Structural biology: FaPy lesions and DNA mutations.

with an analogous, simple hapten that the authors call phosphoryltriazolylethylamine (pTze) (Fig. 1). The antibodies did not bind pSeror pThr-conjugated BSA and had approximately one-tenth the affinity for pTyr (versus pHis) in a dodecapeptide based on the histone H4 sequence. The latter potential complication was neatly dealt with by immunodepletion of the pTyr-conjugated protein with commercially available pTyr antibodies, which have no affinity for pHis, a strategy that should be effective in the interrogation of native eukaryotic proteomes. In addition, the amino acid sequences flanking the histidine phosphorylation sites in all four of the proteins examined vary widely, indicating that recognition of the pHis epitope is context independent and likely to be applicable to the detection of other histidine-phosphorylated proteins. As evidence that the antibodies will prove to be useful tools, the authors showed that they were able to measure the kinetics of endogenous histidine autophosphorylation of bacterially overexpressed Pts1 (of the sugar-phosphoenolpyruvate (PEP) phosphotransferase system) using PEP as a substrate. The antibodies were also used to investigate the mechanism of regulation of PEP synthetase (PpsA) by a-ketoglutarate. Most importantly, the authors demonstrated the ability of the antibodies to detect and immunoprecipitate endogenously expressed and phosphorylated pHis-containing proteins in Escherichia coli. The work of Kee et al.4 is undeniably a major advance for the field of phosphohistidine research. There was considerable doubt as to whether pan-pHis antibodies could actually be generated. Given the chemical lability of the phosphoramidate and the probable nature of the noncovalent interactions involved in a pHis antibody– antigen complex, it was quite likely that the phosphoryl group would be transferred from the pHis residue to nucleophilic residues within the antibodies’ antigen-binding regions, thereafter leading to dissociation of the complex. Phosphoryl relay, usually from ATP to a histidine residue and then on to a second acceptor protein residue, is a common biological role for pHis5. Thus, even as a proof of concept, the work is very important. There are some challenges to maximizing the broader impact of this research—in particular, making a limited supply of polyclonal pan-pHis antibodies available to the scientific community. However, if this can be accomplished in the short term, and monoclonal antibodies can be generated in due course, this research will affect the entire life sciences community. The availability of pan-pHis antibodies promises to substantially accelerate a wealth of new findings in fundamental cell signaling and regulation and may ultimately result in new approaches to the treatment of diseases such as cancer. Scientists, who have not considered looking for histidine phosphorylation in the past because it was too hard, will now start to think differently. Panning for phosphohistidine could turn up some real nuggets. ■