Fabrication and characterization of bioactive composite coatings on Mg-Zn-Ca alloy by MAO/sol-gel.

High corrosion rate and accumulation of hydrogen gas upon degradation impede magnesium alloys' clinical application as implants. In this work, micro-arc oxidation (MAO) was used to fabricate a porous coating on magnesium alloys as an intermediate layer to enhance the bonding strength of propolis layer. Then the composite coatings were fabricated using sol-gel method by dipping sample into the solution containing propolis and polylactic acid at 40 °C. The corrosion resistance of the samples was determined based on potentiodynamic polarization experiments and immersion tests. Biocompatibility was designed by observing the attachment and growth of wharton's jelly-derived mesenchymal stem cells (WJCs) on substrates with MAO coating and substrates with composite coatings. The results showed that, compared with that of Mg-Zn-Ca alloy, the corrosion current density of the samples with composite coatings decreased from 5.37 × 10⁻5 to 1.10 × 10⁻6 A/cm² and the corrosion potential increased by 240 mV. Composite coatings exhibit homogeneous corrosion behavior and can promote WJCs cell adhesion and proliferation. In the meantime, pH value was relatively stable during the immersion tests, which may be significant for cellular survival. In conclusion, our results indicate that composite coatings on Mg-Zn-Ca alloy fabricated by MAO/sol-gel method provide a new type bioactive material.

Receptor activation: what does the rhodopsin structure tell us?

G-protein-coupled receptors (GPCRs) are a large family of seven-transmembrane-helix proteins that mediate responses to hormones, neurotransmitters and, in the case of rhodopsin, photons. The recent determination of the structure of rhodopsin at atomic resolution opens avenues to a deeper understanding of GPCR activation and transmembrane signaling. Data from previous crosslinking, spin labeling and scanning accessibility experiments on rhodopsin have been mapped onto the high-resolution structure. These data correlate well and are consistent with the structure, and suggest that activation by light opens a cleft at the cytoplasmic end of the seven-helix bundle of rhodopsin. Furthermore, lessons learned from rhodopsin might also apply to other members of this essential family of receptors. (For an animation of the crystal structure of rhodopsin see http://archive.bmn.com/supp/tips/tips2211a.html)

An activation switch in the ligand binding pocket of the C5a receptor.

Although agonists are thought to occupy binding pockets within the seven-helix core of serpentine receptors, the topography of these binding pockets and the conformational changes responsible for receptor activation are poorly understood. To identify the ligand binding pocket in the receptor for complement factor 5a (C5aR), we assessed binding affinities of hexapeptide ligands, each mutated at a single position, for seven mutant C5aRs, each mutated at a single position in the putative ligand binding site. In ChaW (an antagonist) and W5Cha (an agonist), the side chains at position 5 are tryptophan and cyclohexylalanine, respectively. Comparisons of binding affinities indicated that the hexapeptide residue at this position interacts with two C5aR residues, Ile-116 (helix III) and Val-286 (helix VII); in a C5aR model these two side chains point toward one another. Both the I116A and the V286A mutations markedly increased binding affinity of W5Cha but not that of ChaW. Moreover, ChaW, the antagonist hexapeptide, acted as a full agonist on the I116A mutant. These results argue that C5aR residues Ile-116 and Val-286 interact with the side chain at position 5 of the hexapeptide ligand to form an activation switch. Based on this and previous work, we present a docking model for the hexapeptide within the C5aR binding pocket. We propose that agonists induce a small change in the relative orientations of helices III and VII and that these helices work together to allow movement of helix VI away from the receptor core, thereby triggering G protein activation.

Structure. Rhodopsin sees the light.

Members of the seven transmembrane receptor superfamily bind a remarkable variety of ligands, from neurotransmitters to odorants, and activate a spectacular array of G protein signaling molecules. These G-protein coupled receptors (GPCRs) are important in many cellular functions and so there has been great interest in elucidating how they transmit their signals to the interior of the cell after activation by ligand. As Bourne and Meng explain in their Perspective, the molecular movements of activated GPCRs are becoming clear now that the first crystal structure of a GPCR (rhodopsin, the light-trapping receptor found in the retina of the eye) has been reported (Palczweski et al.).

Similar structures and shared switch mechanisms of the beta2-adrenoceptor and the parathyroid hormone receptor. Zn(II) bridges between helices III and VI block activation.

The seven transmembrane helices of serpentine receptors comprise a conserved switch that relays signals from extracellular stimuli to heterotrimeric G proteins on the cytoplasmic face of the membrane. By substituting histidines for residues at the cytoplasmic ends of helices III and VI in retinal rhodopsin, we engineered a metal-binding site whose occupancy by Zn(II) prevented the receptor from activating a retinal G protein, Gt (Sheikh, S. P., Zvyaga, T. A. , Lichtarge, O., Sakmar, T. P., and Bourne, H. R. (1996) Nature 383, 347-350). Now we report engineering of metal-binding sites bridging the cytoplasmic ends of these two helices in two other serpentine receptors, the beta2-adrenoreceptor and the parathyroid hormone receptor; occupancy of the metal-binding site by Zn(II) markedly impairs the ability of each receptor to mediate ligand-dependent activation of Gs, the stimulatory regulator of adenylyl cyclase. We infer that these two receptors share with rhodopsin a common three-dimensional architecture and an activation switch that requires movement, relative to one another, of helices III and VI; these inferences are surprising in the case of the parathyroid hormone receptor, a receptor that contains seven stretches of hydrophobic sequence but whose amino acid sequence otherwise shows no apparent similarity to those of receptors in the rhodopsin family. These findings highlight the evolutionary conservation of the switch mechanism of serpentine receptors and help to constrain models of how the switch works.

C5a receptor activation. Genetic identification of critical residues in four transmembrane helices.

Hormones and sensory stimuli activate serpentine receptors, transmembrane switches that relay signals to heterotrimeric guanine nucleotide-binding proteins (G proteins). To understand the switch mechanism, we subjected 93 amino acids in transmembrane helices III, V, VI, and VII of the human chemoattractant C5a receptor to random saturation mutagenesis. A yeast selection identified 121 functioning mutant receptors, containing a total of 523 amino acid substitutions. Conserved hydrophobic residues are located on helix surfaces that face other helices in a modeled seven-helix bundle (Baldwin, J. M., Schertler, G. F., and Unger, V. M. (1997) J. Mol. Biol. 272, 144-164), whereas surfaces predicted to contact the surrounding lipid tolerate many substitutions. Our analysis identified 25 amino acid positions resistant to nonconservative substitutions. These appear to comprise two distinct components of the receptor switch, a surface at or near the extracellular membrane interface and a core cluster in the cytoplasmic half of the bundle. Twenty-one of the 121 mutant receptors exhibit constitutive activity. Amino acids substitutions in these activated receptors predominate in helices III and VI; other activating mutations truncate the receptor near the extracellular end of helix VI. These results identify key elements of a general mechanism for the serpentine receptor switch.

Evaluating docked complexes with the HINT exponential function and empirical atomic hydrophobicities.

Methods that predict geometries of ligands binding to receptor molecules can facilitate ligand discovery and yield information on the factors governing complementarity. Here, the use of atomic hydrophobicities in evaluating binding modes has been examined with four ligand-receptor complexes of known structure. In each system, hundreds of hypothetical binding orientations were generated with DOCK and evaluated using the HINT (Hydropathic INTeractions) exponential function and atomic hydrophobic constants. In three of the four systems, the experimental binding mode received the best HINT score; in the fourth system, the experimental binding mode scored only slightly lower than a similar, apparently reasonable orientation. The HINT function may be generally useful as a scoring method in molecular docking.

Orientational sampling and rigid-body minimization in molecular docking.

The biological activities of proteins depend on specific molecular recognition and binding. Computational methods for predicting binding modes can facilitate the discovery and design of ligands and yield information on the factors governing complementarity. The DOCK suite of programs has been applied to several systems; here, the degree of orientational sampling required to reproduce and identify known binding modes, with and without rigid-body energy minimization, is investigated for four complexes. There is a tradeoff between sampling and minimization. The known binding modes can be identified with intensive sampling alone (10,000 to 20,000 orientations generated per system) or with moderate sampling combined with minimization. Optimization improves energies significantly, particularly when steric clashes are present, and brings many orientations closer to the experimentally observed position. Whether or not minimization is performed, however, sampling must be sufficient to find at least one structure in the vicinity of the presumed true binding mode. Hybrid approaches combining docking and minimization are promising and will become more viable with the use of faster algorithms and the judicious selection of fewer orientations for minimization.