Sequence Variation in the Response Element Determines Binding by the Transcription Factor p73.

How the sequence of a response element affects the binding of a transcription factor and, ultimately, the differential rate of transcription of genes under its control is not well-understood. In the case of the p73 transcription factor, it binds to >200 response elements to trigger developmental, cell arrest, and apoptotic pathways. The p73 response elements match the 20 bp consensus sequence of the p53 response elements that are formed by two 10 bp half-sites, where each half-site is an inverted repeat of two 5 bp quarter-sites. Using sedimentation velocity and fluorescence anisotropy experiments, we studied how systematic variations in the sequence of a half-site response element modify the DNA binding affinity of the p73 DNA-binding domain. We observed that each nucleotide position in the response element has a different influence in determining the binding of the p73 DNA-binding domain. The cytosine in the fourth position of each quarter-site is the largest determinant of DNA binding, followed by the nucleotide in the fifth position, and last, the first three positions show a slight regulatory preference for purines. Together with previous structural and functional results, our data suggest a hierarchical model of binding in which some nucleotide positions in the response element are more important than others in determining the binding of the transcription factor.

Transactivation specificity is conserved among p53 family proteins and depends on a response element sequence code.

Structural and biochemical studies have demonstrated that p73, p63 and p53 recognize DNA with identical amino acids and similar binding affinity. Here, measuring transactivation activity for a large number of response elements (REs) in yeast and human cell lines, we show that p53 family proteins also have overlapping transactivation profiles. We identified mutations at conserved amino acids of loops L1 and L3 in the DNA-binding domain that tune the transactivation potential nearly equally in p73, p63 and p53. For example, the mutant S139F in p73 has higher transactivation potential towards selected REs, enhanced DNA-binding cooperativity in vitro and a flexible loop L1 as seen in the crystal structure of the protein-DNA complex. By studying, how variations in the RE sequence affect transactivation specificity, we discovered a RE-transactivation code that predicts enhanced transactivation; this correlation is stronger for promoters of genes associated with apoptosis.

Structural studies on mechanisms to activate mutant p53.

The design of a broad-spectrum cancer drug would provide enormous clinical benefits to treat cancer patients. Most of cancerous cells have a mutation in the p53 gene that results in an inactive mutant p53 protein. For this reason, p53 is a prime target for the development of a broad-spectrum cancer drug. To provide the atomic information to rationally design a drug to recover p53 activity is the main goal of the structural studies on mutant p53. We review three mechanisms that influence p53 activity and provide information about how reactivation of mutant p53 can be achieved: stabilization of the active conformation of the DNA-binding domain of the protein, suppression of missense mutations in the DNA-binding domain by a second-site mutation, and increased transactivation.

Structure of p73 DNA-binding domain tetramer modulates p73 transactivation.

The transcription factor p73 triggers developmental pathways and overlaps stress-induced p53 transcriptional pathways. How p53-family response elements determine and regulate transcriptional specificity remains an unsolved problem. In this work, we have determined the first crystal structures of p73 DNA-binding domain tetramer bound to response elements with spacers of different length. The structure and function of the adaptable tetramer are determined by the distance between two half-sites. The structures with zero and one base-pair spacers show compact p73 DNA-binding domain tetramers with large tetramerization interfaces; a two base-pair spacer results in DNA unwinding and a smaller tetramerization interface, whereas a four base-pair spacer hinders tetramerization. Functionally, p73 is more sensitive to spacer length than p53, with one base-pair spacer reducing 90% of transactivation activity and longer spacers reducing transactivation to basal levels. Our results establish the quaternary structure of the p73 DNA-binding domain required as a scaffold to promote transactivation.

Crystal structures of the DNA-binding domain tetramer of the p53 tumor suppressor family member p73 bound to different full-site response elements.

How cells choose between developmental pathways remains a fundamental biological question. In the case of the p53 protein family, its three transcription factors (p73, p63, and p53) each trigger a gene expression pattern that leads to specific cellular pathways. At the same time, these transcription factors recognize the same response element (RE) consensus sequences, and their transactivation of target genes overlaps. We aimed to understand target gene selectivity at the molecular level by determining the crystal structures of the p73 DNA-binding domain (DBD) in complex with full-site REs that vary in sequence. We report two structures of the p73 DBD bound as a tetramer to 20-bp full-site REs based on two distinct quarter-sites: GAACA and GAACC. Our study confirms that the DNA-binding residues are conserved within the p53 family, whereas the dimerization and tetramerization interfaces diverge. Moreover, a conserved lysine residue in loop L1 of the DBD senses the presence of guanines in positions 2 and 3 of the quarter-site RE, whereas a conserved arginine in loop 3 adapts to changes in position 5. Sequence variations in the RE elicit a p73 conformational response that might explain target gene specificity.

EEC- and ADULT-associated TP63 mutations exhibit functional heterogeneity toward P63 responsive sequences.

TP63 germ-line mutations are responsible for a group of human ectodermal dysplasia syndromes, underlining the key role of P63 in the development of ectoderm-derived tissues. Here, we report the identification of two TP63 alleles, G134V (p.Gly173Val) and insR155 (p.Thr193_Tyr194insArg), associated to ADULT and EEC syndromes, respectively. These alleles, along with previously identified G134D (p.Gly173Asp) and R204W (p.Arg243Trp), were functionally characterized in yeast, studied in a mammalian cell line and modeled based on the crystal structure of the P63 DNA-binding domain. Although the p.Arg243Trp mutant showed both complete loss of transactivation function and ability to interfere over wild-type P63, the impact of p.Gly173Asp, p.Gly173Val, and p.Thr193_Tyr194insArg varied depending on the response element (RE) tested. Interestingly, p.Gly173Asp and p.Gly173Val mutants were characterized by a severe defect in transactivation along with interfering ability on two DN-P63α-specific REs derived from genes closely related to the clinical manifestations of the TP63-associated syndromes, namely PERP and COL18A1. The modeling of the mutations supported the distinct functional effect of each mutant. The present results highlight the importance of integrating different functional endpoints that take in account the features of P63 proteins' target sequences to examine the impact of TP63 mutations and the associated clinical variability.

Asymmetric DNA recognition by the OkrAI endonuclease, an isoschizomer of BamHI.

Restriction enzymes share little or no sequence homology with the exception of isoschizomers, or enzymes that recognize and cleave the same DNA sequence. We present here the structure of a BamHI isoschizomer, OkrAI, bound to the same DNA sequence (TATGGATCCATA) as that cocrystallized with BamHI. We show that OkrAI is a more minimal version of BamHI, lacking not only the N- and C-terminal helices but also an internal 3(10) helix and containing ß-strands that are shorter than those in BamHI. Despite these structural differences, OkrAI recognizes the DNA in a remarkably similar manner to BamHI, including asymmetric contacts via C-terminal 'arms' that appear to 'compete' for the minor groove. However, the arms are shorter than in BamHI. We observe similar DNA-binding affinities between OkrAI and BamHI but OkrAI has higher star activity (at 37°C) compared to BamHI. Together, the OkrAI and BamHI structures offer a rare opportunity to compare two restriction enzymes that work on exactly the same DNA substrate.

The tetramer of p53 in the absence of DNA forms a relaxed quaternary state.

p53 is a tetrameric multidomain protein that triggers the anticancer cellular response to stress. We have calculated a three-dimensional reconstruction of full-length human p53 in the absence of DNA using single-particle electron microscopy. The reconstruction of DNA-free full-length p53 shows a square-shaped structure with four distinct domains and a hollow center. In comparison with the known compacted DNA-bound full-length p53 structures, the DNA-free p53 tetramer adopts a relaxed conformation with separated monomers and oligomerization interfaces different from those of the DNA-bound conformation.

Deciphering the mechanism of p73 recognition of p53 response elements using the crystal structure of p73-DNA complexes and computational studies.

p73 belongs to p53 family transcription factor activating more than 50% of cell fate p53 target genes involved in cell cycle, apoptosis, DNA damage response alongside neuronal system development and differentiation by binding to 20-bp response elements (REs) having sequence motif (PPPC-A/T-T/A-GYYY) where P-purines and Y-pyrimidines with each 10-bp separated by minimum 0 to 13-bp spacer. The promiscuous nature of recognizing both cell fate and development genes and the underlying RE selectivity mechanism by p73 is not well understood. Here, we report the molecular details of p73 recognizing the REs using the crystal structure of p73 DNA binding domain (DBD) in complex with 12 base pair DNA sequence 5'-cAGGCATGCCTg-3' and molecular dynamics simulations with six different p53 natural promoter sequences. Each 20-base pair natural promoter forms a different major/minor groove due to the presence of nucleotides A/T, A/C, G/G, T/T and G/T at positions 3, 8, 13, 18 uniquely recognized by p73 key residues Lys138 and Arg268. The loops L1 and L3 bearing these residues influence inter-and intra-dimer interfaces interactions and hence p73 forms a unique tetramer with each natural promoter sequence. Structural features of the DNA and the spacing between half-sites influence p73 tetramerization and its transactivation function.

Nanodiscs versus macrodiscs for NMR of membrane proteins.

It is challenging to find membrane mimics that stabilize the native structures, dynamics, and functions of membrane proteins. In a recent advance, nanodiscs have been shown to provide a bilayer environment compatible with solution NMR. We show that increasing the lipid to "belt" peptide ratio expands their diameter, slows their reorientation rate, and allows the protein-containing discs to be aligned in a magnetic field for oriented sample solid-state NMR. The spectroscopic properties of membrane proteins with one to seven transmembrane helices in q = 0.1 isotropic bicelles, ~10 nm diameter isotropic nanodiscs, ~30 nm diameter magnetically aligned macrodiscs, and q = 5 magnetically aligned bicelles are compared.

Domain structure of separase and its binding to securin as determined by EM.

After the degradation of its inhibitor securin, separase initiates chromosome segregation during the metaphase-to-anaphase transition by cleaving cohesin. Here we present a density map at a resolution of 25 A of negatively stained separase-securin complex. Based on labeling data and sequence analysis, we propose a model for the structure of separase, consisting of 26 ARM repeats, an unstructured region of 280 residues and two caspase-like domains, with securin binding to the ARM repeats.

Projection map of aquaporin-9 at 7 A resolution.

Aquaporin-9, an aquaglyceroporin present in diverse tissues, is unique among aquaporins because it is not only permeable to water, urea and glycerol, but also allows passage of larger uncharged solutes. Single particle analysis of negatively stained recombinant rat aquaporin-9 revealed a particle size characteristic of the tetrameric organization of all members of the aquaporin family. Reconstitution of aquaporin-9 into two-dimensional crystals enabled us to calculate a projection map at 7 A resolution. The projection structure indicates a tetrameric structure, similar to GlpF, with each square-like monomer forming a pore. A comparison of the pore-lining residues between the crystal structure of GlpF and a homology model of aquaporin-9 locates substitutions in these residues predominantly to the hydrophobic edge of the tripathic pore of GlpF, providing first insights into the structural basis for the broader substrate specificity of aquaporin-9.

Spiked Genes: A Method to Introduce Random Point Nucleotide Mutations Evenly throughout an Entire Gene Using a Complete Set of Spiked Oligonucleotides for the Assembly.

In vitro mutagenesis methods have revolutionized biological research and the biotechnology industry. In this study, we describe a mutagenesis method based on synthesizing a gene using a complete set of forward and reverse spiked oligonucleotides that have been modified to introduce a low ratio of mutant nucleotides at each position. This novel mutagenesis scheme named "Spiked Genes" yields a library of clones with an enhanced mutation distribution due to its unbiased nucleotide incorporation. Using the far-red fluorescent protein emKate as a model, we demonstrated that Spiked Genes yields richer libraries than those obtained via enzymatic methods. We obtained a library without bias toward any nucleotide or base pair and with even mutations, transitions, and transversion frequencies. Compared with enzymatic methods, the proposed synthetic approach for the creation of gene libraries represents an improved strategy for screening protein variants and does not require a starting template.

Molecular architecture of tumor suppressor p53.

p53 is a transcription factor central to cellular DNA metabolism that controls cellular responses to DNA damage. p53 activity, finely regulated, integrates the information from several pathways to preserve the cell's genetic information. Great attention has been given to the structural determination of p53 domains and its cancerous mutants because 50% of cancer cases present mutations in p53 that hinder its activity resulting in uncontrolled cell reproduction. We enumerate the multiple studies carried to elucidate the structure of p53 domains and we highlight their main findings. The ultimate goal of the reviewed structural efforts is to understand p53 function at atomic level with the aim to overcome cancer by reversing p53 mutant activity to its normal function.

A view of consecutive binding events from structures of tetrameric endonuclease SfiI bound to DNA.

Many reactions in cells proceed via the sequestration of two DNA molecules in a synaptic complex. SfiI is a member of a growing family of restriction enzymes that can bind and cleave two DNA sites simultaneously. We present here the structures of tetrameric SfiI in complex with cognate DNA. The structures reveal two different binding states of SfiI: one with both DNA-binding sites fully occupied and the other with fully and partially occupied sites. These two states provide details on how SfiI recognizes and cleaves its target DNA sites, and gives insight into sequential binding events. The SfiI recognition sequence (GGCCNNNN[downward arrow]NGGCC) is a subset of the recognition sequence of BglI (GCCNNNN[downward arrow]NGGC), and both enzymes cleave their target DNAs to leave 3-base 3' overhangs. We show that even though SfiI is a tetramer and BglI is a dimer, and there is little sequence similarity between the two enzymes, their modes of DNA recognition are unusually similar.

Energetic and structural considerations for the mechanism of protein sliding along DNA in the nonspecific BamHI-DNA complex.

The molecular mechanism by which DNA-binding proteins find their specific binding sites is still unclear. To gain insights into structural and energetic elements of this mechanism, we used the crystal structure of the nonspecific BamHI-DNA complex as a template to study the dominant electrostatic interaction in the nonspecific association of protein with DNA, and the possible sliding pathways that could be sustained by such an interaction. Based on calculations using the nonlinear Poisson-Boltzmann method and Brownian dynamics, a model is proposed for the initial nonspecific binding of BamHI to B-form DNA that differs from that seen in the crystal structure of the nonspecific complex. The model is electrostatically favorable, and the salt dependence as well as other thermodynamic parameters calculated for this model are in good agreement with experimental results. Several residues in BamHI are identified for their important contribution to the energy in the nonspecific binding model, and specific mutagenesis experiments are proposed to test the model on this basis. We show that a favorable sliding pathway of the protein along DNA is helical.

Crystallization of restriction endonuclease SfiI in complex with DNA.

The SfiI endonuclease from Streptomyces fimbriatus (EC 3.1.21.4) is a tetrameric enzyme that binds simultaneously to two recognition sites and cleaves both sites concertedly. It serves as a good model system for studying both specificity and cooperative DNA binding. Crystals of the enzyme were obtained by the hanging-drop vapor-diffusion method in complex with a 21-mer oligonucleotide. The crystals are trigonal, with unit-cell parameters a = b = 85.7, c = 202.6 A, and diffract to 2.6 A resolution on a rotating-anode X-ray generator. Preliminary X-ray analysis reveals the space group to be either P3(1)21 or P3(2)21. Interestingly, the crystals change to space group P6(1)22, with unit-cell parameters a = b = 85.5, c = 419.6 A, when the selenomethionyl (SeMet) derivative of the enzyme is co-crystallized with the same DNA. Phase information is currently being derived from this SeMet SfiI-DNA complex.