Crystal structures and mutagenesis of PPP-family ser/thr protein phosphatases elucidate the selectivity of cantharidin and novel norcantharidin-based inhibitors of PP5C.

Cantharidin is a natural toxin and an active constituent in a traditional Chinese medicine used to treat tumors. Cantharidin acts as a semi-selective inhibitor of PPP-family ser/thr protein phosphatases. Despite sharing a common catalytic mechanism and marked structural similarity with PP1C, PP2AC and PP5C, human PP4C was found to be insensitive to the inhibitory activity of cantharidin. To explore the molecular basis for this selectivity, we synthesized and tested novel C5/C6-derivatives designed from quantum-based modeling of the interactions revealed in the co-crystal structures of PP5C in complex with cantharidin. Structure-activity relationship studies and analysis of high-resolution (1.25Å) PP5C-inhibitor co-crystal structures reveal close contacts between the inhibitor bridgehead oxygen and both a catalytic metal ion and a non-catalytic phenylalanine residue, the latter of which is substituted by tryptophan in PP4C. Quantum chemistry calculations predicted that steric clashes with the bulkier tryptophan side chain in PP4C would force all cantharidin-based inhibitors into an unfavorable binding mode, disrupting the strong coordination of active site metal ions observed in the PP5C co-crystal structures, thereby rendering PP4C insensitive to the inhibitors. This prediction was confirmed by inhibition studies employing native human PP4C. Mutation of PP5C (F446W) and PP1C (F257W), to mimic the PP4C active site, resulted in markedly suppressed sensitivity to cantharidin. These observations provide insight into the structural basis for the natural selectivity of cantharidin and provide an avenue for PP4C deselection. The novel crystal structures also provide insight into interactions that provide increased selectivity of the C5/C6 modifications for PP5C versus other PPP-family phosphatases.

The "drought-inducible" histone H1s of tobacco play no role in male sterility linked to alterations in H1 variants.

Tobacco ( Nicotiana tabacum L.) has two major H1 variants (H1A and H1B), which account for over 80% of chromatin linker histones, and four minor variants: H1C, H1D, H1E and H1F. We have shown previously [M. Prymakowska-Bosak et al. (1999) Plant Cell 11:2317-2329] that reversal of the natural proportion of major to minor H1 variants in transgenic tobacco plants results in a characteristic male-sterility phenotype identical to that occurring in many plant species subjected to water deficit at the time of male meiosis. It has been proposed by others that the drought-induced arrest of male gametophyte development is linked to decreased sugar delivery to reproductive tissues. Within the family of angiosperm H1s there is a well-defined class of minor H1 variants named "drought inducible" because some of its members have been shown to be induced by water deficit. We have identified and cloned the tobacco H1C gene, which, based on sequence similarity, represents a "drought-inducible" minor H1 variant. Analysis of the un-translated mRNA and promoter regions of H1C suggests a regulation by sucrose concentration. Antisense silencing of H1C and its close homologue H1D in plants that do not express H1A and H1B does not affect the characteristic H1A(-)/ H1B(-) male-sterility phenotype. Silencing of H1C and H1D also has no effect on growth and development of plants. Our findings demonstrate that H1C and H1D are dispensable for normal growth and development of tobacco, and that the compensatory up-regulation of "drought-inducible" H1s observed in H1A(-)/ H1B(-) plants is not the direct cause of male sterility linked to alterations in H1 variants.