Action at a Distance: Amino Acid Substitutions That Affect Binding of the Phosphorylated CheY Response Regulator and Catalysis of Dephosphorylation Can Be Far from the CheZ Phosphatase Active Site

Abstract

Transient protein phosphorylation is a common means to accomplish signal transduction. Phosphorylation-mediated signaling in microorganisms often involves the detection of a stimulus by a sensor kinase, followed by the transfer of a phosphoryl group to a response regulator protein. In bacterial chemotaxis, one of the best-studied examples of such a twocomponent regulatory system (36, 37), extracellular stimuli control the autophosphorylation of the CheA sensor kinase with subsequent phosphotransfer to the cytoplasmic response regulator CheY. The phosphorylation state of CheY then dictates the direction or duration of flagellar rotation and thus swimming behavior.The concentration of phosphorylated CheY (CheYp) at any given time is a function of the rates of both phosphorylation and dephosphorylation. The kinetics of phosphoryl group addition and removal set an upper bound on how quickly a cell can respond to a stimulus. During chemotaxis, bacteria integrate information about their chemical environment and make split-second responses that determine whether to continue on their current course or change direction (29). Accordingly,

Deep mutational scanning: a new style of protein science

“…Changes to amino acids that are distant from binding or active sites can have drastic effects on the thermodynamic stability or enzymatic activity of a protein 1 . Highly conservative mutations, whose consequences can be difficult to predict, may be neutral, deleterious or hyper-activating 2,3 .…”

Mutational Analysis of an Intrinsically Disordered Region in the E. coli Phosphatase CheZ, a Regulator of Chemotaxis

“…Robert Bourret AND Ruth Silversmith (University of North Carolina Chapel Hill, Department of Microbiology and Immunology). E. coli strains used in chemotaxis studies are described in Boesch et al, 2000, andFreeman et al, 2011. E. coli RP5231 (∆cheYZ strain) was transformed with plasmid pRS3 which contains an operon expressing both CheZ and CheY.…”

“…The "wild-type" CheZ on pRS3 contains an inadvertent E134K mutation which reduces activity in chemotaxis swarm assay by 20% (Boesch et al, 2000). The mutator strain used in suppressor screen (NR9458) is described in Schaaper and Cornacchio, 1992. Swarm Assay: The swarm assay (Wolfe and Berg, 1989) was performed essentially as previously described (Boesch et al, 2000;Freeman et al, 2011). To measure chemotaxis activity, bacteria from plated colonies were spotted onto a swarm agar plate (1% [wt/vol] tryptone, 0.5% [wt/vol] NaCl, 0.3% [wt/vol] BactoAgar) using a sterile toothpick.…”

“…A model of association of CheY-P with CheZ involves initial binding of CheY-P to the CheZC helix. The CheY-P is then "reeled in" to allow interaction with CheZN so that catalysis can occur (see schematic representation in Figure 2, and Silversmith, 2005;Freeman et al, 2011). This "reeling in" involves localization of the bound CheY-P in the vicinity of the binding surface on CheZN.…”

“…The CheZ mutant suppressor screen was carried out using an established procedure (Boesch et al, 2000;Freeman et al, 2011). Plasmid pRS3 (carrying wild type cheYZ) was transformed and grown in E. coli NR9458 which carries the mutD5 allele (exhibits mutation frequencies 50-100 times higher than wild-type when the bacteria are grown in minimal media).…”