Genome sequencing projects have provided researchers with a complete inventory of the predicted proteins produced by eukaryotic and prokaryotic organisms. Assignment of functions to these proteins represents one of the principal challenges for the field of proteomics. Activity-based protein profiling (ABPP) has emerged as a powerful chemical proteomic strategy to characterize enzyme function directly in native biological systems on a global scale. Here, we review the basic technology of ABPP, the enzyme classes addressable by this method, and the biological discoveries attributable to its application.
The central role of protein kinases in signal transduction pathways has generated intense interest in targeting these enzymes for a wide range of therapeutic indications. Here we report a method for identifying and quantifying protein kinases in any biological sample or tissue from any species. The procedure relies on acyl phosphate-containing nucleotides, prepared from a biotin derivative and ATP or ADP. The acyl phosphate probes react selectively and covalently at the ATP binding sites of at least 75% of the known human protein kinases. Biotinylated peptide fragments from labeled proteomes are captured and then sequenced and identified using a mass spectrometry-based analysis platform to determine the kinases present and their relative levels. Further, direct competition between the probes and inhibitors can be assessed to determine inhibitor potency and selectivity against native protein kinases, as well as hundreds of other ATPases. The ability to broadly profile kinase activities in native proteomes offers an exciting prospect for both target discovery and inhibitor selectivity profiling.
Summary Protein kinases are intensely studied mediators of cellular signaling, yet important questions remain regarding their regulation and in vivo properties. Here we use a probe-based chemoprotemics platform to profile several well studied kinase inhibitors against more than 200 kinases in native cell proteomes and reveal new biological targets for some of these inhibitors. Several striking differences were identified between native and recombinant kinase inhibitory profiles, in particular, for the Raf kinases. The native kinase binding profiles presented here closely mirror the cellular activity of these inhibitors, even when the inhibition profiles differ dramatically from recombinant assay results. Additionally, Raf activation events could be detected upon live cell treatment with inhibitors. These studies highlight the complexities of protein kinase behavior in the cellular context and demonstrate that profiling with only recombinant/purified enzymes can be misleading.
The solution structure of Co‚Bleomycin (CoBLM) A2 green (the hydroperoxide form of CoBLM) complexed with the self-complementary oligonucleotide d(CCAGGCCTGG) with a cleavage site at C6 has been determined by 2D NMR spectroscopic methods and molecular dynamics calculations. Intermolecular NOEs (60 between CoBLM A2 green and DNA) and intramolecular NOEs (61 within CoBLM A2 green) have defined the position and orientation of CoBLM A2 green with respect to its single binding site in the duplex. CoBLM A2 green is a stable analog of the activated BLM, the Fe 3+ hydroperoxide (Sam, J. W.; Tang, X.-J.; Peisach, J. J. Am. Chem. Soc. 1994, 116, 5250-5256). These studies have provided the first structural insight into the mode of binding of the bithiazole tail of CoBLM A2 green to DNA, the basis for specificity of its cleavage at pyrimidines (Py) in d(G-Py) sequences, and the orientation of its terminal oxygen of the hydroperoxide relative to the 4′ carbon hydrogen bond being cleaved in the DNA. The bithiazole tail inserts 3′ to the C6 cleavage site from the minor groove. The terminal thiazolium ring is completely stacked between the bases of G14 and G15, while the penultimate thiazolium ring is only partially stacked between the bases of C6 and C7. The bithiazole tail thus binds via a partial intercalation mode and the DNA is unwound by 13°over the (G5‚C16)∼(C6‚G15)∼(C7‚G14)∼(T8‚A13) steps. No specific interactions between the bithiazole tail and the DNA have been identified, and thus, this interaction does not define the BLM's cleavage specificity but its binding affinity. The metal binding domain and the peptide linker region of CoBLM A2 green bind within the minor groove of the duplex and define the basis for its specificity of DNA cleavage. The 4-amino group and the N3 of the pyrimidine ring of CoBLM A2 green form specific hydrogen bonds with the N3 and the 2-amino group, respectively, of the G5 in the duplex and provide an unusual example of a minor groove base triplelike interaction. A basis for the preference for G over A, 5′ to the Py cleavage site, is thus established. The metal binding domain and the valeryl moiety in the linker have a conformation strikingly similar to that defined in the free CoBLM A2 green (Wu, W.; Vanderwall, D. E.; Lui, S. M.; Tang, X.-J.; Turner, C. J.; Kozarich, J. W.; Stubbe, J. ). The most remarkable feature of this structure is the observation of the proton associated with the hydroperoxide of CoBLM A2 green and its observed intermolecular NOEs to the minor groove protons of C6 and C7 of the duplex. Thus this structure provides a rare snapshot of an analog of a reactive intermediate poised to initiate the hydrogen atom abstraction event. The molecular modeling reveals that the distal oxygen of the hydroperoxide is 2.5 Å from the 4′-hydrogen of C6. A number of additional intramolecular hydrogen bonds between the hydroperoxide ligand and the peptide linker region are also proposed, which appear to play a key role in positioning the reactive intermediate near the hydrogen atom being abstr...
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