Linked Life Data

12484754

Source:http://linkedlifedata.com/resource/pubmed/id/12484754

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
51
pubmed:dateCreated
2002-12-17
pubmed:abstractText
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.
pubmed:grant
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Dec
pubmed:issn
0006-2960
pubmed:author
pubmed:issnType
Print
pubmed:day
24
pubmed:volume
41
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
15173-80
pubmed:dateRevised
2007-11-14
pubmed:meshHeading
pubmed:year
2002
pubmed:articleTitle
Effect of phosphorylation on the interdomain interaction of the response regulator, NarL.
pubmed:affiliation
Institute of Molecular Biology, University of Oregon, Eugene, Oregon 97403, USA.
pubmed:publicationType
Journal Article, Research Support, U.S. Gov't, P.H.S.