Site-directed ligand discovery.

We report a strategy (called "tethering") to discover low molecular weight ligands ( approximately 250 Da) that bind weakly to targeted sites on proteins through an intermediary disulfide tether. A native or engineered cysteine in a protein is allowed to react reversibly with a small library of disulfide-containing molecules ( approximately 1,200 compounds) at concentrations typically used in drug screening (10 to 200 microM). The cysteine-captured ligands, which are readily identified by MS, are among the most stable complexes, even though in the absence of the covalent tether the ligands may bind very weakly. This method was applied to generate a potent inhibitor for thymidylate synthase, an essential enzyme in pyrimidine metabolism with therapeutic applications in cancer and infectious diseases. The affinity of the untethered ligand (K(i) approximately 1 mM) was improved 3,000-fold by synthesis of a small set of analogs with the aid of crystallographic structures of the tethered complex. Such site-directed ligand discovery allows one to nucleate drug design from a spatially targeted lead fragment.

The advantages of ambiguous orientation.

Recent studies have revealed that although it is possible for certain transcription factors to bind in an orientated fashion on DNA, they have not evolved to do so. Rather, they rely on contacts with other proteins to precisely define their mode of binding.

A structural snapshot of base-pair opening in DNA.

The response of double-helical DNA to torsional stress may be a driving force for many processes acting on DNA. The 1.55-A crystal structure of a duplex DNA oligonucleotide d(CCAGGCCTGG)(2) with an engineered crosslink in the minor groove between the central guanine bases depicts how the duplex can accommodate such torsional stress. We have captured in the same crystal two rather different conformational states. One duplex contains a strained crosslink that is stabilized by calcium ion binding in the major groove, directly opposite the crosslink. For the other duplex, the strain in the crosslink is relieved through partial rupture of a base pair and partial extrusion of a cytosine accompanied by helix bending. The sequence used is the target sequence for the HaeIII methylase, and this partially flipped cytosine is the same nucleotide targeted for extrusion by the enzyme. Molecular dynamics simulations of these structures show an increased mobility for the partially flipped-out cytosine.

The orientation of the AP-1 heterodimer on DNA strongly affects transcriptional potency.

Activation of gene transcription in eukaryotes requires the cooperative assembly of an initiation complex containing many protein subunits. The necessity that these components contact each other and the promoter/enhancer in defined ways suggests that their spatial arrangement might influence the activation response. Indeed, growing evidence indicates that DNA architecture can profoundly affect transcriptional potency. Much less is known about the influence of protein architecture on transcriptional activation. Here, we examine the architectural dependence of activator function through the analysis of matched pairs of AP-1*DNA complexes differing only in their orientation. Mutation of a critical Arg residue in the basic-leucine zipper domain of either Fos or Jun yielded single point-mutant heterodimers that bind DNA in a single defined orientation, as determined directly by native chemical ligation/affinity cleavage; by contrast, the corresponding wild-type protein binds DNA as a roughly equal mixture of two isomeric orientations, which are related by subunit interchange. The stereochemistry of the point-mutant heterodimers could be switched by inversion of a C*G base pair in the center of the AP-1 site, thus providing access to both fixed orientational isomers. Yeast reporter gene assays consistently revealed that one orientational isomer activates transcription at least 10-fold more strongly than the other. These results suggest that protein architecture, especially the spatial relationship of the activation domain to the promoter, can exert a powerful influence on activator potency.

The leucine zipper domain controls the orientation of AP-1 in the NFAT.AP-1.DNA complex.

BACKGROUND: Heterologous transcription factors bound to adjacent sites in eukaryotic promoters often exhibit cooperative behavior. In most instances, the molecular basis for this cooperativity is poorly understood. Our efforts have been directed toward elucidation of the mechanism of cooperativity between NFAT and AP-1, two proteins that coordinately direct expression of the T-cell growth factor interleukin-2 (IL-2). RESULTS: We have previously shown that NFAT1 orients the two subunits of AP-1, c-Jun and c-Fos, on DNA through direct protein-protein interactions. In the present study, we have constructed cJun-cFos chimeric proteins and determined their orientation using a novel affinity-cleavage technology based on chemical ligation. We find that, in the presence of NFAT, the chimeric heterodimer binds in such a way as to preserve the orientation of the AP-1 leucine zipper, but not that of the basic region. CONCLUSIONS: Protein-protein interactions between NFAT and the leucine zipper of AP-1 enable the two proteins to bind DNA cooperatively and coordinately regulate the IL-2 promoter. The chemical ligation technology presented here provides a powerful strategy for affinity cleavage studies, including those using recombinant proteins.