Conformational Shifts In A Chemoreceptor Helical Hairpin Control Kinase Signaling In Escherichia Coli

Escherichia coli is one of the most important bacterial infectious diseases that threaten human food safety and public health.E.coli has chemotaxis function and it is one of the essential factors for bacterial infection causing disease.Although the molecular mechanisms of receptor arrays for accurate sensing of the chemical gradient in the environment is well studies,our understanding on how the signaling units interact with each other and the mechanism of signaling uniting on modulating the Che A kinase are still lacking.Therefore,in order to better understand how the cytoplamic tip of Tsr receptor modulates Che A kinase,we constructed a full spectrum of amino acids replacements at the E385-R388 positions of Tsr receptors.These two residues have been suggested to form a salt-bridge in the receptor trimer of dimers structure.We studied the signaling properties of the amino acid replacement receptors.This study shed lights on our further understanding of the interrelationship of chemotaxis with bacterial virulence factors and the molecular mechanisms of chemical signal transduction.The main findings of our studies are as follows.1.Chemotactic signaling properties of Tsr-E385 and Tsr-R388 mutantsWe constructed a full set of amino acid substitutions at Tsr E385 position using the full codon mutation in p PA114 plasmid.Twelve of the Tsr-E385*mutant receptors retained some serine chemotaxis function in UU2612.Seven of the E385*receptors were unable to support serine chemotaxis in the soft agar assay.We measured the expression levels of all mutant receptors and all of them are expressed at or near the wild-type level.Therefore,the defects in supporting chemotaxis was not due to expression of structure stability of the mutant receptors,but was caused by functional changes in the mutant protein.Similarly,we also constructed a full set of amino acid substitutions at Tsr R388 position,using plasmid p RR53.Most Tsr-R388*mutant receptors abolished serine chemotaxis in soft agar assays.All R388*mutant receptors also showed stable native expression level.We also measure trimer formation of the Tsr E385*and R388*mutant receptors.All of the mutant receptors were able to form trimers,which suggest that the interaction of the side chains of the glutamic acid and arginine at these two position is not critical for trimer assembly or stability.2.Signaling properties of E385*and R388*receptors in FRET assaysWe used FRET assays to characterize the signaling transduction properties of the mutant receptors.We first measured the signaling behavior of the mutant receptors in UU2567,a strain lacking both Che R and Che B.In this strain,the receptors will maintain its modification state as QEQEE.We also measured FRET behaviors in UU2700,which contains both Che R and Che B.Receptors in UU2700 can be covalently modified by these two enzymes and are generally present in mixed modification states.OFF-shifted output than that of QEQEE receptors.Wild-type Tsr receptor in UU2567 strain showed moderate kinase activity and intermediate sensitivity in response to serine stimuli.Most of the mutant receptors exhibited kinase activities and serine-response behaviors in both UU2567 and UU2700 strains,albeit at various levels and sensitivities.It suggests that the mutant receptors were still capable kinase control and their signaling output is amenable to at least some sensory adaptation control.We also assessed the signaling properties of the mutant receptors in host strains containing only one of the Che R or Che B,further characterized the effects of the adaption enzymes on the mutant receptors.Receptors with OFF-shifted signaling output should be a preferred substrate for Che R enzyme,whereas receptors with ON-shifted signaling output should be a better substrate for Che B.Therefore,the modification patterns of the receptors can indirectly reflect the signaling states of the receptors.We examined modification states of the mutant receptors in host strains containing only one of the modification enzymes and assessed the final modification states of the receptors in these two hosts.We categorized signaling and Che R/Che B substrate preference properties of Tsr E385*and R388*receptors into two general structure-function classes.One is that the response threshold equilibrium is altered,such as ON-shifted and OFF-shifted.Another is that the responses were aberrant or showing no output control.The mutants in the first category suggest receptor tip structure and interaction being altered for equilibrium shift,whereas the second category indicating drastic structure change introduced by those amino acid replacements.3.Signaling Properties of OFF-Shifted or ON-Shifted mutant receptors in adaptation sufficient and deficient hostsThe E385A,V,T and R388A,V,T,M,F receptors showed OFF-shifted signaling properties.Those OFF-shifted properties are illustrated by extensive Che R modification in the methylation assays and reduced kinase activity and/or serine response threshold in the FRET assays.The E385S,Q and R388G,W receptors on the other hand demonstrated ON-shifted signaling properties.They are illustrated by wild-type like modification patterns by both Che B and Che R in the methylation assays and full kinase activities,elevated K_1/2 serine responses in the FRET assays.In the Che RB deficient strain,OFF-shifted receptors at both E385*and R388*residues had substantially reduced response cooperativities.However,the ON-shifted E385S,Q receptors also had low Hill ecoefficiencies,whereas the ON-shifted R388G,W receptors had wild-type response cooperativities.In contrast,in the Che RB+strain,both ON-and OFF-shifted Tsr-E385*mutants demonstrated sensory adaptation(E385A,S,V,T,Q)when assayed for adaptation to a K_1/2 serine stimulus,however the output-shifted R388*mutants(R388A,G,T,W)showed less apparent adaptation in these assays.Both groups of receptors showed wild-type kinase activities in the Che RB+host,suggesting that the mutant receptors are capable of achieving adaptational output control.Another group OFF-shifted receptors(R388V,M,F)produced some kinase activity in the Che RB-host but failed to fully recover that activity following an attractant stimulus.Response to attractant and the following adaptation response in these receptors appear to regulate both the kinase activities and the signaling array size.In order to understand whether changes in signal team size play a role in kinase modulation,the R388V,M,F receptors are assayed in a Che RB deficient strain:UU2869.This strain has a mutant Che W(W-X3)that disrupts array interface connections among core units.In UU2869 strain,WT Tsr showed normal kinase activity,responded to serine with lower threshold,but much lower cooperativity.In contrast,the R388V,M,F receptors could not stimulate kinase activity in UU2869 strain,which suggests that these receptors are trapped in an OFF state after the attractant stimuli.4.Signaling properties of mutant Tsr receptors with poor output controlIn the Che RB deficient host,the Tsr-E385N and G receptors showed significantly slow,low-cooperativity responses to a serine stimulus at the K_1/2 concentration and slow recovery of kinase activity after the removal of serine.However,these mutant receptors showed wild-type like behaviors in the UU2869 host(Che W X3 host).It suggests that array connectivity is probably a critical factor.In the Che RB deficient host,another group of receptors(E385F,Y,W,H)displayed noncooperative,partial kinase-control responses.The behaviors of E385Y and E385W best demonstrate the atypical responses of the group,further these two receptors showed no adaptation behaviors even in the Che RB+host.In the UU2869 host(Che RB deficient and Che W X3 host),the E385Y receptor gained complete control of kinase activity,whereas the E385W receptor completely lost all kinase activity.These results further suggest that network connections between the core signaling complexes play critical roles for these receptors to fully modulate the Che A kinase activity.The E385I,L,M receptors could stimulate kinase activity in the Che RB deficient host,but failed to turn off the kinase activity in response to serine stimuli,even at 100 millimolar of serine.Their signaling properties in the Che RB+host is also unusual.For example,both the E385I and M receptors demonstrated high kinase activities in the Che RB+host,even though E385I is a superb substrate to Che R,whereas the E385M is a poor Che R substrate.We term these mutant receptors as“inverted”because their kinase activities increased in the Che RB+host,the opposite of the wild-type behavior.5.Signaling properties of locked output mutant Tsr receptorsThe R388D,E and E385P receptors could not stimulate kinase activity in either Che RB deficient or Che RB+host strains.We tested these mutants in UU3406 strain,which produces a chimeric Che A-YFP protein,to assay these mutants'ability to assemble signaling complexes.These mutant receptors all showed wild-type like cellular clusters in UU3406 host.Further,the R388D,E receptors are demonstrated to be extremely good substrate for Che R,which is consistent with receptors in a signaling off state.Therefore,the locked off signaling properties of these locked-OFF mutant receptors are not due to failure to assemble signaling complexes,but most likely reflects the signaling states of these mutant receptors.The E385K,R,D,C receptors in the other hand illustrated locked-ON state:stimulate Che A kinase even in the presence of high concentration of serine.These locked-ON receptors also showed high kinase activity in the Che RB+host and did not abate their locked signaling states even in the presence of the adaptation system.The signaling properties of the charge reversal mutants:E385K,R and R388D,E receptors,were more extreme than the other amino acid replacements.The charge reversal substitutions probably produce destabilization of local structure due to charge repulsion between the side chains of these two positions.If that is the main structural change introduced,double charge reversal substitutions at both positions probably could produce closer to wild-type like signaling properties.In the Che RB deficient strain,all four double mutant receptors did show more appropriate kinase activity and kinase suppression in response to serine stimuli.However,none of the four double mutant receptors demonstrated sensory adaptation,which explain their loss of serine chemotaxis in soft agar tests.In conclusion,our results suggest that chemoeffector stimuli and adaptationalmodifications influence the cooperative connections between core signaling units.Thisarray remodeling process may involve activity-dependent changes in the relative strengths of interface 1 and 2 interactions between the Che W and Che A.P5 components of receptor core signaling complexes.The mutant signaling defects suggested that the chemoreceptor tip operates as a two-state device with discrete active and inactive conformations.