Effect of FKBP65, a putative elastin chaperone, on the coacervation of tropoelastin in vitro.

FKBP65 is a protein of the endoplasmic reticulum that is relatively abundant in elastin-producing cells and is associated with tropoelastin in the secretory pathway. To test an earlier suggestion by Davis and co-workers that FKBP65 could act as an intracellular chaperone for elastin, we obtained recombinant FKBP65 (rFKBP65) by expressing it in E. coli and examined its effect on the coacervation characteristics of chicken aorta tropoelastin (TE) using an in vitro turbidimetric assay. Our results reveal that rFKBP65 markedly promotes the initiation of coacervation of TE without significantly affecting the temperature of onset of coacervation. This effect shows saturation at a 1:2 molar ratio of TE to rFKBP65. By contrast, FKBP12, a peptidyl prolyl isomerase, has a negligible effect on TE coacervation. Moreover, the effect of rFKBP65 on TE coacervation is unaffected by the addition of rapamycin, an inhibitor of peptidyl prolyl isomerase (PPIase) activity. These observations rule out the involvement of the PPIase activity of rFKBP65 in modulating the coacervation of TE. Additional experiments using a polypeptide model of TE showed that rFKBP65, while promoting coacervation, may retard the maturation of this model polypeptide into larger aggregates. Based on these results, we suggest that FKBP65 may act as an elastin chaperone in vivo by controlling both the coacervation and the maturation stages of its self-assembly into fibrils.

Expression and spectroscopic characterization of a large fragment of the mu-opioid receptor.

We report here a procedure for the production in Escherichia coli and subsequent purification and characterization of an 80-residue fragment of the human mu-opioid receptor. The fragment ('TM2-3'), which comprises the second and third transmembrane segments as well as the first extracellular loop of the receptor, was expressed as a fusion with glutathione-S-transferase. The fusion protein, which accumulated in insoluble inclusion bodies, was solubilized with N-lauroylsarcosine, and TM2-3 was obtained by thrombin cleavage of the fusion protein followed by reversed-phase HPLC purification. CD spectroscopy of TM2-3 in lysophosphatidylcholine micelles showed that TM2-3 adopts approximately 50% alpha-helical structure in this environment, with the remainder consisting of disordered and/or beta-structure. This is consistent with the assumption of an alpha-helical structure by the two membrane-spanning regions and a nonhelical structure in the loop region of TM2-3. Fluorescence spectroscopy and fluorescence quenching experiments suggested that the extracellular loop lies near the surface of the lysophosphatidylcholine micelle. Our work shows that the study of large receptor fragments is a technically accessible approach to the study of the structural properties of the mu-opioid receptor and, possibly, other G-protein-coupled receptors as well.

Role of metal ions in ligand-receptor interaction: insights from structural studies.

Experimental data indicate that metal ions such as Na(+), Ca(2+) and Mg(2+), which are present in millimolar concentrations in the extracellular environment, modulate binding of ligands to plasma membrane receptors. Here, we briefly review structural studies that demonstrate that various types of ligands, including peptide hormones and drugs, bind metal ions, in particular Ca(2+), in the lipid milieu. We propose that the metal ion-bound forms of ligands represent their bioactive conformations. With a view to understanding the mechanism of modulation of ligand-receptor interactions by metal ions, we have computed a homology model of the mu-opioid receptor, a G protein-coupled receptor (GPCR), and performed docking of specific agonist and antagonist ligands in the receptor. This resulted in the formation of a ligand-metal ion-receptor (ternary) complex which accounted for the data on the structure-activity relationships of ligands and mutation data on the receptor. Based on experimental and modeling data, we have proposed a general mechanism of activation of GPCRs by their corresponding ligands wherein metal ions play a pivotal role. Studies on overexpressed segments of mu-receptor are in progress to verify the above proposal.

Identification of small molecule chemical inhibitors of the collagen-specific chaperone Hsp47.

Hsp47 is a collagen-specific molecular chaperone whose activity has been implicated in the pathogenesis of fibrotic diseases. Here, we describe the development of an assay for screening libraries of chemical compounds for inhibitors of Hsp47. A preliminary screen of 2080 compounds identified four that demonstrated inhibitory activity against Hsp47 in vitro, with IC(50) values ranging from 3 to 27 muM. Compounds identified through this method may provide the basis for development of novel antifibrotic therapeutics.

Mapping Hsp47 binding site(s) using CNBr peptides derived from type I and type II collagen.

As a crucial molecular chaperone in collagen biosynthesis, Hsp47 interacts with the nascent form as well as the mature triple-helical form of procollagen. The location(s) of Hsp47 binding sites on the collagen molecule are, as yet, unknown. We have examined the substrate specificity of Hsp47 in vitro using well-characterized CNBr peptide fragments of type I and type II collagen along with radiolabeled, recombinant Hsp47. Interaction of these peptides with Hsp47 bound to collagen-coated microtiter wells showed several binding sites for Hsp47 along the length of the alpha1 and alpha2 chains of type I collagen and the alpha1 chain of type II collagen, with the N-terminal regions showing the strongest affinities. The latter observation was also supported by the results of a ligand-blot assay. Except for two peptides in the alpha2(I) chain, peptides that showed substantial binding to Hsp47 did so in their triple-helical and not random-coil form. Unlike earlier studies that used peptide models for collagen, the results obtained here on fragments of type I and type II collagen identify, for the first time, binding of Hsp47 to specific regions of the collagen molecule. They also point to additional structural requirements for Hsp47 binding besides the known preference for third-position Arg residues and the triple-helical conformation.