The tumor-suppressor protein p53 is among the most effective of the cell's natural defenses against cancer. In response to cellular stress, p53 binds as a tetramer to diverse DNA targets containing two decameric half-sites, thereby activating the expression of genes involved in cell-cycle arrest or apoptosis. Here we present high-resolution crystal structures of sequence-specific complexes between the core domain of human p53 and different DNA half-sites. In all structures, four p53 molecules self-assemble on two DNA half-sites to form a tetramer that is a dimer of dimers, stabilized by protein-protein and base-stacking interactions. The protein-DNA interface varies as a function of the specific base sequence in correlation with the measured binding affinities of the complexes. The new data establish a structural framework for understanding the mechanisms of specificity, affinity, and cooperativity of DNA binding by p53 and suggest a model for its regulation by regions outside the sequence-specific DNA binding domain.

p53 binds as a tetramer to DNA targets consisting of two decameric half-sites separated by a variable spacer. Here we present high-resolution crystal structures of complexes between p53 core-domain tetramers and DNA targets consisting of contiguous half-sites. In contrast to previously reported p53-DNA complexes that display standard Watson-Crick base pairs, the newly reported structures exhibit non-canonical Hoogsteen base-pairing geometry at the central A/T doublet of each half-site. Structural and computational analyses demonstrate that the Hoogsteen geometry distinctly modulates the B-DNA helix in terms of local shape and electrostatic potential which together with the contiguous DNA configuration results in enhanced protein-DNA and protein-protein interactions compared to non-contiguous half-sites. Our results suggest a mechanism, which relates spacer length to protein-DNA binding affinity. Our findings also expand the current understanding of protein-DNA recognition and establish the structural and chemical properties of Hoogsteen base pairs as the basis for a novel mode of sequence readout.

To gain insight into the structural basis of DNA bending by adenine-thymine tracts (A-tracts) and their role in DNA recognition by gene-regulatory proteins, we have determined the crystal structure of the high-affinity DNA target of the cancer-associated human papillomavirus E2 protein. The three independent B-DNA molecules of the crystal structure determined at 2.2-Å resolution are examples of A-tract-containing helices where the global direction and magnitude of curvature are in accord with solution data, thereby providing insights, at the base pair level, into the mechanism of DNA bending by such sequence motifs. A comparative analysis of E2-DNA conformations with respect to other structural and biochemical studies demonstrates that (i) the A-tract structure of the core region, which is not contacted by the protein, is critical for the formation of the high-affinity sequence-specific protein-DNA complex, and (ii) differential binding affinity is regulated by the intrinsic structure and deformability encoded in the base sequence of the DNA target.A-tract ͉ papillomavirus E2-DNA target ͉ transcriptional control T he relationship between DNA sequence, structure, and function has been studied and discussed extensively for the last 20 years. A particular effort has been directed toward the structural elucidation of short runs of four to six adeninethymine residues, known as A-tracts, in an attempt to reveal the structural basis of DNA curvature induced by such sequence motifs when they are inserted in phase with the helical periodicity (1, 2). Despite numerous efforts, including x-ray crystallographic and solution studies, the structural basis of A-tractinduced bending has remained enigmatic (3). Because no single structure could explain the whole phenomenon, it was necessary to rely on models, several of which had been proposed (4). They generally conform to the gel migration data, which suggest that the center of curvature is toward the minor groove of the A-tracts and toward the major groove of the intervening general sequences (5). However, they differ substantially in the details of the stereochemical origin of curvature. This issue is of particular biological significance, as sequence-dependent DNA curvature or bending is an important determinant of DNA recognition by proteins (6).The E2 regulatory system of human papillomaviruses provides an example where sequence-specific binding of proteins to A-tracts is crucial to the organism's life cycle. The E2 proteins from all viral strains activate or repress transcription in a context-dependent manner and are required for the initiation of replication in vivo. Their function depends on sequence-specific binding to a highly conserved 12-bp sequence of the general form ACCGNNNNCGGT, where N4 is variable (7,8). However, the E2 binding sites in the human papillomavirus (HPV) genomes, including the cancer-related strains HPV-16 and HPV-18 (9), exhibit a further level of specificity in the interaction with their cognate E2 proteins. This specificity is manifested by th...

The emergence of bacterial multidrug resistance to antibiotics threatens to cause regression to the preantibiotic era. Here we present the crystal structure of the large ribosomal subunit from Staphylococcus aureus, a versatile Gram-positive aggressive pathogen, and its complexes with the known antibiotics linezolid and telithromycin, as well as with a new, highly potent pleuromutilin derivative, BC-3205. These crystal structures shed light on specific structural motifs of the S. aureus ribosome and the binding modes of the aforementioned antibiotics. Moreover, by analyzing the ribosome structure and comparing it with those of nonpathogenic bacterial models, we identified some unique internal and peripheral structural motifs that may be potential candidates for improving known antibiotics and for use in the design of selective antibiotic drugs against S. aureus.

Reaction of the electron-rich, bulky tridentate PNP ligand (2,6-bis-(di-tert-butylphosphinomethyl)pyridine) with Ru(PPh3)3Cl2 at 65 °C resulted in formation of a solution containing the dinitrogen monomeric Ru(II) complex 1a and the N2-bridged dinuclear Ru(II) complex 1b, which can be interconverted. Passing argon through the solution results in formation of pure 1b. The Ru(II) hydride dinitrogen complex 2 was obtained by the reaction of complex 1b with 2 equiv of NaBEt3H. Complex 1b reacted with 4 equiv of AgOCOCF3 to yield [Ru(PNP)(CF3COO)2], 3. The Ru(II) carbonyl hydride complex 4 was obtained by the reaction of PNP and Ru2(OAc)4 in methanol as a result of O−H activation and decarbonylation of methanol. Complexes 1b, 2, and 4 were structurally characterized by X-ray crystallography. Complexes 1b and 2 catalyze the dehydrogenation of secondary alcohols to the corresponding ketones with good yields and high selectivity accompanied with the evolution of dihydrogen in a homogeneous system without a need for a hydrogen acceptor. The presumed intermediate Ru dihydride complex is generated in situ by reaction 1b or 2 with a base (1 equiv for each Ru−Cl bond), and the reaction can proceed in the absence of excess base or acid.