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.
Highlights
Lular studies of Human prolactin (hPRL) signaling provide a molecular basis for the hPRL role in breast cancer proliferation (6 –10)
The affinity of hPRL for hPRLrECD decreases 500-fold as the pH decreases from pH 8 to 6, whereas the binding of hGH is unchanged over this same pH range [23]
A primary contribution of residues in the hPRLrECD was initially excluded given the lack of pH dependence for hGH binding to the same receptor molecule
Summary
Recombinant Protein Purification, and Cell Culture—A modified vector, pT7L, was used to express and purify WT and site-directed mutants of hPRL from the BL21(DE3) strain of Escherichia coli as described previously [24]. In the final round of modeling considering all the SPR, the known protonation constants (and cooperativity constants) for unbound (i.e. free) hPRL and hPRLrECD were fixed, whereas the bound state pKa values were varied. As our intention for performing the thermodynamic modeling is only to identify the overall changes in protonation constants upon complexation required to explain the SPR results and because the simulations have proven insensitive to the relatively minor changes in pKa values between 25 and 35 °C, the extensive effort required for experimental measurement of the exact site-specific protonation constants for all His mutations at both temperatures is not warranted
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