title: Analysis of ATP binding to CheA containing tryptophan substitutions near the active site.
abstract: Signal transduction in the chemotaxis system of Escherichia coli involves an autophosphorylating protein histidine kinase, CheA. At the active site of CheA, phenylalanine residues 455 and 459 occupy positions near the ATP-binding pocket, immediately adjacent to one of the hinge regions of a loop that undergoes an ATP-induced conformational change ("lid closure") that has been characterized previously in X-ray crystal structures [Bilwes et al. (2001) Nat. Struct. Biol. 8, 353-360]. We generated versions of CheA carrying F455W and F459W replacements and investigated whether the fluorescence properties of the introduced tryptophan side chains were affected by nucleotide binding in a manner that would provide a signal for investigating the dynamics of active site events, such as ATP binding and lid closure. Our results indicate that CheA(F455W) is useful in this regard, but CheA(F459W) is not. CheA(F455W) retained full catalytic activity and exhibited easily monitored fluorescence changes upon binding nucleotide: we observed a 25-30% decrease in CheA(F455W) fluorescence emission intensity at 330 nm upon binding ATP in the absence of Mg(2+); in the presence of Mg(2+), the emission spectrum of the CheA(F455W):ATP complex was red-shifted by 5 nm and exhibited an increased intensity (approximately 20% higher at 345 nm relative to that of uncomplexed CheA(F455W)). Different fluorescence changes were observed when two ATP analogues (ADPNP and ADPCP) were used instead of ATP and when Mn(2+) or Ca(2+) was used in place of Mg(2+). We exploited the fluorescence changes induced by Mg(2+)-ATP to explore the kinetics and mechanism of nucleotide binding by CheA(F455W). In the absence of Mg(2+), binding appears to involve a simple one-step equilibrium (k(assn) = 0.7 microM(-1) s(-1) and k(dissn) = 270 s(-1) at 4 degrees C). In the presence of Mg(2+), the binding mechanism involves at least two steps: (i) rapid, relatively weak binding followed by (ii) a rapid, reversible step (k(forward) = 300 s(-1) and k(reverse) =15 s(-1) at 4 degrees C) that enhanced the overall affinity of the complex and generated an increase in W455 fluorescence. This second step could reflect a conformational change at the CheA active site, such as lid closure. These results provide the first insight into the dynamics of nucleotide binding and substrate-induced conformational changes at the active site of a protein histidine kinase.
authors.affiliation: Department of Cell Biology & Molecular Genetics, University of Maryland, College Park, Maryland 20742, USA. rs224@umail.umd.edu
authors.forename: Richard C
authors.initials: RC
authors.lastname: Stewart
mesh_terms: D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism*; D019943:Amino Acid Substitution / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites / Q000235:genetics; D002118:Calcium / Q000737:chemistry; D002413:Cations, Divalent / Q000737:chemistry; D002633:Chemotaxis / Q000235:genetics; D004926:Escherichia coli / Q000166:cytology / Q000201:enzymology / Q000235:genetics; D029968:Escherichia coli Proteins; D000071677:Histidine Kinase; D008274:Magnesium / Q000737:chemistry; D008345:Manganese / Q000737:chemistry; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000072236:Methyl-Accepting Chemotaxis Proteins; D016297:Mutagenesis, Site-Directed; D010649:Phenylalanine / Q000235:genetics; D011485:Protein Binding; D011487:Protein Conformation; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics; D014364:Tryptophan / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D002413:Cations, Divalent; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D000072236:Methyl-Accepting Chemotaxis Proteins; D008345:Manganese; D010649:Phenylalanine; D014364:Tryptophan; D000255:Adenosine Triphosphate; D011494:Protein Kinases; D000071677:Histidine Kinase; C000607608:cheA protein, E coli; D008274:Magnesium; D002118:Calcium