Histidine phosphorylation is an important and ubiquitous post-translational modification. Histidine undergoes phosphorylation on either of the nitrogens in its imidazole side chain, giving rise to 1- and 3- phosphohistidine (pHis) isomers, each having a phosphoramidate linkage that is labile at high temperatures and low pH, in contrast with stable phosphomonoester protein modifications. While all organisms routinely use pHis as an enzyme intermediate, prokaryotes, lower eukaryotes and plants also use it for signal transduction. However, research to uncover additional roles for pHis in higher eukaryotes is still at a nascent stage. Since the discovery of pHis in 1962, progress in this field has been relatively slow, in part due to a lack of the tools and techniques necessary to study this labile modification. However, in the past ten years the development of phosphoproteomic techniques to detect phosphohistidine (pHis), and methods to synthesize stable pHis analogues, which enabled the development of anti-phosphohistidine (pHis) antibodies, have accelerated our understanding. Recent studies that employed anti-pHis antibodies and other advanced techniques have contributed to a rapid expansion in our knowledge of histidine phosphorylation. In this review, we examine the varied roles of pHis-containing proteins from a chemical and structural perspective, and present an overview of recent developments in pHis proteomics and antibody development.

Human nucleoside diphosphate (NDP) kinase A is a ‘house-keeping’ enzyme essential for the synthesis of nonadenine nucleoside (and deoxynucleoside) 5′-triphosphate. It is involved in complex cellular regulatory functions including the control of metastatic tumour dissemination. The mutation S120G has been identified in high-grade neuroblastomas. We have shown previously that this mutant has a folding defect: the urea-denatured protein could not refold in vitro . A molten globule folding intermediate accumulated, whereas the wild-type protein folded and associated into active hexamers. In the present study, we report that autophosphorylation of the protein corrected the folding defect. The phosphorylated S120G mutant NDP kinase, either autophosphorylated with ATP as donor, or chemically prosphorylated by phosphoramidate, refolded and associated quickly with high yield. Nucleotide binding had only a small effect. ADP and the non-hydrolysable ATP analogue 5′-adenyly-limido-diphosphate did not promote refolding. ATP-promoted refolding was strongly inhibited by ADP, indicating protein dephosphorylation. Our findings explain why the mutant enzyme is produced in mammalian cells and in Escherichia coli in a soluble form and is active, despite the folding defect of the S120G mutant observed in vitro . We generated an inactive mutant kinase by replacing the essential active-site histidine residue at position 118 with an asparagine residue, which abrogates the autophosphorylation. The double mutant H118N/S120G was expressed in inclusion bodies in E. coli . Its renaturation stops at a folding intermediate and cannot be reactivated by ATP in vitro . The transfection of cells with this double mutant might be a good model to study the cellular effects of folding intermediates.