Contribution of individual histidines to the global stability of human prolactin.
A member of the family of hematopoietic cytokines human prolactin (hPRL) is a 23k kDa polypeptide hormone, which displays pH dependence in its structural and functional properties. The binding affinity of hPRL for the extracellular domain of its receptor decreases 500-fold over the relatively narrow, physiologic pH range from 8 to 6; whereas, the affinity of human growth hormone (hGH), its closest evolutionary cousin, does not. Similarly, the structural stability of hPRL decreases from 7.6 to 5.6 kcal/mol from pH 8 to 6, respectively, whereas the stability of hGH is slightly increased over this same pH range. hPRL contains nine histidines, compared with hGH's three, and they are likely responsible for hPRL's pH-dependent behavior. We have systematically mutated each of hPRL's histidines to alanine and measured the effect on pH-dependent global stability. Surprisingly, a vast majority of these mutations stabilize the native protein, by as much as 2-3 kcal/mol. Changes in the overall pH dependence to hPRL global stability can be rationalized according to the predominant structural interactions of individual histidines in the hPRL tertiary structure. Using double mutant cycles, we detect large interaction free energies within a cluster of nearby histidines, which are both stabilizing and destabilizing to the native state. Finally, by comparing the structural locations of hPRL's nine histidines with their homologous residues in hGH, we speculate on the evolutionary role of replacing structurally stabilizing residues with histidine to introduce pH dependence to cytokine function.
Keeler
Camille
C
Department of Laboratory Medicine, Yale University, New Haven, Connecticut 06520-8035, USA.
Tettamanzi
M Cristina
MC
Meshack
Syrus
S
Hodsdon
Michael E
ME
D000409:Alanine / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006639:Histidine / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D019382:Human Growth Hormone / Q000737:chemistry; D006801:Humans; D006863:Hydrogen-Ion Concentration; D016018:Least-Squares Analysis; D008958:Models, Molecular; D009154:Mutation; D011388:Prolactin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D055550:Protein Stability
D019382:Human Growth Hormone; D006639:Histidine; D011388:Prolactin; D000409:Alanine