Structural studies on sinorhizobium meliloti DctD related to ATP binding and activation

by Park, Sungdae.

Sinorhizobium meliloti DctD activates transcription at the dctA promoter by catalyzing the transformation of closed complex of E?54 (RNA polymerase/sigma-54) into open ones in response to the phosphorylation status of DctB, a C4-dicarboxylate sensor. DctD is ?54-dependent transcriptional activator, and consists of three functional domains: an N-terminal, two-component receiver domain; a central AAA+ ATPase domain; and a C-terminal DNA binding domain. Since the DctB and DctD proteins form a two-component regulatory system, it is easy to hypothesize a general outline of signal transduction for these proteins. But detailed molecular mechanisms underlying twocomponent signal transduction have only recently begun to emerge from research in several labs. As a step toward understanding molecular events involved in the DctB/DctD signaling system in Sinorhizobium meliloti, I have investigated its several aspects. Tryptic digestion experiments of DctD with or without ATP showed that unphosphorylated 44 kD has the ability to bind ATP, and, upon ATP binding, undergoes conformational changes, which is registered in changes in the pattern of tryptic digestion. By modeling the decay of the 44 kD peptide at varying amounts of ATP as a combination of two first order processes; one with and one without ATP being bound, it was possible to estimate an equilibrium dissociation constant of approximately 0.7 mM for ATP. Subsequent pre-steady state kinetic studies of mant-ATP binding to DctD gave the rough estimation of an equilibrium dissociation constant of 0.87 mM. The results of both iii experiments suggest that the receiver domain does not play its role simply by preventing the AAA+ ATPase domain from binding ATP. Crystallographic studies on the receiver domains of DctD and its variant, E121K, revealed another important feature of this signaling system. The crystal structure of E121KNL obtained with or without Mg 2+ and BeF3-1 showed that, in the active state mimicked by Mg 2+- BeF3-1, E121KNL has changes induced in the active site similar to those seen in FixJN~P, Spo0AN~P and CheY-BeF3-1, and assumes a dimer interface dramatically different from that of the off-state E121KNL. Along with the results of previous crystallographic and genetic studies, the following ‘working model’ for signal transduction in DctD can be proposed. In the ‘offstate’, the receiver domain and coiled-coil linker form a dimer that inhibits oligomerization of the AAA+ ATPase domain. In this conformation the receiver domain cannot be phosphorylated or bind Mg 2+ and BeF3-1. The binding of Mg 2+ and BeF3-1 stabilizes an alternative dimeric conformation in which the ?4-?5-?5 interface is replaced with an ?4-?5 interface, thereby repositioning the phosphorylation site (Asp55) in the subunits by ~20 Å. Reorienting the receiver domains relieves inhibition of the AAA+ ATPase domain by at least allowing or even stimulating it to oligomerize and activate transcription at the dctA promoter. Since ~4.5 % of two-component receiver domains are predicted to have coiled-coil linkers found in DctD, switching between alternate dimeric states as seen in DctD may be a common mechanism used by a subgroup of twocomponent regulatory systems. iv