Biomedical subjects

Zippora Shakked

Publications and source records attributed to Zippora Shakked.

8 recordsLinked to original sources

PubMed2007

RUN-CBFbeta interaction in C. elegans: computational prediction and experimental verification.

The Runt domain proteins are eukaryotic transcription factors that regulate major developmental pathways. All members of this family contain a highly-conserved sequence-specific DNA binding domain: the Runt domain (RD). Structural and biochemical studies have shown that the Runt domain undergoes a conformational transition upon binding to DNA and that this process is regulated by an unrelated partner protein CBFbeta that enhances the DNA binding affinity of RD. Most of the reported studies on the Runt domain transcription factors were performed on proteins from mammals and Drosophila whereas very little has been known about the C. elegans RD protein, RUN, which provides the simplest model system for understanding the function of this class of transcription factors. We performed computational studies on RD domains from various species including C. elegans, Drosophila, and human, using the atom-atom contact surface area scoring method. The scoring analysis indicates that the DNA binding regulation of the C. elegans RD protein (CeRD) occurs via its interaction with a CBFbeta-like partner, as found for the human proteins, whereas a different mode of regulation may occur in the Drosophila system. Sequence, secondary structure and fold analyses of a putative CBFbeta protein identified in the C. elegans genome, CeCBFbeta, sharing a 22% identity with the human protein, predict a similar structure of this protein to that of the human CBFbeta protein. We produced the C. elegans proteins CeRD and CeCBFbeta in bacteria and confirmed their physical interaction as well as cross interactions with the corresponding human proteins. We also confirmed the structural similarity of CBFbeta and CeCBFbeta by circular dichroism analysis. The combined results suggest that a similar mechanism of regulation operates for the human and the C. elegans RD proteins despite the low sequence identity between their CBFbeta proteins and the evolutionary distance between the two systems.

Amino Acid Sequence↗

PubMed2006

Structural basis of DNA recognition by p53 tetramers.

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.

Apoptosis↗

PubMed2005

Molecular flexibility in ab initio drug docking to DNA: binding-site and binding-mode transitions in all-atom Monte Carlo simulations.

The dynamics of biological processes depend on the structure and flexibility of the interacting molecules. In particular, the conformational diversity of DNA allows for large deformations upon binding. Drug-DNA interactions are of high pharmaceutical interest since the mode of action of anticancer, antiviral, antibacterial and other drugs is directly associated with their binding to DNA. A reliable prediction of drug-DNA binding at the atomic level by molecular docking methods provides the basis for the design of new drug compounds. Here, we propose a novel Monte Carlo (MC) algorithm for drug-DNA docking that accounts for the molecular flexibility of both constituents and samples the docking geometry without any prior binding-site selection. The binding of the antimalarial drug methylene blue at the DNA minor groove with a preference of binding to AT-rich over GC-rich base sequences is obtained in MC simulations in accordance with experimental data. In addition, the transition between two drug-DNA-binding modes, intercalation and minor-groove binding, has been achieved in dependence on the DNA base sequence. The reliable ab initio prediction of drug-DNA binding achieved by our new MC docking algorithm is an important step towards a realistic description of the structure and dynamics of molecular recognition in biological systems.

Algorithms↗

PubMed2005

Structures of the DNA-binding site of Runt-domain transcription regulators.

Runt-domain (RD) proteins are transcription factors that play fundamental roles in various developmental pathways. They bind specifically to DNA sequences of the general form PyGPyGGTPy (Py = pyrimidine), through which they regulate transcription of target genes. The DNA duplex TCTGCGGTC/TGACCGCAG, incorporating the binding site for the RD transcription factors (bold), was crystallized in space group P4(3). X-ray analysis of two crystals diffracting to 1.7 and 2.0 angstroms resolution, which had slight variations in their unit-cell parameters, revealed two distinct conformations of the A-DNA helix. The two crystal structures possessed several structure and hydration features that had previously been observed in A-DNA duplexes. A comparative analysis of the present A-DNA structures and those of previously reported B-DNA crystal structures of RD-binding sites in free and protein-bound states showed the various duplexes to display several common features. Within this series, the present A-DNA duplexes adopt two conformations along the pathway from the canonical A-DNA to the B-DNA forms and the protein-bound helices display conformational features that are intermediate between those of the current A-DNA structures and that of the B-DNA-type helix of the free RD target. Based on these data and energy considerations, it is likely that the propensity of the RD-binding site to adopt the A-DNA or B-DNA conformation in solution depends on the sequence context and environmental conditions, and that the transition from either DNA form to the protein-bound conformation involves a small energy barrier.

Base Sequence↗

PubMed2005

Structural and energetic origins of sequence-specific DNA bending: Monte Carlo simulations of papillomavirus E2-DNA binding sites.

DNA bending is an important structural feature for indirect readout in protein-DNA recognition. The binding of papillomavirus E2 transcription factors to their DNA binding sites is associated with DNA bending, providing an attractive model system to study the origins of sequence-specific DNA bending. The consensus E2 target is of the general form ACCGN(4)CGGT with a variable four base pair region. We applied a new all-atom Monte Carlo (MC) algorithm that combines effective sampling with fast conformational equilibration. The resulting MC ensembles resemble the corresponding high-resolution crystal structures very well. Distinct bending is observed for the E2-DNA binding site with a central AATT linker in contrast to an essentially straight DNA with a central ACGT linker. Contributions of specific base pair steps to the overall bending are shown in terms of local structural parameters. The analysis of conformational substates provides new insights into the energetic origins of intrinsic DNA bending.

Algorithms↗

PubMed2004

Predicting indirect readout effects in protein-DNA interactions.

Recognition of DNA by proteins relies on direct interactions with specific DNA-functional groups, along with indirect effects that reflect variable energetics in the response of DNA sequences to twisting and bending distortions induced by proteins. Predicting indirect readout requires knowledge of the variations in DNA curvature and flexibility in the affected region, which we have determined for a series of DNA-binding sites for the E2 regulatory protein by using the cyclization kinetics method. We examined 16 sites containing different noncontacted spacer sequences, which vary by more than three orders of magnitude in binding affinity. For 15 of these sites, the variation in affinity was predicted within a factor of 3, by using experimental curvature and flexibility values and a statistical mechanical theory. The sole exception was traced to differential magnesium ion binding.

Base Sequence↗

PubMed2002

DNA binding and 3'-5' exonuclease activity in the murine alternatively-spliced p53 protein.

In this study we show that the naturally occurring C-terminally alternative spliced p53 (referred to as AS-p53) is active as a sequence-specific DNA binding protein as well as a 3'-5'-exonuclease in the presence of Mg2+ ions. The two activities are positively correlated as the sequence-specific DNA target is more efficiently degraded than a non-specific target. In contrast, a mutated AS-p53 protein that is deficient in DNA binding lacks exonuclease activity. The use of modified p53 binding sites, where the 3'-phosphate is replaced by a phosphorothioate group, enabled the inhibition of DNA degradation under the binding conditions. We demonstrate that AS-p53 interacts with its specific DNA target by two distinct binding modes: a high-affinity mode characterized by a low-mobility protein-DNA complex at the nanomolar range, and a low-affinity mode shown by a high-mobility complex at the micromolar range. Comparison of the data on the natural and the modified p53 binding sites suggests that the high-affinity mode is related to AS-p53 function as a transcription factor and that the low-affinity mode is associated with its exonuclease activity. The implications of these findings to a specific cellular role of AS-p53 are discussed.

Alternative Splicing↗

PubMed2002

DNA recognition by the RUNX1 transcription factor is mediated by an allosteric transition in the RUNT domain and by DNA bending.

The Runt domain proteins are transcription regulators of major developmental pathways. Here we present the crystal structures of the Runt domain (RD) of the human protein RUNX1 and its DNA binding site in their free states and compare them with the published crystal structures of RD bound to DNA and to the partner protein CBFbeta. We demonstrate that (1) RD undergoes an allosteric transition upon DNA binding, which is further stabilized by CBFbeta, and that (2) the free DNA target adopts a bent-helical conformation compatible with that of the complex. These findings elucidate the mechanism by which CBFbeta enhances RD binding to DNA as well as the role of the intrinsic conformation of the DNA target in the recognition process.

Allosteric Regulation↗