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 |
|---|---|---|---|
| 1CEY | 1995-02-07 | ASSIGNMENTS, SECONDARY STRUCTURE, GLOBAL FOLD, AND DYNAMICS OF CHEMOTAXIS Y PROTEIN USING THREE-AND FOUR-DIMENSIONAL HETERONUCLEAR (13C,15N) NMR SPECTROSCOPY | |
| 2LP4 | 2012-10-24 | Solution structure of P1-CheY/P2 complex in bacterial chemotaxis | |
| 1CYE | 1995-02-07 | THREE DIMENSIONAL STRUCTURE OF CHEMOTACTIC CHE Y PROTEIN IN AQUEOUS SOLUTION BY NUCLEAR MAGNETIC RESONANCE METHODS | |
| 1DJM | 2000-04-05 | SOLUTION STRUCTURE OF BEF3-ACTIVATED CHEY FROM ESCHERICHIA COLI | |
| 1JBE | 2001-08-08 | 1.08 | 1.08 A Structure of apo-Chey reveals meta-active conformation |
| 3RVQ | 2012-05-09 | 1.15 | Structure of the CheY-Mn2+ Complex with substitutions at 59 and 89: N59D E89K |
| 3RVK | 2012-05-09 | 1.16 | Structure of the CheY-Mn2+ Complex with substitutions at 59 and 89: N59D E89Q |
| 3RVM | 2012-05-09 | 1.45 | Structure of the CheY-Mn2+ Complex with substitutions at 59 and 89: N59D and E89R |
| 1U8T | 2004-10-05 | 1.5 | Crystal structure of CheY D13K Y106W alone and in complex with a FliM peptide |
| 3RVL | 2012-05-09 | 1.55 | Structure of the CheY-BeF3 Complex with substitutions at 59 and 89: N59D and E89R |
| 3RVO | 2012-05-09 | 1.55 | Structure of CheY-Mn2+ Complex with substitutions at 59 and 89: N59D E89Y |
| 3OO0 | 2011-08-31 | 1.55 | Structure of apo CheY A113P |
| 6TG7 | 2019-12-04 | 1.65 | Crystal structure of the CheY in presence of magnesium |
| 3CHY | 1993-01-15 | 1.66 | CRYSTAL STRUCTURE OF ESCHERICHIA COLI CHEY REFINED AT 1.7-ANGSTROM RESOLUTION |
| 3OO1 | 2011-08-31 | 1.7 | Structure of E. Coli CheY mutant A113P in the absence of Sulfate |
| 3OLV | 2011-08-31 | 1.7 | Structural and functional effects of substitution at position T+1 in CheY: CheYA88V-BeF3-Mg complex |
| 1E6M | 2000-09-21 | 1.7 | TWO-COMPONENT SIGNAL TRANSDUCTION SYSTEM D57A MUTANT OF CHEY |
| 2ID9 | 2007-09-25 | 1.75 | 1.85 A Structure of T87I/Y106W Phosphono-CheY |
| 2ID7 | 2007-09-25 | 1.75 | 1.75 A Structure of T87I Phosphono-CheY |
| 1CHN | 1994-07-31 | 1.76 | MAGNESIUM BINDING TO THE BACTERIAL CHEMOTAXIS PROTEIN CHEY RESULTS IN LARGE CONFORMATIONAL CHANGES INVOLVING ITS FUNCTIONAL SURFACE |
| 2CHF | 1994-04-30 | 1.8 | STRUCTURE OF THE MG2+-BOUND FORM OF CHEY AND THE MECHANISM OF PHOSPHORYL TRANSFER IN BACTERIAL CHEMOTAXIS |
| 2CHE | 1994-04-30 | 1.8 | STRUCTURE OF THE MG2+-BOUND FORM OF CHEY AND MECHANISM OF PHOSPHORYL TRANSFER IN BACTERIAL CHEMOTAXIS |
| 1C4W | 2000-05-08 | 1.84 | 1.9 A STRUCTURE OF A-THIOPHOSPHONATE MODIFIED CHEY D57C |
| 1E6L | 2001-03-05 | 1.9 | Two-component signal transduction system D13A mutant of CheY |
| 1UDR | 1997-11-19 | 1.9 | CHEY MUTANT WITH LYS 91 REPLACED BY ASP, LYS 92 REPLACED BY ALA, ILE 96 REPLACED BY LYS AND ALA 98 REPLACED BY LEU (STABILIZING MUTATIONS IN HELIX 4) |
| 1YMV | 1996-04-03 | 1.9 | SIGNAL TRANSDUCTION PROTEIN CHEY MUTANT WITH PHE 14 REPLACED BY GLY, SER 15 REPLACED BY GLY, AND MET 17 REPLACED BY GLY |
| 1D4Z | 1999-10-14 | 1.9 | CRYSTAL STRUCTURE OF CHEY-95IV, A HYPERACTIVE CHEY MUTANT |
| 5DKF | 2016-03-09 | 1.94 | Reaction of phosphorylated CheY with imidazole 3 of 3 |
| 5DGC | 2016-03-09 | 1.94 | Reaction of phosphorylated CheY with imidazole 2 of 3 |
| 3FFW | 2009-09-22 | 2.0 | Crystal Structure of CheY triple mutant F14Q, N59K, E89Y complexed with BeF3- and Mn2+ |
| 2FMH | 2006-05-23 | 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) |
| 2FKA | 2006-05-23 | 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) |
| 2FMK | 2006-05-23 | 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) |
| 3F7N | 2009-09-22 | 2.0 | Crystal Structure of CheY triple mutant F14E, N59M, E89L complexed with BeF3- and Mn2+ |
| 5CHY | 1996-12-07 | 2.0 | STRUCTURE OF CHEMOTAXIS PROTEIN CHEY |
| 3FGZ | 2009-09-22 | 2.0 | Crystal Structure of CheY triple mutant F14E, N59M, E89R complexed with BeF3- and Mn2+ |
| 2FLW | 2006-05-23 | 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) |
| 1E6K | 2001-03-05 | 2.0 | Two-component signal transduction system D12A mutant of CheY |
| 2IDM | 2007-09-25 | 2.0 | 2.00 A Structure of T87I/Y106W Phosphono-CheY |
| 1EAY | 1998-07-15 | 2.0 | CHEY-BINDING (P2) DOMAIN OF CHEA IN COMPLEX WITH CHEY FROM ESCHERICHIA COLI |
| 2FMF | 2006-05-23 | 2.0 | Crystal structure of CheY in complex with CheZ 200-214 solved from a F432 crystal grown in Hepes (pH 7.5) |
| 3FFX | 2009-09-22 | 2.01 | Crystal Structure of CheY triple mutant F14E, N59R, E89H complexed with BeF3- and Mn2+ |
| 3OLY | 2011-08-31 | 2.05 | Structural and functional effects of substitution at position T+1 in CheY: CheYA88M-BeF3-Mn complex |
| 1VLZ | 1995-07-10 | 2.05 | UNCOUPLED PHOSPHORYLATION AND ACTIVATION IN BACTERIAL CHEMOTAXIS: THE 2.1 ANGSTROM STRUCTURE OF A THREONINE TO ISOLEUCINE MUTANT AT POSITION 87 OF CHEY |
| 5D2C | 2016-03-09 | 2.06 | Reaction of phosphorylated CheY with imidazole 1 of 3 |
| 2FLK | 2006-05-23 | 2.1 | Crystal structure of CheY in complex with CheZ(200-214) solved from a F432 crystal grown in CAPS (pH 10.5) |
| 3MYY | 2011-05-11 | 2.1 | Structure of E. Coli CheY mutant A113P bound to Beryllium fluoride |
| 1FFG | 2001-01-17 | 2.1 | CHEY-BINDING DOMAIN OF CHEA IN COMPLEX WITH CHEY AT 2.1 A RESOLUTION |
| 3OLX | 2011-08-31 | 2.1 | Structural and functional effects of substitution at position T+1 in CheY: CheYA88S-BeF3-Mn complex |
| 3RVJ | 2012-05-09 | 2.1 | Structure of the CheY-BeF3 Complex with substitutions at 59 and 89: N59D and E89Q |
| 3RVR | 2012-05-09 | 2.1 | Structure of the CheYN59D/E89R Molybdate complex |
| 3RVS | 2012-05-09 | 2.1 | Structure of the CheYN59D/E89R Tungstate complex |
| 1AB6 | 1998-02-04 | 2.2 | STRUCTURE OF CHEY MUTANT F14N, V86T |
| 3FFT | 2009-09-22 | 2.21 | Crystal Structure of CheY double mutant F14E, E89R complexed with BeF3- and Mn2+ |
| 1F4V | 2001-01-17 | 2.22 | CRYSTAL STRUCTURE OF ACTIVATED CHEY BOUND TO THE N-TERMINUS OF FLIM |
| 1HEY | 1995-07-10 | 2.24 | INVESTIGATING THE STRUCTURAL DETERMINANTS OF THE P21-LIKE TRIPHOSPHATE AND MG2+ BINDING SITE |
| 3RVN | 2012-05-09 | 2.25 | Structure of the CheY-BeF3 Complex with substitutions at 59 and 89: N59D and E89Y |
| 1EHC | 1997-05-15 | 2.26 | STRUCTURE OF SIGNAL TRANSDUCTION PROTEIN CHEY |
| 2FMI | 2006-05-23 | 2.3 | Crystal structure of CheY in complex with CheZ 200-214 solved from a F432 crystal grown in Tris (pH 8.4) |
| 3OLW | 2011-08-31 | 2.3 | Structural and functional effects of substitution at position T+1 in CheY: CheYA88T-BeF3-Mn complex |
| 1YMU | 1996-04-03 | 2.3 | SIGNAL TRANSDUCTION PROTEIN CHEY MUTANT WITH MET 17 REPLACED BY GLY (M17G) |
| 6CHY | 1996-12-07 | 2.33 | STRUCTURE OF CHEMOTAXIS PROTEIN CHEY |
| 1FQW | 2001-07-18 | 2.37 | CRYSTAL STRUCTURE OF ACTIVATED CHEY |
| 1AB5 | 1998-02-04 | 2.4 | STRUCTURE OF CHEY MUTANT F14N, V21T |
| 1FFS | 2001-01-17 | 2.4 | CHEY-BINDING DOMAIN OF CHEA IN COMPLEX WITH CHEY FROM CRYSTALS SOAKED IN ACETYL PHOSPHATE |
| 3RVP | 2012-05-09 | 2.4 | Structure of the CheY-BeF3 Complex with substitutions at 59 and 89: N59D and E89K |
| 1ZDM | 2005-04-26 | 2.4 | Crystal Structure of Activated CheY Bound to Xe |
| 2B1J | 2006-09-26 | 2.4 | Crystal Structure of Unphosphorylated CheY Bound to the N-Terminus of FliM |
| 2PL9 | 2008-01-15 | 2.6 | Crystal Structure of CheY-Mg(2+)-BeF(3)(-) in Complex with CheZ(C19) Peptide solved from a P2(1)2(1)2 Crystal |
| 1BDJ | 1999-05-11 | 2.68 | COMPLEX STRUCTURE OF HPT DOMAIN AND CHEY |
| 2PMC | 2008-01-15 | 2.69 | Crystal Structure of CheY-Mg(2+) in Complex with CheZ(C15) Peptide solved from a P1 Crystal |
| 1FFW | 2001-01-17 | 2.7 | CHEY-BINDING DOMAIN OF CHEA IN COMPLEX WITH CHEY WITH A BOUND IMIDO DIPHOSPHATE |
| 2CHY | 1990-07-15 | 2.7 | THREE-DIMENSIONAL STRUCTURE OF CHEY, THE RESPONSE REGULATOR OF BACTERIAL CHEMOTAXIS |
| 1MIH | 2003-04-08 | 2.7 | A ROLE FOR CHEY GLU 89 IN CHEZ-MEDIATED DEPHOSPHORYLATION OF THE E. COLI CHEMOTAXIS RESPONSE REGULATOR CHEY |
| 1KMI | 2002-07-24 | 2.9 | CRYSTAL STRUCTURE OF AN E.COLI CHEMOTAXIS PROTEIN, CHEZ |
| 1A0O | 1998-12-30 | 2.95 | CHEY-BINDING DOMAIN OF CHEA IN COMPLEX WITH CHEY |
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 |