Effect of phosphorylation on the interdomain interaction of the response regulator, NarL.
DNA binding by the effector domain (NarLC) of the response regulator, NarL, is modulated by the phosphorylation state of the receiver domain (NarLN). The receiver domain appears to block the site of DNA binding in the nonphosphorylated state. Phosphorylation is proposed to disrupt this interaction, causing the effector domain to be released and free to bind DNA (Baikalov, I., Schroder, I., Kaczor-Grzeskowiak, M., Grzeskowiak, K., Gunsalus, R. P., and Dickerson, R. E. (1996) Biochemistry 35, 11053-61). To better understand this modulation, we analyzed the interaction between the two domains in the absence of a polypeptide linkage. Using multidimensional NMR, we mapped chemical shift changes that occurred during a titration between the two isolated domains. Specific residues in NarLC exhibit large chemical shift changes upon the addition of NarLN. These residues are primarily at the interface between the two domains as seen in the crystal structure. Using the residues with the largest chemical shift changes, we observed a dissociation constant of 88 +/- 7 microM. In the presence of 10 mM MgCl(2), the affinity is reduced 4-fold to about 350 microM. This work shows that the domains interact in trans and that this interaction, while fairly weak, provides a way to monitor the energetics of domain-domain interaction in this system. Phosphorylation of NarLN by a small-molecule phosphate donor, phosphoramidate, decreases this interaction about 25-fold from the nonphosphorylated sample. The results support the model that the mechanism of activation of NarL involves a disruption of the interdomain interface and suggests that the linker is not necessary for the transmission of signal across the domain interface. The linker does play a role in increasing the local concentration of the domains and therefore increasing the amount of closed conformation with respect to the open conformation. We estimate the levels of open conformation to be low (about 1%) in the nonphosphorylated state in the absence of magnesium ion and much higher in the phospho state (near 50%). This modulation of the open or active state via the interaction at the interface may also be applicable to other multidomain response regulator proteins.
Eldridge
Aimee M
AM
Institute of Molecular Biology, University of Oregon, Eugene, Oregon 97403, USA.
Kang
Hyun-Seo
HS
Johnson
Eric
E
Gunsalus
Robert
R
Dahlquist
Frederick W
FW
D002633:Chemotaxis; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism*; D008954:Models, Biological; D008956:Models, Chemical; D008958:Models, Molecular; D009586:Nitrogen Fixation; D019906:Nuclear Magnetic Resonance, Biomolecular; D010766:Phosphorylation; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D013816:Thermodynamics; D017186:Titrimetry
D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; C069780:NarL protein, E coli