Enhanced Affinities and Specificities of Consolidated Ligands for the Src Homology (SH) 3 and SH2 Domains of Abelson Protein-tyrosine Kinase

Abstract

The possible interrelationships between multiple domains of proteins involved in intracellular signal transduction are complex and not easily investigated. We have synthesized a series of bivalent consolidated ligands, which interact simultaneously with the SH2 and SH3 domain of Abelson kinase in a SH(32) dual domain construct, a portion of native Abelson kinase. Affinities were measured by quenching of intrinsic tryptophan fluorescence. Consolidated ligands have enhanced affinity and specificity compared to monovalent equivalents. Affinity is also dependent on the length of the linker joining the two parts, with an optimum distance similar to that expected from structural models of Abl (SH(32). These results suggest that consolidated ligands may be generally useful reagents for probing structural and functional activities of multidomain proteins. Src homology (SH)1 domains are building blocks in many proteins involved in intracellular signal transduction. Detailed understanding of the pathways involving these domains is complicated by the substantial range of individual specificities in SH2 and SH3 domains, and their combination into large proteins that can form multiple homo-and heteromolecular associations. The reductionist approach of studying individual domains has been very successful (1-3). Nonetheless, the interactions between the domains are still poorly understood. These interactions are likely to be of significance in explaining more fully the complete activities of the signal-transducing complexes. A detailed structural picture of the inter-and intramolecular organization of the domains is then a significant objective. Several cases of multiple SH2 and SH3 domaincontaining constructs have been studied structurally (Lck SH(32) (Ref. 4), Grb2 SH(323) (Ref. 5), and Abl SH(32) (Ref. 6)), but there are technical limitations to current structural approaches. In the crystalline state, packing forces may be of the same magnitude as the weak interdomain forces, so there are limits to the interpretation of diffraction studies. Solution studies by NMR are only applicable in a molecular mass range less than ϳ30 kDa. For NMR, time-averaged NOE constraints in rapidly exchanging conformations are not readily interpretable. As a complement to direct structural methods, we propose the investigation of such multidomain complexes using "consolidated" ligands. These ligands, having multiple binding portions, may be expected to bind with high affinity and specificity when a linker between the two affine segments is of the correct length, and there is little affinity of the linker itself. The consolidated ligands do not necessarily resemble any natural ligand. Such ligands are demonstrated here, binding to the SH(32) of Abelson kinase. It is reasonable to assume that consolidated ligands for dual SH2, dual SH3, or multiple combinations with other ligands can be produced using a similar approach.The proposed consolidated ligands are similar in concept to affinity reagents, with the modification that the second functionalit...

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