A molecular mechanism for the "digital" response of p53 to stress.

The tumor suppressor protein p53 accumulates in response to cellular stress and consequently orchestrates the expression of multiple genes in a p53-level and time-dependent manner to overcome stress consequences, for which a molecular mechanism is currently unknown. Previously, we reported that DNA torsional flexibility distinguishes among p53 response elements (REs) and that transactivation at basal p53 levels is correlated with p53 REs flexibility. Here, we calculated the flexibility of ~200 p53 REs. By connecting functional outcomes of p53-target genes' activation to the calculated flexibility of their REs, we show that genes known to belong to pathways that are activated rapidly upon stress contain REs that are significantly more flexible relative to REs of genes known to be involved in pathways that are activated later in the response to stress. The global structural properties of several p53 REs belonging to different pathways were experimentally validated. Additionally, reporter-gene expression driven by flexible p53 REs occurred at lower p53 levels and with faster rates than expression from rigid REs. Furthermore, analysis of published endogenous mRNA levels of p53-target genes as a function of REs' flexibility showed that early versus late genes differ significantly in their flexibility properties of their REs and that highly flexible p53 REs enable high-activation level exclusively to early-response genes. Overall, we demonstrate that DNA flexibility of p53 REs contributes significantly to functional selectivity in the p53 system by facilitating the initial steps of p53-dependent target-genes expression, thereby contributing to survival versus death decisions in the p53 system.

On the Origins of Enzymes: Phosphate-Binding Polypeptides Mediate Phosphoryl Transfer to Synthesize Adenosine Triphosphate.

Reactions involving the transfer of a phosphoryl (-PO32-) group are fundamental to cellular metabolism. These reactions are catalyzed by enzymes, often large and complex, belonging to the phosphate-binding loop (P-loop) nucleoside triphosphatase (NTPase) superfamily. Due to their critical importance in life, it is reasonable to assume that phosphoryl-transfer reactions were also crucial in the pre-LUCA (last universal common ancestor) world and mediated by precursors that were simpler, in terms of their sequence and structure, relative to their modern-day enzyme counterparts. Here, we demonstrate that short phosphate-binding polypeptides (∼50 residues) comprising a single, ancestrally inferred, P-loop or Walker A motif mediate the reversible transfer of a phosphoryl group between two adenosine diphosphate molecules to synthesize adenosine triphosphate and adenosine monophosphate. This activity, although rudimentary, bears resemblance to that of adenylate kinase (a P-loop NTPase enzyme). The polypeptides, dubbed as "P-loop prototypes", thus relate to contemporary P-loop NTPases in terms of their sequence and function, and yet, given their simplicity, serve as plausible representatives of the early "founder enzymes" involved in proto-metabolic pathways.

Helicase-like functions in phosphate loop containing beta-alpha polypeptides.

The P-loop Walker A motif underlies hundreds of essential enzyme families that bind nucleotide triphosphates (NTPs) and mediate phosphoryl transfer (P-loop NTPases), including the earliest DNA/RNA helicases, translocases, and recombinases. What were the primordial precursors of these enzymes? Could these large and complex proteins emerge from simple polypeptides? Previously, we showed that P-loops embedded in simple ßα repeat proteins bind NTPs but also, unexpectedly so, ssDNA and RNA. Here, we extend beyond the purely biophysical function of ligand binding to demonstrate rudimentary helicase-like activities. We further constructed simple 40-residue polypeptides comprising just one ß-(P-loop)-α element. Despite their simplicity, these P-loop prototypes confer functions such as strand separation and exchange. Foremost, these polypeptides unwind dsDNA, and upon addition of NTPs, or inorganic polyphosphates, release the bound ssDNA strands to allow reformation of dsDNA. Binding kinetics and low-resolution structural analyses indicate that activity is mediated by oligomeric forms spanning from dimers to high-order assemblies. The latter are reminiscent of extant P-loop recombinases such as RecA. Overall, these P-loop prototypes compose a plausible description of the sequence, structure, and function of the earliest P-loop NTPases. They also indicate that multifunctionality and dynamic assembly were key in endowing short polypeptides with elaborate, evolutionarily relevant functions.

On the emergence of P-Loop NTPase and Rossmann enzymes from a Beta-Alpha-Beta ancestral fragment.

This article is dedicated to the memory of Michael G. Rossmann. Dating back to the last universal common ancestor, P-loop NTPases and Rossmanns comprise the most ubiquitous and diverse enzyme lineages. Despite similarities in their overall architecture and phosphate binding motif, a lack of sequence identity and some fundamental structural differences currently designates them as independent emergences. We systematically searched for structure and sequence elements shared by both lineages. We detected homologous segments that span the first ßαß motif of both lineages, including the phosphate binding loop and a conserved aspartate at the tip of ß2. The latter ligates the catalytic metal in P-loop NTPases, while in Rossmanns it binds the nucleotide's ribose moiety. Tubulin, a Rossmann GTPase, demonstrates the potential of the ß2-Asp to take either one of these two roles. While convergence cannot be completely ruled out, we show that both lineages likely emerged from a common ßαß segment that comprises the core of these enzyme families to this very day.

Differential susceptibility & replication potential of Vero E6, BHK-21, RD, A-549, C6/36 cells & Aedes aegypti mosquitoes to three strains of chikungunya virus.

Background & objectives: Chikungunya virus (CHIKV), a mosquito-borne arthritogenic virus causes infections ranging from febrile illness to debilitating polyarthralgia in humans. Re-emergence of the virus has affected millions of people in Africa and Asia since 2004. During the outbreak, a new lineage of the virus has evolved as an adaptation for enhanced replication and transmission by Aedes albopictus mosquito. A study was designed to compare the susceptibility of four vertebrate cell lines, namely Vero E6 (African green monkey kidney), BHK-21 (Baby hamster kidney), RD (human rhabdomyosarcoma), A-549 (human alveolar basal epithelial cell) and C6/36 (Ae. albopictus) to Asian genotype and two lineages of East, Central and South African (E1:A226 and E1:A226V) of CHIKV. Methods: One-step growth kinetics of different CHIKV strains was carried out in the above five cell lines to determine the growth kinetics and virus yield. Virus titre was determined by 50 per cent tissue culture infectious dose assay and titres were calculated by the Reed and Muench formula. Growth and virus yield of the three strains in Ae. aegypti mosquitoes was studied by intrathoracic inoculation and virus titration in Vero E6 cell line. Results: Virus titration showed Vero E6, C6/36 and BHK-21 cell lines are high virus yielding with all the three lineages while RD and A-549 yielded low virus titres. C6/36 cell line was the most sensitive and yielded the maximum titre. Ae. aegypti mosquitoes, when inoculated with high titre virus, yielded an almost equal growth with the three strains while rapid growth of E1:A226V and Asian strain was observed with 1 log virus. Interpretation & conclusions: C6/36 cell line was found to be the most sensitive and high yielding for CHIKV irrespective of lineages while Vero E6 and BHK-21 cell lines yielded high titres and may find application for vaccine/diagnostic development. Infection of Ae. aegypti mosquitoes with the three CHIKV strains gave almost identical pattern of growth.

New Insights into the Role of DNA Shape on Its Recognition by p53 Proteins.

The tumor suppressor p53 acts as a transcription factor recognizing diverse DNA response elements (REs). Previous structural studies of p53-DNA complexes revealed non-canonical Hoogsteen geometry of A/T base pairs at conserved CATG motifs leading to changes in DNA shape and its interface with p53. To study the effects of DNA shape on binding characteristics, we designed REs with modified base pairs "locked" into either Hoogsteen or Watson-Crick form. Here we present crystal structures of these complexes and their thermodynamic and kinetic parameters, demonstrating that complexes with Hoogsteen base pairs are stabilized relative to those with all-Watson-Crick base pairs. CATG motifs are abundant in p53REs such as GADD45 and p53R2 related to cell-cycle arrest and DNA repair. The high-resolution structures of these complexes validate their propensity to adopt the unique Hoogsteen-induced structure, thus providing insights into the functional role of DNA shape and broadening the mechanisms that contribute to DNA recognition by proteins.

Diverse p53/DNA binding modes expand the repertoire of p53 response elements.

The tumor suppressor protein p53 acts as a transcription factor, binding sequence-specifically to defined DNA sites, thereby activating the expression of genes leading to diverse cellular outcomes. Canonical p53 response elements (REs) are made of two decameric half-sites separated by a variable number of base pairs (spacers). Fifty percent of all validated p53 REs contain spacers between 1 and 18 bp; however, their functional significance is unclear at present. Here, we show that p53 forms two different tetrameric complexes with consensus or natural REs, both with long spacers: a fully specific complex where two p53 dimers bind to two specific half-sites, and a hemispecific complex where one dimer binds to a specific half-site and the second binds to an adjacent spacer sequence. The two types of complexes have comparable binding affinity and specificity, as judged from binding competition against bulk genomic DNA. Structural analysis of the p53 REs in solution shows that these sites are not bent in both their free and p53-bound states when the two half-sites are either abutting or separated by spacers. Cell-based assay supports the physiological relevance of our findings. We propose that p53 REs with long spacers comprise separate specific half-sites that can lead to several different tetrameric complexes. This finding expands the universe of p53 binding sites and demonstrates that even isolated p53 half-sites can form tetrameric complexes. Moreover, it explains the manner in which p53 binds to clusters of more than one canonical binding site, common in many natural REs.