No abstract

A molecular model of the human melanin-concentrating hormone (MCH) peptide was constructed and docked into a helical, bacteriorhodopsin-based model of the recently identified human MCH receptor. From this hormone-receptor complex, potential sites of agonist-receptor interaction were identified, and site-directed mutagenesis was used to substitute residues predicted to reside within the receptor binding pocket. Substitution of Asp(123)(3.32) in the third transmembrane domain of the receptor resulted in a loss of detectable (125)I-MCH binding and of MCH-stimulated Ca(2+) flux; cell surface expression of the mutant receptor was not affected. Arg(11) and Arg(14) of the MCH ligand were identified as potential sites of interaction with Asp(123)(3.32). [Ala(14)]-MCH was equipotent to native MCH in its ability to bind to and activate the wild-type MCH receptor, whereas [Ala(11)]-MCH displayed a 3000-fold reduction in binding affinity and a complete loss of measurable functional activity. Furthermore, [Lys(11)]-MCH and [D-Arg(11)]-MCH displayed reduced affinity for the receptor. [Lys(11)]-MCH was observed to be a partial agonist, eliciting approximately 67% of the native peptide's activity in a Ca(2+) flux assay, and [D-Arg(11)]-MCH was determined to be a functional antagonist with a K(b) valve of 15.8 microM. These data provide evidence that a basic moiety with specific stereochemical requirements at this site is needed for receptor activation. We conclude that both Asp(123)(3.32) in the MCH receptor and Arg(11) in the MCH peptide are required for the formation of the MCH peptide/receptor complex and propose that they form a direct interaction that is critical for receptor function.

No abstract

We probed the substrate specificity of a recombinant noncovalent complex of the full-length hepatitis C virus (HCV) NS3 serine protease and NS4A cofactor, using a series of small synthetic peptides derived from the three trans-cleavage sites of the HCV nonstructural protein sequence. We observed a distinct cleavage site preference exhibited by the enzyme complex. The values of the turnover number (k cat) for the most efficient NS4A/4B, 4B/5A, and 5A/5B peptide substrates were 1.6, 11, and 8 min ؊1 , respectively, and the values for the corresponding Michaelis-Menten constants (K m) were 280, 160, and 16 M, providing catalytic efficiency values (k cat /K m) of 92, 1,130, and 8,300 M ؊1 s ؊1. An alanine-scanning study for an NS5A/5B substrate (P6P4) revealed that P1 Cys and P3 Val were critical. Finally, substitutions at the scissile P1 Cys residue by homocysteine (Hcy), S-methylcysteine (Mcy), Ala, S-ethylcysteine (Ecy), Thr, Met, D-Cys, Ser, and penicillamine (Pen) produced progressively less efficient substrates, revealing a stringent stereochemical requirement for a Cys residue at this position.

A thermodynamic analysis using isothermal titration calorimetry (ITC) has been performed to examine the binding interaction between the SH2 (Src homology 2) domain of growth factor receptor binding protein 2 (Grb2-SH2) and one of its phosphotyrosine (pY) polypeptide ligands. Interaction of the Shc-derived phosphotyrosine hexapeptide Ac-SpYVNVQ-NH2 with Grb2-SH2 was both enthalpically and entropically favorable (DeltaH = -7.55 kcal mol-1, -TDeltaS = -1.46 kcal mol-1 , DeltaG = -9.01 kcal mol-1, T = 20 degrees C). ITC experiments using five alanine-substituted peptides were performed to examine the role of each side chain in binding. The results were consistent with homology models of the Grb2-SH2-Shc hexapeptide complex which identified several possible hydrogen bonds between Grb2-SH2 and the phosphotyrosine and conserved asparagine(+2) side chains of the Shc hexapeptide. These studies also demonstrated that the hydrophobic valine(+1) side chain contributes significantly to the favorable entropic component of binding. The thermodynamic and structural data are consistent with a Grb2-SH2 recognition motif of pY-hydrophobic-N-X (where X is any amino acid residue). The measured heat capacity of binding (DeltaCp = -146 cal mol-1 K-1) was very similar to computed values using semiempirical estimates (DeltaCp = -106 to -193 cal mol-1 K-1) derived from apolar and polar accessible surface area values calculated from several homology models of the Grb2-SH2-Shc hexapeptide complex. The homology model which most closely reproduced the measured DeltaCp value is also the model which had the lowest RMS deviation from the subsequently determined crystal structure. Calculations based on the thermodynamic data and these semiempirical estimates indicated that the binding event involves burial of nearly comparable apolar (677 A2) and polar (609 A2) surface areas.