Abstract
In 2015, monoclonal antibodies (mAbs) that selectively recognize the 1-pHis or 3-pHis isoforms of phosphohistidine were developed by immunizing rabbits with degenerate Ala/Gly peptides containing the nonhydrolyzable phosphohistidine (pHis) analog- phosphotriazolylalanine (pTza). Here, we report structures of five rabbit mAbs bound to cognate pTza peptides: SC1-1 and SC50-3 that recognize 1-pHis, and their 3-pHis-specific counterparts, SC39-4, SC44-8, and SC56-2. These cocrystal structures provide insights into the binding modes of the pTza phosphate group that are distinct for the 1- and 3-pHis mAbs with the selectivity arising from specific contacts with the phosphate group and triazolyl ring. The mode of phosphate recognition in the 3-pHis mAbs recapitulates the Walker A motif, as present in kinases. The complementarity-determining regions (CDRs) of four of the Fabs interact with the peptide backbone rather than peptide side chains, thus conferring sequence independence, whereas SC44-8 shows a proclivity for binding a GpHAGA motif mediated by a sterically complementary CDRL3 loop. Specific hydrogen bonding with the triazolyl ring precludes recognition of pTyr and other phosphoamino acids by these mAbs. Kinetic binding experiments reveal that the affinity of pHis mAbs for pHis and pTza peptides is submicromolar. Bound pHis mAbs also shield the pHis peptides from rapid dephosphorylation. The epitope-paratope interactions illustrate how these anti-pHis antibodies are useful for a wide range of research techniques and this structural information can be utilized to improve the specificity and affinity of these antibodies toward a variety of pHis substrates to understand the role of histidine phosphorylation in healthy and diseased states.
Highlights
Phosphorylation is a crucial posttranslational modification that extends the functionality and versatility of the cellular proteome [1]
We report structures of five rabbit monoclonal antibodies (mAbs) bound to cognate pTza peptides: SC1-1 and SC50-3 that recognize 1-pHis, and their 3-phosph ohistidine (3-pHis)–specific counterparts, SC394, SC44-8, and SC56-2. These cocrystal structures provide insights into the binding modes of the pTza phosphate group that are distinct for the 1- and 3-pHis mAbs with the selectivity arising from specific contacts with the phosphate group and triazolyl ring
As an approach to understand the interaction of anti-pHis mAbs with pHis substrates in atomic detail, we solved the cocrystal structures of five different pHis Fabs bound to their cognate pTza peptides using X-ray crystallography (SI Appendix, Table S1)
Summary
Phosphorylation is a crucial posttranslational modification that extends the functionality and versatility of the cellular proteome [1]. Two-component systems in bacteria, fungi, and plants use His phosphorylation in coupling environmental signals to the cellular outcomes that include virulence, survival, and quorum sensing [11,12,13] His phosphorylation in eukaryotes plays a vital role in regulating cellular processes, such as nucleotide homeostasis, ion channel regulation, or G protein signaling [14,15,16]. We developed monoclonal antibodies (mAbs) against the 1-pHis and 3-pHis modifications using stable phosphotriazolylalanine mimetics as immunogens These antibodies are promising tools to uncover the role of pHis in eukaryotic cells. The percentage of pHis uncovered in these studies is relatively more than that of the well-studied pTyr modification (8%) These findings make it all the more important to understand the role of His phosphorylation in both prokaryotes and eukaryotes. Development of reagents that are specific for pHis modifications are needed to validate the largescale phosphoproteomic substrate identifications and explore functional significance of pHis modifications
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