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:7592655Additional 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
| 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 |
|---|---|---|---|
| 5D2C | 2015-08-05T00:00:00+0000 | 2.06 | Reaction of phosphorylated CheY with imidazole 1 of 3 |
| 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 |
| 2CHE | 1994-01-17T00:00:00+0000 | 1.8 | STRUCTURE OF THE MG2+-BOUND FORM OF CHEY AND MECHANISM OF PHOSPHORYL TRANSFER IN BACTERIAL CHEMOTAXIS |
| 2CHF | 1994-01-17T00:00:00+0000 | 1.8 | STRUCTURE OF THE MG2+-BOUND FORM OF CHEY AND THE MECHANISM OF PHOSPHORYL TRANSFER IN BACTERIAL CHEMOTAXIS |
| 2CHY | 1990-05-17T00:00:00+0000 | 2.7 | THREE-DIMENSIONAL STRUCTURE OF CHEY, THE RESPONSE REGULATOR OF BACTERIAL CHEMOTAXIS |
| 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) |
| 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) |
| 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) |
Relevant UniProtKB Entries
| Percent Identity | Matching Chains | Protein | Accession | Entry Name |
|---|---|---|---|---|
| 100.0 | Chemotaxis protein CheY | P0AE68 | CHEY_ECO57 | |
| 100.0 | Chemotaxis protein CheY | P0AE67 | CHEY_ECOLI | |
| 100.0 | Chemotaxis protein CheY | P0AE69 | CHEY_SHIFL | |
| 99.2 | Chemotaxis protein CheY | Q8FGP6 | CHEY_ECOL6 | |
| 97.7 | Chemotaxis protein CheY | P0A2D6 | CHEY_SALTI | |
| 97.7 | Chemotaxis protein CheY | P0A2D5 | CHEY_SALTY | |
| 94.5 | Chemotaxis protein CheY | Q9FAD7 | CHEY_ENTCL | |
| 91.4 | Chemotaxis protein CheY | Q93P00 | CHEY_YEREN | |
| 90.6 | Chemotaxis protein CheY | Q8D0P1 | CHEY_YERPE |