagen) containing a His 6 tag at the C-terminal end as described (20,24 and PRLR ECDs were overexpressed in 0.5-to 1-liter cultures of E. coli BL21(DE3) or M15(REP4), respectively, and purified as described previously (20). Briefly, pro- Nter hPRL FOR 5Ј GA GAT ATA CAT ATG GCA CAG CAT CCA CCA TAC TGT CCC GGC GGG G 3Ј REV 5Ј C CCC GCC GGG ACA G TA TGG TGG ATG CTG TGC CAT ATG TAT ATC TC 3Ј T14P hPRL FOR 5Ј CGA TGC CAG GTG CCT CTT CGA GAC CTG TTT GAC C 3Ј REV 5Ј GGT CAA ACA GGT CTC GAA GAG GCA CCT GGC ATC G 3Ј PGGA Nter FOR 5Ј CAT CCA CCA TAC TGT CGA AAC CAG CCA GCC CGA TGC CAG GTG 3Ј REV 5Ј CAC CTG GCA TCG GGC TGG CTG GTT TCG ACA GTA TGG TGG ATG 3Ј PGGA-T14P PGGA FOR 5Ј CGA TGC CAG GTG CCT CTT CGA GAC CTG TTT GAC C 3Ј REV 5Ј GGT CAA ACA GGT CTC GAA GAG GCA CCT GGC ATC G 3Ј Full oPL PGGA-T14P FOR 5Ј GT CGA AAC CAG CCA GGC AAA TGC CAG ATC CCT CTT CGA GAC C 3Ј REV 5Ј G GTC TCG AAG AGG GAT CTG GCA TTT GCG TGG CTG GTT TCG AC 3Ј teins were overexpressed as insoluble inclusion bodies, which were solubilized in 8 M urea (5 min at 55°C, then 2 h at room temperature) and refolded by continuous dialysis (72 h, 4°C) against 100 volumes of 25 mM NH 4 HCO 3 , pH 8.6.…”

“…Although titration of hPRL by hPRL-ECD in NMR experiments suggested changes in chemical shifts at high receptor/ hormone ratio (24), the lack of structural data for the 1:2 complex between PRL and its receptor has remained detrimental to our understanding of this hormonal system. For example, it has been impossible to this date to evaluate the putative structural changes occurring in the intermediate 1:1 hormone⅐receptor complex upon interaction with the second receptor.…”

“…Although the formation of 1:2 complexes has been demonstrated by various approaches, including gel filtration, native electrophoresis, and surface plasmon resonance, interaction at binding site 2 appears to be too transient to stabilize ternary complexes (20,24,26). To circumvent this problem, we aimed at generating an affinity-matured hPRL variant able to form stable 1:2 complexes.…”

S179D prolactin: Antagonistic agony!

“…Because phosphorylation occurs when the hormone is in the process of disulfide bond rearrangement (disulfide isomerase and glutathione are also present in the secretory granules (Lorenson and Jacobs, 1984;Greenan et al, 1990)) from the oligomeric to monomeric state, sites in the molecule that would normally be internal in the monomeric version of unmodified PRL are available to the kinase. Serine 179 is on the hydrophobic side of helix 4 and would therefore be in the hydrophobic core of monomeric unmodified PRL (Teilum et al, 2005). Thus, it is presumed that phosphorylation of serine 179 results in a different conformation than monomeric unmodified PRL (see below for work on the molecular mimic in this regard).…”