Chemically induced dimerization provides a general way to gain control over intracellular processes. Typically, FK506-binding protein (FKBP) domains are fused to a signaling domain of interest, allowing crosslinking to be initiated by addition of a bivalent FKBP ligand. In the course of protein engineering studies on human FKBP, we discovered that a single point mutation in the ligandbinding site (Phe-36 3 Met) converts the normally monomeric protein into a ligand-reversible dimer. Two-hybrid, gel filtration, analytical ultracentrifugation, and x-ray crystallographic studies show that the mutant (FM) forms discrete homodimers with micromolar affinity that can be completely dissociated within minutes by addition of monomeric synthetic ligands. These unexpected properties form the basis for a ''reverse dimerization'' regulatory system involving F M fusion proteins, in which association is the ground state and addition of ligand abolishes interactions. We have used this strategy to rapidly and reversibly aggregate fusion proteins in different cellular compartments, and to provide an off switch for transcription. Reiterated FM domains should be generally useful as conditional aggregation domains (CADs) to control intracellular events where rapid, reversible dissolution of interactions is required. Our results also suggest that dimerization is a latent property of the FKBP fold: the crystal structure reveals a remarkably complementary interaction between the monomer binding sites, with only subtle changes in side-chain disposition accounting for the dramatic change in quaternary structure.

Targeted disruption of the pp60 src (Src) gene has implicated this tyrosine kinase in osteoclast-mediated bone resorption and as a therapeutic target for the treatment of osteoporosis and other bone-related diseases. Herein we describe the discovery of a nonpeptide inhibitor (AP22408) of Src that demonstrates in vivo antiresorptive activity. Based on a cocrystal structure of the noncatalytic Src homology 2 (SH2) domain of Src complexed with citrate [in the phosphotyrosine (pTyr) binding pocket], we designed 3,4-diphosphonophenylalanine (Dpp) as a pTyr mimic. In addition to its design to bind Src SH2, the Dpp moiety exhibits bone-targeting properties that confer osteoclast selectivity, hence minimizing possible undesired effects on other cells that have Src-dependent activities. The chemical structure AP22408 also illustrates a bicyclic template to replace the post-pTyr sequence of cognate Src SH2 phosphopeptides such as Ac-pTyr-Glu-Glu-Ile (1). An x-ray structure of AP22408 complexed with Lck (S164C) SH2 confirmed molecular interactions of both the Dpp and bicyclic template of AP22408 as predicted from molecular modeling. Relative to the cognate phosphopeptide, AP22408 exhibits significantly increased Src SH2 binding affinity (IC50 ‫؍‬ 0.30 M for AP22408 and 5.5 M for 1). Furthermore, AP22408 inhibits rabbit osteoclast-mediated resorption of dentine in a cellular assay, exhibits bone-targeting properties based on a hydroxyapatite adsorption assay, and demonstrates in vivo antiresorptive activity in a parathyroid hormone-induced rat model.

Using structure-based design and protein mutagenesis we have remodeled the FKBP12 ligand binding site to include a sizable, hydrophobic specificity pocket. This mutant (F36V-FKBP) is capable of binding, with low or subnanomolar affinities, novel synthetic ligands possessing designed substituents that sterically prevent binding to the wild-type protein. Using binding and structural analysis of bumped compounds, we show here that the pocket is highly promiscuous-capable of binding a range of hydrophobic alkyl and aryl moieties with comparable affinity. Ligand affinity therefore appears largely insensitive to the degree of occupancy or quality of packing of the pocket. NMR spectroscopic analysis indicates that similar ligands can adopt radically different binding modes, thus complicating the interpretation of structure-activity relationships.

The tyrosine kinase pp60src (Src) is the prototypical member of a family of proteins that participate in a broad array of cellular signal transduction processes, including cell growth, differentiation, survival, adhesion, and migration. Abnormal Src family kinase (SFK) signaling has been linked to several disease states, including osteoporosis and cancer metastases. Src has thus emerged as a molecular target for the discovery of small-molecule inhibitors that regulate Src kinase activity by binding to the ATP pocket within the catalytic domain. Here, we present crystal structures of the kinase domain of Src in complex with two purine-based inhibitors: AP23451, a small-molecule inhibitor designed to inhibit Src-dependent bone resorption, and AP23464, a small-molecule inhibitor designed to inhibit the Src-dependent metastatic spread of cancer. In each case, a trisubstituted purine template core was elaborated using structure-based drug design to yield a potent Src kinase inhibitor. These structures represent early examples of high affinity purine-based Src family kinase-inhibitor complexes, and they provide a detailed view of the specific protein-ligand interactions that lead to potent inhibition of Src. In particular, the 3-hydroxyphenethyl N9 substituent of AP23464 forms unique interactions with the protein that are critical to the picomolar affinity of this compound for Src. The comparison of these new structures with two relevant kinase-inhibitor complexes provides a structural basis for the observed kinase inhibitory selectivity. Further comparisons reveal a concerted induced-fit movement between the N-and C-terminal lobes of the kinase that correlates with the affinity of the ligand. Binding of the most potent inhibitor, AP23464, results in the largest inducedfit movement, which can be directly linked to interactions of the hydrophenethyl N9 substituent with a region at the interface between the two lobes. A less pronounced induced-fit movement is also observed in the Src-AP23451 complex. These new structures illustrate how the combination of structural, computational, and medicinal chemistry can be used to rationalize the process of developing high affinity, selective tyrosine kinase inhibitors as potential therapeutic agents. Received 2 October 2005, revised and accepted for publication 11 October 2005The protein tyrosine kinase pp60src (Src) is the prototypical member of a family of non-receptor kinases, termed the Src family kinases (SFKs), which participate in a broad spectrum of cellular processes, including cell growth, differentiation, survival, adhesion, and migration (1-4). Src was identified as the transforming element of the Rous oncogenic retrovirus, exhibiting its transforming properties through a constitutively active mutant form of the protein effecting hyperactivation of kinase-dependent substrate protein phosphorylation and subsequent aberrant cellular signaling processes (5). Not surprisingly, SFK-dependent signal transduction mechanisms have been studied in a wide variety of cell types. S...

We propose that, the binding site for the complement subcomponent Clq on immunoglobulin G involves the last two (C-terminal) beta-strands of the C gamma 2 domain. This region contains a large number of accessible and highly conserved charged residues and charge is postulated as an important component of the Clq-IgG interaction. The conclusions are reached on the basis of accessibility and sequence conservation analyses of C gamma 2 amino acid residues, the use of specific inhibitors and chemical modification studies.