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.

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.

Analysis of site-specific histidine protonation in human prolactin.

The structural and functional properties of human prolactin (hPRL), a 23 kDa protein hormone and cytokine, are pH-dependent. The dissociation rate constant for binding to the extracellular domain of the hPRL receptor increases nearly 500-fold over the relatively narrow and physiologic range from pH 8 to 6. As the apparent midpoint for this transition occurs around pH 6.5, we have looked toward histidine residues as a potential biophysical origin of the behavior. hPRL has a surprising number of nine histidines, nearly all of which are present on the protein surface. Using NMR spectroscopy, we have monitored site-specific proton binding to eight of these nine residues and derived equilibrium dissociation constants. During this analysis, a thermodynamic interaction between a localized triplet of three histidines (H27, H30, and H180) became apparent, which was subsequently confirmed by site-directed mutagenesis. After consideration of multiple potential models, we present statistical support for the existence of two negative cooperativity constants, one linking protonation of residues H30 and H180 with a magnitude of approximately 0.1 and the other weaker interaction between residues H27 and H30. Additionally, mutation of any of these three histidines to alanine stabilizes the folded protein relative to the chemically denatured state. A detailed understanding of these complex protonation reactions will aid in elucidating the biophysical mechanism of pH-dependent regulation of hPRL's structural and functional properties.

Solution structure and backbone dynamics of an N-terminal ubiquitin-like domain in the GLUT4-regulating protein, TUG.

The GLUT4-regulating protein, TUG, functions to retain GLUT4-containing membrane vesicles intracellularly and, in response to insulin stimulation, releases these vesicles to the cellular exocytic machinery for translocation to the plasma membrane. As part of our on going effort to describe the molecular basis for TUG function, we have determined the tertiary structure and characterized the backbone dynamics for an N-terminal ubiquitin-like domain (TUG-UBL1) using NMR spectroscopy. A well-ordered conformation is observed for residues 10-83 of full-length TUG, and confirms a beta-grasp or ubiquitin-like topology. Although not required for in vitro association with GLUT4, the functional role of the TUG-UBL1 domain has not yet been described. We undertook a limited literature review of similar N-terminal UBL domains and noted that a majority participate in protein-protein interactions, generally functioning as adaptor modules to physically associate the over all activity of the protein with a specific cellular process, such as the ubiquitin-proteasome pathway. In consistent fashion, TUG-UBL1 is not expected to participate in a covalent protein modification reaction as it lacks the characteristic C-terminal diglycine ("GG") motif required for conjugation to an acceptor lysine, and also lacks the three most common acceptor lysine residues involved in polyubiquitination. Additionally, analysis of the TUG-UBL1 molecular surface reveals a lack of conservation of the "Ile-44 hydrophobic face" typically involved in ubiquitin recognition. Instead, we speculate on the possible significance of a concentrated area of negative electrostatic potential with increased backbone mobility, both of which are features suggestive of a potential protein-protein interaction site.

Withanolides from Salpichroa origanifolia.

Five new withanolides, 5 alpha,6 alpha:22,26:24,25-triepoxy-16 alpha,26-dihydroxy-18(13-->17)-abeo-ergosta-2,13-dien-1-one (salpichrolide N, 1), 5 alpha,6 alpha:22,26:24,25-triepoxi-15 alpha,26-dihydroxyergosta-2,16-dien-1-one (salpichrolide L, 2), 5 alpha,6 alpha:22,26-diepoxi-24,25,26-trihydroxy-17(13-->18)-abeo-ergosta-2,13,15,17-tetraen-1-one (salpichrolide M, 3a), 5 alpha,6 alpha:22,25:22,26-triepoxy-24-hydroxy-17(13-->18)-abeo-ergosta-2,13,15,17-tetraen-1-one (salpichrolide J, 4), and 5 alpha,6 alpha:22,26-diepoxy-22,24,25-trihydroxy-17(13-->18)-abeo-ergosta-2,13,15,17-tetraen-1-one (salpichrolide K, 5), were isolated from the leaves of Salpichroa origanifolia and characterized by a combination of spectroscopic (1D and 2D NMR, MS) and chemical methods.

Antifeedant activity of withanolides from Salpichroa origanifolia on Musca domestica.

The antifeedant effect of several salpichrolides on larvae of Musca domestica was investigated. Three naturally occurring compounds, salpichrolide A (1), salpichrolide C (2), and salpichrolide G (3), previously isolated from Salpichroa origanifolia, and two known (4, 6) and three new (5, 7, 8) synthetic analogues were tested. The maximal effect on development was observed for salpichrolide A (1), while salpichrolide G (3) was the most toxic. The content of the salpichrolides in S. origanifolia was monitored by HPLC during plant development, reaching a maximum during summer.

A rare injury to the upper limb: elbow dislocation combined with Galeazzi fracture-dislocation.

The authors describe a rare instance of elbow dislocation associated with Galeazzi fracture-dislocation in a 16 year-old patient injured in a motor vehicle accident. They analyze the modalities of the trauma, the type of treatment employed, and the result after 2 years.