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Title 

Physical evidence for a phosphorylation-dependent conformational change in the enhancer-binding protein NtrC

Authors 

In Gyu HwangT ThorgeissonJong hui LeeS KustuYeon Kyun Shin

Publisher 

National Academy of Sciences

Issue Date 

1999

Citation 

Proceedings of the National Academy of Sciences of the United States of America, vol. 96, no. 9, pp. 4880-4885

Keywords 

nitrogen regulatory protein cregulator proteinunclassified drugconformational transitionenzyme activitynonhumanoligomerizationpriority journalprotein conformationprotein domain

Abstract 

The bacterial enhancer-binding protein nitrogen regulatory protein C (NtrC) activates transcription by σ54-containing RNA polymerase in a reaction that depends on ATP hydrolysis. Phosphorylation of an aspartate residue in the N-terminal receiver domain of NtrC induces oligomerization of the protein and activates the ATPase activity, which is a function of its central output domain. To study the role of the receiver domain of NtrC, which is known to act positively, we isolated mutant forms of the protein carrying single cysteine residues and derivatized them with a sulfhydryl- specific nitroxide reagent for electron paramagnetic resonance studies. Single cysteines were placed at four positions at which we had obtained constitutive amino acid substitutions, those that yield activity without phosphorylation. In only one case, derivatized C86 in α-helix 4 of the receiver domain, did the motion of the side chain become dramatically slower upon phosphorylation. Importantly, derivatized NtrC(D86C) (NtrC(D86C*)) activated transcription normally. Additional experiments indicated that the spectral change observed upon phosphorylation of NtrC(D86C*) was due to interdomain interactions rather than a conformational change within the N- terminal domain itself. These interactions did not appear to occur within a monomer. Although it is not clear whether the spectral change seen upon phosphorylation of NtrC(D86C*) is due to an interaction that occurs within a dimer of NtrC or requires the formation of higher-order oligomers, the change indicated that α-helix 4 of the receiver domain probably plays an important role in communication with the remainder of the protein.

ISSN 

0027-8424

Appears in Collections

1. Journal Articles > Journal Articles

Registered Date

2017-04-19


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