CRK is a human homolog of chichen v-Crk, which is an adaptor protein. The SH2 domain of CRK binds to several tyrosine-phosphorylated proteins, including the epidermal growth factor receptor, p130(Cas), Shc, and paxillin. The SH3 domain, in turn, binds to cytosolic proteins of 135-145, 160, 180, and 220 kDa. We screened expression libraries by Far Western blotting, using CRK SH3 as a probe, and identified partial cDNA sequences of four distinct proteins, including C3G, DOCK180, EPS15, and clone ST12. The consensus sequence of the CRK SH3 binding sites as deduced from their amino acid sequences was Pro+3-Pro+2-X+1-Leu0-Pro-1-X-2-Lys-3. The interaction of the CRK SH3 domain with the DOCK180 peptide was examined with an optical biosensor, based on the principles of surface plasmon resonance. A low dissociation constant of the order of 10(-7) resulted from a high association rate constant (kassoc = 3 x 10(4)) and low dissociation rate constant (kdiss = 3 x 10(-3)). All CRK-binding proteins except clone ST12 also bound to another adaptor protein, Grb2. Mutational analysis revealed that glycine at position +1 of ST12 inhibited the binding to Grb2 while retaining the high affinity binding to CRK SH3. The result suggests that the amino acid at position +1 also contributes to the high affinity binding of the peptides to the SH3 domain of Grb2, but not to that of CRK.

The diversity of the immune response, which can provide a panel of catalytic antibodies with varying degrees of catalytic activity and substrate specificity by immunization with a single hapten, raises the question concerning the extent to which a rationally designed hapten dictates the paratopes for catalytic function in the antigencombining site. We have investigated the biochemical properties within a panel of six hydrolytic catalytic antibodies elicited against a phosphonate transition state analog 3 and have examined the correlation between the functions and the antigen-combining-site structures. Although the individual values for kCM, Km, and Ktsa (the affinity for the transition state analog) of the six antibodies differed substantially, the transition state analysis (kcJkuncai versus K$/ A'tsa) displayed a linear relationship (slope = 0.99) with the four antibodies 6D9, 8D11, 4D5, and 9C10, which have homologous primary amino acid sequences, providing evidence that all of the differential binding energy of the transition state vs the ground state is available for the rate enhancement. This also suggested that these four antibodies catalyze the hydrolysis by variations of the same basic mechanism of transition state stabilization. In antibody 6D9, the difference in free energy between the antibody-catalyzed and uncatalyzed reactions was calculated to be 4.0 kcal/mol, a value that is consistent with the typical binding energy for one hydrogen bond between charged and uncharged groups in enzyme-substrate complexes. On the other hand, antibody 7C8, which has an amino acid sequence different from those of the others, deviated from the linear relationship observed in the transition state analysis, suggesting that catalytic factors other than transition state stabilization, such as a functioning acid or base, are involved in the catalysis. Thus, the difference in the catalytic properties is reflected in the differences in the Fv amino acid sequences. The analysis of the substrate specificity suggested that the catalytic antibodies with highly homologous primary amino acid sequences possess homogeneous binding modes to the substrate or hapten. The three-dimensional molecular model of the antibody 6D9-hapten complex reveals that the phosphonate moiety in the hapten interacts with His (L27d), a catalytic amino acid residue participating in the transition state stabilization in the antibody-catalyzed reactions. This histidine is conserved in the catalytic antibodies 6D9, 8D11, 4B5, and 9C10, and chemical modification by DEPC treatment caused a complete loss of the hydrolytic activity. Although the amino acid sequence of antibody 3G6 is highly homologous to the other four catalytic antibodies, antibody 3G6 has Tyr (L27d), which had reduced activity when modified with tetranitromethane. It is noteworthy that the majority of these catalytic antibodies, generated against a single transition state analog, display high homology in the biochemical and structural properties and catalyze the reaction with the same mechanism ...

Graphical abstract

We examined the subtype-selective binding site of the beta-adrenergic receptors (betaARs). The beta(1)/beta(2)-chimeric receptors showed the importance of the second and seventh transmembrane domains (TM2 and TM7) of the beta(2)AR for the binding of the beta(2)-selective agonists such as formoterol and procaterol. Alanine-substituted mutants of TM7 of the beta(2)AR showed that Tyr(308,) located at the top of TM7, mainly contributed to beta(2) selectivity. However, Tyr(308) interacted with formoterol and procaterol in two different ways. The results of Ala- and Phe-substituted mutants indicated that the phenyl group of Tyr(308) interacted with the phenyl group in the N-substituent of formoterol (hydrophobic interaction), and the hydroxyl group of Tyr(308) interacted with the protonated amine of procaterol (hydrophilic interaction). In contrast to beta(2)AR, TM2 is a major determinant that beta(1)-selective agonists such as denopamine and T-0509 bound the beta(1)AR with high affinity. Three amino acids (Leu(110), Thr(117), and Val(120)) in TM2 of the beta(1)AR were identified as major determinants for beta(1)-selective binding of these agonists. Three-dimensional models built on the basis of the predicted structure of rhodopsin showed that Tyr(308) of the beta(2)AR covered the binding pocket formed by TM2 and TM7 from the upper side, and Thr(117) of the beta(1)AR located in the middle of the binding pocket to provide a hydrogen bonding for the beta(1)-selective agonists. These data indicate that TM2 and TM7 of the betaAR formed the binding pocket that binds the betaAR subtype-selective agonists with high affinity.