The p53 tumor suppressor protein binds to DNA as a dimer of dimers to regulate transcription of genes that mediate responses to cellular stress. We have prepared a cross-linked trapped p53 core domain dimer bound to decamer DNA and have determined its structure by x-ray crystallography to 2.3 Å resolution. The p53 core domain subunits bind nearly symmetrically to opposite faces of the DNA in a head-to-head fashion with a loophelix motif making sequence-specific DNA contacts and bending the DNA by about 20°at the site of protein dimerization. Protein subunit interactions occur over the central DNA minor groove and involve residues from a zinc-binding region. Analysis of tumor derived p53 mutations reveals that the dimerization interface represents a third hot spot for mutation that also includes residues associated with DNA contact and protein stability. Residues associated with p53 dimer formation on DNA are poorly conserved in the p63 and p73 paralogs, possibly contributing to their functional differences. We have used the dimeric protein-DNA complex to model a dimer of p53 dimers bound to icosamer DNA that is consistent with solution bending data and suggests that p53 core domain dimer-dimer contacts are less frequently mutated in human cancer than intra-dimer contacts.The p53 tumor suppressor responds to cellular stresses such as DNA damage, in part, by binding to DNA and regulating the transcription of genes involved in cell cycle arrest, apoptosis, or senescence (1). The p53 protein forms a homotetramer, or dimer of dimers (2), with each subunit containing an N-terminal transactivation domain, a DNA-binding core domain (p53DBD) that is the site of the vast majority of tumor-derived substitution mutations, a tetramerization domain structured as a dimer of dimers (2-4), and a C-terminal regulatory domain (5, 6). p53 response elements contain two decamer sequences with the consensus PuPuPuC(A/T)͉(A/T)GPyPyPy (where Pu indicates a purine and Py indicates a pyrimidine) (7) with anywhere from 0 (icosamer) to 20 base pairs between them, although an icosamer is more commonly found in vivo (8). While individual p53DBD subunits can interact with a pentamer sequence, lower order complexes in the presence of a icosamer sequence are not observed, demonstrating that cooperative p53DBD interactions occur on DNA (9, 10). Other studies also reveal that p53DBD exists in both a latent and active DNAbinding form that can be detected in vivo (11)(12)(13). This is consistent with structural studies showing that nascent p53DBD forms dimers that are incompatible with simultaneous DNA binding (14), suggesting that the p53DBD, in the context of full-length p53, changes quaternary structure for DNA binding as a dimer of dimers. The functional importance of the p53 dimer is further supported by the observation that replacement of the p53 tetramerization domain with the dimerization domain of GCN4 affords near wild type p53 tumor suppression activity in vivo (15).The only molecular insights into the mode of DNA recognition by p53 were ...

HIV-1 reverse transcriptase (RT) undergoes a series of conformational changes during viral replication and is a central target for antiretroviral therapy. The intrinsic flexibility of RT can provide novel allosteric sites for inhibition. Crystals of RT that diffract X-rays to better than 2 Å resolution facilitated the probing of RT for new druggable sites using fragment screening by X-ray crystallography. A total of 775 fragments were grouped into 143 cocktails, which were soaked into crystals of RT in complex with the non-nucleoside drug rilpivirine (TMC278). Seven new sites were discovered, including the Incoming Nucleotide Binding, Knuckles, NNRTI Adjacent, and 399 sites, located in the polymerase region of RT, and the 428, RNase H Primer Grip Adjacent, and 507 sites, located in the RNase H region. Three of these sites—Knuckles, NNRTI Adjacent, and Incoming Nucleotide Binding—are inhibitory and provide opportunities for discovery of new anti-AIDS drugs.

HIV-1 reverse transcriptase (RT) is a primary target for anti-AIDS drugs. Structures of HIV-1 RT, usually determined at ∼2.5–3.0 Å resolution, are important for understanding enzyme function and mechanisms of drug resistance in addition to being helpful in the design of RT inhibitors. Despite hundreds of attempts, it was not possible to obtain the structure of a complex of HIV-1 RT with TMC278, a nonnucleoside RT inhibitor (NNRTI) in advanced clinical trials. A systematic and iterative protein crystal engineering approach was developed to optimize RT for obtaining crystals in complexes with TMC278 and other NNRTIs that diffract X-rays to 1.8 Å resolution. Another form of engineered RT was optimized to produce a high-resolution apo-RT crystal form, reported here at 1.85 Å resolution, with a distinct RT conformation. Engineered RTs were mutagenized using a new, flexible and cost effective method called methylated overlap-extension ligation independent cloning. Our analysis suggests that reducing the solvent content, increasing lattice contacts, and stabilizing the internal low-energy conformations of RT are critical for the growth of crystals that diffract to high resolution. The new RTs enable rapid crystallization and yield high-resolution structures that are useful in designing/developing new anti-AIDS drugs.

The tumor suppressor p53 regulates downstream genes in response to many cellular stresses and is frequently mutated in human cancers. Here, we report the use of a crosslinking strategy to trap a tetrameric p53 DNA binding domain (p53DBD) bound to DNA and the X-ray crystal structure of the protein/DNA complex. The structure reveals that two p53DBD dimers bind to B form DNA with no relative twist and that a p53 tetramer can bind to DNA without introducing significant DNA bending. The numerous dimer-dimer interactions involve several strictly conserved residues thus suggesting a molecular basis for p53DBD-DNA binding cooperativity. Surface residue conservation of the p53DBD tetramer bound to DNA highlights possible regions of other p53 domain or p53 cofactor interactions.