Potential Regulatory Role of Competitive Encounter Complexes in Paralogous Phosphotransferase Systems

• Published in: Journal of molecular biology 2019-05, Vol.431 (12), p.2331-2342
• Main Authors: Strickland, Madeleine, Kale, Seyit, Strub, Marie-Paule, Schwieters, Charles D., Liu, Jian, Peterkofsky, Alan, Tjandra, Nico
• Format: Article
• Language: English
• Subjects: encounter complex; Enzyme I; Escherichia coli - chemistry; Escherichia coli - metabolism; Escherichia coli Proteins - chemistry; Escherichia coli Proteins - metabolism; Humans; Models, Molecular; nitrogen phosphotransferase system (PTSNtr); NMR; paramagnetic relaxation enhancement (PRE); Peptide Fragments - chemistry; Peptide Fragments - metabolism; Phosphate-Binding Proteins - chemistry; Phosphate-Binding Proteins - metabolism; Phosphoenolpyruvate Sugar Phosphotransferase System - chemistry; Phosphoenolpyruvate Sugar Phosphotransferase System - metabolism; Phosphotransferases - chemistry; Phosphotransferases - metabolism; Protein Domains
• ISSN: 0022-2836; 1089-8638
• DOI: 10.1016/j.jmb.2019.04.040

There are two paralogous Escherichia coli phosphotransferase systems, one for sugar import (PTSsugar) and one for nitrogen regulation (PTSNtr), that utilize proteins enzyme Isugar (EIsugar) and HPr, and enzyme INtr (EINtr) and NPr, respectively. The enzyme I proteins have similar folds, as do their substrates HPr and NPr, yet they show strict specificity for their cognate partner both in stereospecific protein–protein complex formation and in reversible phosphotransfer. Here, we investigate the mechanism of specific EINtr:NPr complex formation by the study of transient encounter complexes. NMR paramagnetic relaxation enhancement experiments demonstrated transient encounter complexes of EINtr not only with the expected partner, NPr, but also with the unexpected partner, HPr. HPr occupies transient sites on EINtr but is unable to complete stereospecific complex formation. By occupying the non-productive transient sites, HPr promotes NPr transient interaction to productive sites closer to the stereospecific binding site and actually enhances specific complex formation between NPr and EINtr. The cellular level of HPr is approximately 150 times higher than that of NPr. Thus, our finding suggests a potential mechanism for cross-regulation of enzyme activity through formation of competitive encounter complexes. [Display omitted] •The N-terminal domain of Enzyme-I (EINNtr) binds phosphotransfer substrate NPr.•EINNtr forms encounter complexes not only with NPr but also with non-substrate HPr.•HPr is 150-fold more abundant than NPr in E. coli cells.•HPr outcompetes NPr encounters, funneling NPr to the specific binding site.•Competitive encounters could represent a general mechanism of cross-regulation.