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
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
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
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
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
3RVL 2012-05-09 1.55 Structure of the CheY-BeF3 Complex with substitutions at 59 and 89: N59D and E89R
3CHY 1993-01-15 1.66 CRYSTAL STRUCTURE OF ESCHERICHIA COLI CHEY REFINED AT 1.7-ANGSTROM RESOLUTION
3OLV 2011-08-31 1.7 Structural and functional effects of substitution at position T+1 in CheY: CheYA88V-BeF3-Mg complex
3OO1 2011-08-31 1.7 Structure of E. Coli CheY mutant A113P in the absence of Sulfate
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
2CHE 1994-04-30 1.8 STRUCTURE OF THE MG2+-BOUND FORM OF CHEY AND MECHANISM OF PHOSPHORYL TRANSFER IN BACTERIAL CHEMOTAXIS
2CHF 1994-04-30 1.8 STRUCTURE OF THE MG2+-BOUND FORM OF CHEY AND THE MECHANISM OF PHOSPHORYL TRANSFER IN BACTERIAL CHEMOTAXIS
1C4W 2000-05-08 1.84 1.9 A STRUCTURE OF A-THIOPHOSPHONATE MODIFIED CHEY D57C
1D4Z 1999-10-14 1.9 CRYSTAL STRUCTURE OF CHEY-95IV, A HYPERACTIVE CHEY MUTANT
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
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)
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
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)
1E6K 2001-03-05 2.0 Two-component signal transduction system D12A mutant of CheY
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)
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)
5CHY 1996-12-07 2.0 STRUCTURE OF CHEMOTAXIS PROTEIN CHEY
3FFW 2009-09-22 2.0 Crystal Structure of CheY triple mutant F14Q, N59K, E89Y complexed with BeF3- and Mn2+
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)
3FGZ 2009-09-22 2.0 Crystal Structure of CheY triple mutant F14E, N59M, E89R complexed with BeF3- and Mn2+
3F7N 2009-09-22 2.0 Crystal Structure of CheY triple mutant F14E, N59M, E89L complexed with BeF3- and Mn2+
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
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
3RVS 2012-05-09 2.1 Structure of the CheYN59D/E89R Tungstate complex
3RVR 2012-05-09 2.1 Structure of the CheYN59D/E89R Molybdate complex
1FFG 2001-01-17 2.1 CHEY-BINDING DOMAIN OF CHEA IN COMPLEX WITH CHEY AT 2.1 A RESOLUTION
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)
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
3MYY 2011-05-11 2.1 Structure of E. Coli CheY mutant A113P bound to Beryllium fluoride
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
1YMU 1996-04-03 2.3 SIGNAL TRANSDUCTION PROTEIN CHEY MUTANT WITH MET 17 REPLACED BY GLY (M17G)
3OLW 2011-08-31 2.3 Structural and functional effects of substitution at position T+1 in CheY: CheYA88T-BeF3-Mn complex
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)
6CHY 1996-12-07 2.33 STRUCTURE OF CHEMOTAXIS PROTEIN CHEY
1FQW 2001-07-18 2.37 CRYSTAL STRUCTURE OF ACTIVATED CHEY
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
1AB5 1998-02-04 2.4 STRUCTURE OF CHEY MUTANT F14N, V21T
2B1J 2006-09-26 2.4 Crystal Structure of Unphosphorylated CheY Bound to the N-Terminus of FliM
1ZDM 2005-04-26 2.4 Crystal Structure of Activated CheY Bound to Xe
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
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
1FFW 2001-01-17 2.7 CHEY-BINDING DOMAIN OF CHEA IN COMPLEX WITH CHEY WITH A BOUND IMIDO DIPHOSPHATE
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