Mutations leading to altered CheA binding cluster on a face of CheY.


Abstract

CheY is the response regulator of Escherichia coli chemotaxis and is one of the best studied response regulators of the two-component signaling system. CheY can receive phosphate from the histidine kinase, CheA. Phospho-CheY interacts with the motor-switch complex to induce clockwise flagellar rotation, thus causing the cell to tumble. We used an enzyme-linked immunosorbent assay to study the direct interaction between the kinase, CheA, and the regulator, CheY. The products of random, suppressor, and site-specific cheY mutants were assayed for their ability to bind CheA. Nine mutants showed altered binding. We sequenced and mapped these point mutations on the crystal structure of CheY, and a high degree of spatial clustering was revealed, indicating that this region of CheY is involved in CheA binding. Interestingly, five of these altered binding mutants were previously defined as being involved in motor-switch binding interactions. This suggested a possible overlap between the motor-switch binding and CheA binding surfaces of CheY. Using CheY (Trp-58) fluorescence quenching, we determined the equilibrium dissociation constants of CheA (124-257) binding for these CheY mutants. The results from the fluorescence quenching are in close agreement with our initial enzyme-linked immunosorbent assay results. Therefore, we propose that the CheA and the motor binding surfaces on CheY partially overlap and that this overlap allows CheY to interact with either the CheA or the flagellar motor, depending on its signaling (phosphorylation) state.

Submission Details

ID: AChvgLiJ

Submitter: Shu-Ching Ou

Submission Date: Feb. 28, 2019, 10:12 a.m.

Version: 1

Publication Details
Shukla D;Matsumura P,J Biol Chem (1995) Mutations leading to altered CheA binding cluster on a face of CheY. PMID:7592655
Additional Information

Study Summary

Number of data points 29
Proteins Chemotaxis protein CheY
Unique complexes 10
Assays/Quantities/Protocols Experimental Assay: Kd ; Derived Quantity: ΔΔG ; Derived Quantity: SD of Kd
Libraries variants for CheY_CheA

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P
 

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)


Relevant PDB Entries

Structure ID Release Date Resolution Structure Title
5DGC 2015-08-27T00:00:00+0000 1.94 Reaction of phosphorylated CheY with imidazole 2 of 3
5DKF 2015-09-03T00:00:00+0000 1.94 Reaction of phosphorylated CheY with imidazole 3 of 3
5D2C 2015-08-05T00:00:00+0000 2.06 Reaction of phosphorylated CheY with imidazole 1 of 3
2FKA 2006-01-04T00:00:00+0000 2.0 Crystal structure of Mg(2+) and BeF(3)(-)-bound CheY in complex with CheZ(200-214) solved from a F432 crystal grown in CAPS (pH 10.5)
2FLW 2006-01-06T00:00:00+0000 2.0 Crystal structure of Mg2+ and BeF3- ound CheY in complex with CheZ 200-214 solved from a F432 crystal grown in Hepes (pH 7.5)
2FMF 2006-01-09T00:00:00+0000 2.0 Crystal structure of CheY in complex with CheZ 200-214 solved from a F432 crystal grown in Hepes (pH 7.5)
2FMI 2006-01-09T00:00:00+0000 2.3 Crystal structure of CheY in complex with CheZ 200-214 solved from a F432 crystal grown in Tris (pH 8.4)
2FLK 2006-01-06T00:00:00+0000 2.1 Crystal structure of CheY in complex with CheZ(200-214) solved from a F432 crystal grown in CAPS (pH 10.5)
2FMK 2006-01-09T00:00:00+0000 2.0 Crystal structure of Mg2+ and BeF3- bound CheY in complex with CheZ 200-214 solved from a P2(1)2(1)2 crystal grown in MES (pH 6.0)
2FMH 2006-01-09T00:00:00+0000 2.0 Crystal structure of Mg2+ and BeF3- bound CheY in complex with CheZ 200-214 solved from a F432 crystal grown in Tris (pH 8.4)

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
90.6 Chemotaxis protein CheY Q8D0P1 CHEY_YERPE
91.4 Chemotaxis protein CheY Q93P00 CHEY_YEREN
94.5 Chemotaxis protein CheY Q9FAD7 CHEY_ENTCL
97.7 Chemotaxis protein CheY P0A2D5 CHEY_SALTY
97.7 Chemotaxis protein CheY P0A2D6 CHEY_SALTI
99.2 Chemotaxis protein CheY Q8FGP6 CHEY_ECOL6
100.0 Chemotaxis protein CheY P0AE69 CHEY_SHIFL
100.0 Chemotaxis protein CheY P0AE67 CHEY_ECOLI
100.0 Chemotaxis protein CheY P0AE68 CHEY_ECO57