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Delineation of alternative conformational states in Escherichia coli peptide deformylase via thermodynamic studies for the binding of actinonin.

ABSTRACT: We investigated the binding of a naturally occurring antibiotic, actinonin, to the Ni(2+)-reconstituted recombinant form of Escherichia coli peptide deformylase (PDF(Ec)) via isothermal titration microcalorimetry. The binding data conformed to both exothermic and endothermic phases with magnitudes of DeltaG degrees , DeltaH degrees , and TDeltaS degrees being equal to -12, -2.7, and 9.3 kcal/mol and -8.7, 3.9, and 12.6 kcal/mol, respectively. Evidently, although both phases are dominated by favorable entropic changes, the exothermic phase has about 6.7 kcal/mol enthalpic advantage over the endothermic phase. We observed that the removal of bound Ni(2+) from PDF(Ec) abolished the exothermic phase without affecting the endothermic phase, but it was regained upon addition of Zn(2+). In conjunction with metal analysis data, we propose that the recombinant form of PDF(Ec) is expressed in two stable conformational states that yield markedly distinct ITC profiles (i.e., exothermic versus endothermic) upon interaction with actinonin. The existence of two conformational states of PDF(Ec) is further supported by the observation of two distinct and independent transitions during the thermal unfolding of the enzyme. In addition, the thermodynamic data reveal that the formation of the PDF(Ec)-actinonin complex results in the transfer of one H(+) from the enzyme phase to the bulk solvent at pH 6.3. Both exothermic and endothermic phases produce highly negative DeltaC(p) degrees values, but there is no apparent enthalpy-entropy compensation effect upon formation of the PDF(Ec)-actinonin complex. In view of the known structural features of the enzyme, arguments are presented that the alternative conformational states of PDF(Ec) are modulated by the metal ligation at the enzyme site.

SUBMITTER: Berg AK

PROVIDER: S-EPMC2644421 | biostudies-literature | 2009 Feb

REPOSITORIES: biostudies-literature

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Delineation of alternative conformational states in Escherichia coli peptide deformylase via thermodynamic studies for the binding of actinonin.

Berg Alexander K AK Srivastava D K DK

Biochemistry 20090201 7

We investigated the binding of a naturally occurring antibiotic, actinonin, to the Ni(2+)-reconstituted recombinant form of Escherichia coli peptide deformylase (PDF(Ec)) via isothermal titration microcalorimetry. The binding data conformed to both exothermic and endothermic phases with magnitudes of DeltaG degrees , DeltaH degrees , and TDeltaS degrees being equal to -12, -2.7, and 9.3 kcal/mol and -8.7, 3.9, and 12.6 kcal/mol, respectively. Evidently, although both phases are dominated by favo ...[more]

PMID: 19191548

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Project description:Tsr, the serine chemoreceptor in Escherichia coli, transduces signals from a periplasmic ligand-binding site to its cytoplasmic tip, where it controls the activity of the CheA kinase. To function, Tsr forms trimers of homodimers (TODs), which associate in vivo with the CheA kinase and CheW coupling protein. Together, these proteins assemble into extended hexagonal arrays. Here, we use cryo-electron tomography and molecular dynamics simulation to study Tsr in the context of a near-native array, characterizing its signaling-related conformational changes at both the individual dimer and the trimer level. In particular, we show that individual Tsr dimers within a trimer exhibit asymmetric flexibilities that are a function of the signaling state, highlighting the effect of their different protein interactions at the receptor tips. We further reveal that the dimer compactness of the Tsr trimer changes between signaling states, transitioning at the glycine hinge from a compact conformation in the kinase-OFF state to an expanded conformation in the kinase-ON state. Hence, our results support a crucial role for the glycine hinge: to allow the receptor flexibility necessary to achieve different signaling states while also maintaining structural constraints imposed by the membrane and extended array architecture.IMPORTANCE In Escherichia coli, membrane-bound chemoreceptors, the histidine kinase CheA, and coupling protein CheW form highly ordered chemosensory arrays. In core signaling complexes, chemoreceptor trimers of dimers undergo conformational changes, induced by ligand binding and sensory adaptation, which regulate kinase activation. Here, we characterize by cryo-electron tomography the kinase-ON and kinase-OFF conformations of the E. coli serine receptor in its native array context. We found distinctive structural differences between the members of a receptor trimer, which contact different partners in the signaling unit, and structural differences between the ON and OFF signaling complexes. Our results provide new insights into the signaling mechanism of chemoreceptor arrays and suggest an important functional role for a previously postulated flexible region and glycine hinge in the receptor molecule.

Dataset Information

Delineation of alternative conformational states in Escherichia coli peptide deformylase via thermodynamic studies for the binding of actinonin.

Publications

Delineation of alternative conformational states in Escherichia coli peptide deformylase via thermodynamic studies for the binding of actinonin.

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