Crystal structure of the motor domain of centromere-associated protein E in complex with a non-hydrolysable ATP analogue.

Centromere-associated protein E (CENP-E) is a kinesin motor protein essential for mitosis and a new target for anticancer agents with less side effects. To rationally design anticancer drug candidates based on structure, it is important to determine the three-dimensional structure of the CENP-E motor domain bound to its inhibitor. Here, we report the first crystal structure of the CENP-E motor domain in complex with a non-hydrolysable ATP analogue, adenylyl-imidodiphosphate (AMPPNP). Furthermore, the structure is compared with the ADP-bound form of the CENP-E motor domain as well as the AMPPNP-bound forms of other kinesins. This study indicates that helix α4 of CENP-E participates in the slow binding of CENP-E to microtubules. These results will contribute to the development of anticancer drugs targeting CENP-E.

Cryo-EM Structure of the Type IV Pilus Extension ATPase from Enteropathogenic Escherichia coli.

Type 4 pili (T4P) are retractable surface appendages found on numerous bacteria and archaea that play essential roles in various microbial functions, including host colonization by pathogens. An ATPase is required for T4P extension, but the mechanism by which chemical energy is transduced to mechanical energy for pilus extension has not been elucidated. Here, we report the cryo-electron microscopy (cryo-EM) structure of the BfpD ATPase from enteropathogenic Escherichia coli (EPEC) in the presence of either ADP or a mixture of ADP and AMP-PNP. Both structures, solved at 3 Å resolution, reveal the typical toroid shape of AAA+ ATPases and unambiguous 6-fold symmetry. This 6-fold symmetry contrasts with the 2-fold symmetry previously reported for other T4P extension ATPase structures, all of which were from thermophiles and solved by crystallography. In the presence of the nucleotide mixture, BfpD bound exclusively AMP-PNP, and this binding resulted in a modest outward expansion in comparison to the structure in the presence of ADP, suggesting a concerted model for hydrolysis. De novo molecular models reveal a partially open configuration of all subunits where the nucleotide binding site may not be optimally positioned for catalysis. ATPase functional studies reveal modest activity similar to that of other extension ATPases, while calculations indicate that this activity is insufficient to power pilus extension. Our results reveal that, despite similarities in primary sequence and tertiary structure, T4P extension ATPases exhibit divergent quaternary configurations. Our data raise new possibilities regarding the mechanism by which T4P extension ATPases power pilus formation. IMPORTANCE Type 4 pili are hairlike surface appendages on many bacteria and archaea that can be extended and retracted with tremendous force. They play a critical role in disease caused by several deadly human pathogens. Pilus extension is made possible by an enzyme that converts chemical energy to mechanical energy. Here, we describe the three-dimensional structure of such an enzyme from a human pathogen in unprecedented detail, which reveals a mechanism of action that has not been seen previously among enzymes that power type 4 pilus extension.

[Mechanism of the activation of extracellular signal-regulated kinase (ERK) in prostate cancer cell lines with different metastatic potential].

OBJECTIVE: To investigate the mechanism of the activation of signal transduction of ERK induced by purinergic receptor agonist ATP in prostate cancer cells with different metastatic potential. METHODS: Cell counts and MTT method were used to detect the influence of ATP on the growth of 1E8 (metastatic) and 2B4 (non-metastatic) cells derived from human prostate cancer cell line PC3M. The activity of ERK1/2 was analyzed with phosphospecific antibodies directed against the dually phosphorylated, active forms of ERK1/2 (p44/p42) by Western Blot. RESULTS: ATP can significantly inhibit the growth of 1E8 and 2B4 cells in vitro (inhibition rate in the 6th and the 8th day were 54% and 59% for 1E8 and 67% and 39% for 2B4 respectively). ATP activated both ERK1 and ERK2 in 1E8 and 2B4 cells with a time and dose dependent pattern. The activation of ERK1/2 by ATP was blocked by the P2 purinoceptor antagonist, suramin with an inhibitory rate of 82% +/- 9% for 1E8 and an inhibitory rate of 81% +/- 6% for 2B4. The activation of ERK1/2 by ATP can be inhibited by the inhibitor of the upstream kinase MEK- PD098059 with an inhibitory rate of 94% +/- 4% for 1E8 and 91% +/- 4% for 2B4,which suggests a link between the G protein coupled P2 purinoceptor and activation of Ras MEK MAPK pathway. ATP-stimulated ERK activation was sensitive to treatment with G protein modulator pertussis toxin (PTX) with an inhibitory rate of 50% +/- 3% for 1E8 and 51% +/- 4% for 2B4. The activating potential of ATP to ERK1/2 in metastatic 1E8 cells is greater than that to nonmetastatic 2B4 cells, and the response of 1E8 cells to TPA was quite different from the response of 2B4 cells, thus implying a potential signaling mechanism in regulating metastasis phenotypes. CONCLUSION: The metastatic 1E8 and non-metastatic 2B4 cells show differential response to ATP-induced ERK activation. This may provide an instructive clue to cancer metastasis research.

Microscopical localization on adenylate cyclase: a historical review of methodologies.

The histochemistry technique for localizing adenylate cyclase has been developed over the past two decades. Early efforts were directed at overcoming the criticism of the lead capture technique, the inhibition of the enzyme by fixation, and problems associated with the substrate. The introduction of alternative metal ions, strontium and cerium, offered solutions to the criticism of the lead capture technique. The inhibition of the enzyme by the various fixation methods used has been rarely overcome satisfactorily and the use of non-fixed material during incubation is one of the alternatives that has been suggested. The introduction of adenylate (beta-gamma-methylene) diphosphate as an alternative substrate offers a solution to the problems associated with commercially available adenylyl imidodiphosphate. Although no standard medium or method has been accepted by all researchers, the histochemical technique still has a place in the arsenal of the modern cell biologist. The technique localizes the active enzyme, as opposed to the protein, active and nonactive, by immunocytochemistry and the precursors of the protein by in situ hybridization methods.

Measurement of volume injected into individual cells by quantitative fluorescence microscopy.

Pressure microinjection is frequently used to introduce substances into mammalian cells, but precise quantitation of the volume injected into individual cells has been difficult. A simple and reliable procedure for determining the volume injected was developed in order to determine what intracellular concentration of AMP-PNP was necessary to inhibit specific cellular processes. The technique uses fluorescent Lucifer Yellow-labeled dextrans in the microinjection buffer and quantitative fluorescence microscopy to measure the fluorescence intensity of the injected cell. The volume injected is computed from a standard curve derived from the volume and fluorescence of spherical, microscopic droplets of Lucifer Yellow dextran solution. The droplets are ejected from a micropipet into immersion oil where they sink to rest on a siliconized coverslip. For the measurement of fluorescence, an inexpensive photomultiplier system that is attached to a fluorescence microscope is described. The potential uses of this method for other microassays are discussed.

ATP formation from adenyl-5'-yl imidodiphosphate, a nonhydrolyzable ATP analog.

The purity of several preparations of adenyl-5'-yl imidodiphosphate (AMP-PNP) was analyzed using thin layer chromatography and the luciferin-luciferase assay. Three contaminants were identified: adenyl-5'-yl phosphoramidate, phosphorylated AMP-PNP, and ATP. The level of ATP contamination ranged from 0.02% to 0.3% in commercially obtained AMP-PNP preparations, and rose to 10% following incubation of AMP-PNP at 37 degrees C for 3 weeks in aqueous solution. The chemistry of the phosphoramidate bond is reviewed briefly, and evidence for a simple mechanism for the spontaneous formation of ATP from AMP-PNP is presented.

Adenylylimidodiphosphate: effect of contaminants on adenylate cyclase activity.

Commercially available adenylylimidodiphosphate, APP(NH)P, appears to be contaminated with a variety of compounds. The effects of unpurified APP(NH)P on adenylate cyclase activity closely resembled the effects of guanylylimidodiphosphate (GPP(NH)P); that is, APP(NH)P alone caused a time-dependent, quasi-irreversible activation, and the stimulation of adenylate cyclase by APP(NH)P in combination with epinephrine was synergistic, eliminating the need for GTP. The GPP(NH)P-like activity of APP(NH)P could be separated from APP(NH)P by purification of the analogue on DEAE cellulose columns or by paper chromatography. The purified APP(NH)P does not appear to interest at the GTP site.