ABSTRACT: The chemosensory signaling array of bacterial chemotaxis is composed of functional core units containing two receptor trimers of dimers, a homodimeric CheA kinase, and two CheW adaptor proteins. In vitro reconstitutions generate individual, functional core units and larger functional assemblies, including dimers, hexagons, and hexagonal arrays. Such reconstituted complexes have been shown to have both quasi-stable and ultrastable populations that decay with lifetimes of 1-2 days and ∼3 weeks at 22 °C, respectively, where decay results primarily from proteolysis of the bound kinase [Erbse, A. H., and Falke, J. J. (2009) Biochemistry 48, 6975-6987; Slivka, P. F., and Falke, J. J. (2012) Biochemistry 51, 10218-10228]. In this work, we show that the ultrastable population can be destabilized to the quasi-stable level via the introduction of a bulky tryptophan residue at either one of two essential protein-protein interfaces within the core unit: the receptor-kinase contact or kinase-adaptor interface 1. Moreover, we demonstrate that the quasi-stable population can be made ultrastable via the introduction of a disulfide bond that covalently stabilizes the latter interface. The resulting disulfide at least doubles the functional lifetime of the ultrastable population, to ≥5.9 weeks at 22 °C, by protecting the kinase from endogenous and exogenous proteases. Together, these results indicate that the ultrastability of reconstituted core complexes requires well-formed contacts among the receptor, kinase, and adaptor proteins, whereas quasi-stability arises from less perfect contacts that allow slow proteolysis of the bound kinase. Furthermore, the results reveal that ultrastability, and perhaps the size or order of chemosensory complexes and arrays, can be increased by an engineered disulfide bond that covalently cross-links a key interface. Overall, it appears that native ultrastability has evolved to provide an optimal rather than maximal level of kinetic durability, suggesting that altered selective pressure could either increase or decrease the functional lifetime of core complexes.
