Characterization of the papillomavirus alpha(1)E2 peptide unfolded to folded transition upon DNA binding.

Transcriptional regulation depends on sequence-specific binding of regulatory proteins to their responsive elements in viral DNA. The papillomavirus E2 protein binds to DNA through the consensus sequence ACCG-NNNN-CGGT, activating or inhibiting viral replication. Through molecular dynamics simulations we were able to characterize the role of the DNA molecule on E2 binding region (named alpha(1)E2) conformation, acquiring structural insights for previous works suggesting an unfolded to folded transition upon alpha(1)E2 complexation to DNA. Moreover, the results indicate sites to guide the design of alpha(1)E2 synthetic derivatives to inhibit the HPV infection.

ATP and fructose-2,6-bisphosphate regulate skeletal muscle 6-phosphofructo-1-kinase by altering its quaternary structure.

Recently, it has been demonstrated that fructose-2,6-bisphosphate (F2,6BP) protects skeletal muscle 6-phosphofructo-1-kinase (PFK) from thermal inactivation (50 degrees C) and against the deleterious effects of guanidinium hydrochloride (GdmCl). On the other hand, ATP, when added at its inhibitory concentrations, that is, >1 mM, enhanced either the thermal- or GdmCl-induced inactivation of PFK. Moreover, we concluded that these phenomena were probably due to the stabilization of PFK tetrameric structure by F2,6BP, and the dissociation of this structure into dimers induced by ATP. Aimed at elucidating the effects of F2,6BP and ATP on PFK at the structural and functional levels, the present work correlates the effects of these metabolites on the equilibrium between PFK dimers and tetramers to the regulation promoted on the enzyme catalytic activity. We show that ATP present a dual effect on PFK structure, favoring the formation of tetramer at stimulatory concentrations (up to 1 mM), and dissociating tetramers into dimers at inhibitory concentrations (>1 mM). Furthermore, F2,6BP counteracted this later ATP effect at either the structural or catalytic levels. Additionally, the effects of both F2,6BP or ATP on the equilibrium between PFK tetramers and dimers and on the enzyme activity presented a striking parallelism. Therefore, we concluded that modulation of PFK activity by ATP and F2,6BP is due to the effects of these ligands on PFK quaternary structure, altering the oligomeric equilibrium between PFK tetramers and dimers.

Fructose-2,6-bisphosphate counteracts guanidinium chloride-, thermal-, and ATP-induced dissociation of skeletal muscle key glycolytic enzyme 6-phosphofructo-1-kinase: A structural mechanism for PFK allosteric regulation.

Rabbit muscle 6-phosphofructo-1-kinase (PFK) is the key glycolytic enzyme being regulated by diverse molecules and signals. This enzyme may undergo a reversible dissociation from a fully active homotetramer to a quite inactive dimer. There are evidences that some positive and negative modulators of PFK, such as ADP and citrate, may interfere with the enzyme oligomeric structure shifting the tetramer-dimer equilibrium towards opposite orientations, where the negative modulators favor the dissociation of tetramers into dimers and vice versa. PFK is allosterically inhibited by ATP at its physiological range of concentration, an effect counteracted by fructose-2,6-bisphosphate (F2,6BP). However, the structural molecular mechanism by which ATP and F2,6BP regulate PFK is hitherto demonstrated. The present paper aimed at demonstrating that either the ATP-induced inhibition of PFK and the reversion of this inhibition by F2,6BP occur through the same molecular mechanism, i.e., the displacement of the oligomeric equilibrium of the enzyme. This conclusion is arrived assessing the effects of ATP and F2,6BP on PFK inactivation through two distinct ways to dissociate the enzyme: (a) upon incubation at 50 degrees C, or (b) incubating the enzyme with guanidinium hydrochloride (GdmCl). Our results reveal that temperature- and GdmCl-induced inactivation of PFK prove remarkably more effective in the presence 5mM ATP than in the absence of additives. On the other hand, the presence of 100 nM F2,6BP attenuate the effects of both high-temperature exposition and GdmCl on PFK, even in the simultaneous presence of 5mM ATP. These data support the hypothesis that ATP shifts the oligomeric equilibrium of PFK towards the smaller conformations, while F2,6BP acts in the opposite direction. This conclusion leads to important information about the molecular mechanism by which PFK is regulated by these modulators.

Specificity in DNA recognition by a peptide from papillomavirus E2 protein.

The E2 proteins of papillomavirus specifically bind to double-stranded DNA containing the consensus sequence ACCG-N4-CGGT, where N is any nucleotide. Here, we show the binding and recognition of dissimilar DNA sequences by an 18 amino-acid peptide (alpha1E2), which corresponds to the DNA-recognition helix, alpha-helix-1. Isothermal DNA binding assays performed with the DNA consensus sequence show saturable curves with alpha1E2 peptide, and the alpha1E2 peptide is converted to an ordered conformation upon complexation. Measurements performed with non-specific DNA sequence fail to saturate, a behavior characteristic of non-specific binding. Binding of the alpha1E2 peptide to these DNA sequences display a different counter-ion dependence, indicating a dissimilar, sequence-dependent mechanism of interaction. Quantitative stoichiometric measurements revealed the specificity in alpha1E2 peptide recognition of the ACCG half-site, demonstrating capacity for discrimination of nucleic acid bases sequences without the need of a whole protein architecture.

A radioassay for phosphofructokinase-1 activity in cell extracts and purified enzyme.

Phosphofructokinase-1 plays a key role in the regulation of carbohydrate metabolism. Its activity can be used as an indicator of the glycolytic flux in a tissue sample. The method most commonly employed to determine phosphofructokinase-1 activity is based on oxidation of NADH by the use of aldolase, triosephosphate isomerase, and alpha-glycerophosphate dehydrogenase. This method suffers from several disadvantages, including interactions of the auxiliary enzymes with phosphofructokinase-1. Other methods that have been used also require auxiliary enzymes or are less sensitive than a coupled assay. Here, we propose a direct method to determine phosphofructokinase-1 activity, without the use of auxiliary enzymes. This method employs fructose-6-phosphate and ATP labeled with 32P in the gamma position ([gamma-32P]ATP), and leads to the formation of ADP and fructose-1,6-bisphosphate labeled with 32P ([1-32P]fructose-1,6-bisphosphate). Activated charcoal is used to adsorb unreacted [gamma-32P]ATP, and the radioactive product in the supernatant, [1-32P]fructose-1,6-bisphosphate, is analyzed on a liquid scintillation counter. The proposed method is precise and relatively inexpensive, and can be applied to determine phosphofructokinase-1 activity in cellular extracts as well as in the purified enzyme.

Structural genomics of protein phosphatases.

The New York SGX Research Center for Structural Genomics (NYSGXRC) of the NIGMS Protein Structure Initiative (PSI) has applied its high-throughput X-ray crystallographic structure determination platform to systematic studies of all human protein phosphatases and protein phosphatases from biomedically-relevant pathogens. To date, the NYSGXRC has determined structures of 21 distinct protein phosphatases: 14 from human, 2 from mouse, 2 from the pathogen Toxoplasma gondii, 1 from Trypanosoma brucei, the parasite responsible for African sleeping sickness, and 2 from the principal mosquito vector of malaria in Africa, Anopheles gambiae. These structures provide insights into both normal and pathophysiologic processes, including transcriptional regulation, regulation of major signaling pathways, neural development, and type 1 diabetes. In conjunction with the contributions of other international structural genomics consortia, these efforts promise to provide an unprecedented database and materials repository for structure-guided experimental and computational discovery of inhibitors for all classes of protein phosphatases.

Opposing effects of two osmolytes--trehalose and glycerol--on thermal inactivation of rabbit muscle 6-phosphofructo-1-kinase.

Trehalose and glycerol are known as good stabilizers of function and structure of several macromolecules against stress conditions. We previously reported that they have comparable effectiveness on protecting two yeast cytosolic enzymes against thermal inactivation. However, enzyme protection has always been associated to a decrease in catalytic activity at the stabilizing conditions i.e., the presence of the protective molecule. In the present study we tested trehalose and glycerol on thermal protection of the mammalian cytosolic enzyme phosphofructokinase. Here we found that trehalose was able to protect phosphofructokinase against thermal inactivation as well as to promote an activation of its catalytic activity. The enzyme incubated in the presence of 1 M trehalose did not present any significant inactivation within 2 h of incubation at 50 degrees C, contrasting to control experiments where the enzyme was fully inactivated during the same period exhibiting a t0.5 for thermal inactivation of 56+/-5 min. On the other hand, enzyme incubated in the presence of 37.5% (v/v) glycerol was not protected against incubation at 50 degrees C. Indeed, when phosphofructokinase was incubated for 45 min at 50 degrees C in the presence of lower concentrations of glycerol (7.5-25%, v/v), the remaining activity was 2-4 times lower than control. These data show that the compatibility of effects previously shown for trehalose and glycerol with some yeast cytosolic enzymes can not be extended to all globular enzyme system. In the case of phosphofructokinase, we believe that its property of shifting between several different complex oligomers configurations can be influenced by the physicochemical properties of the stabilizing molecules.