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Solution Structures of the DctD Receiver Domain

Introduction Two-component signal transduction is the predominant information-processing mechanism used by bacteria to control adaptive responses to environmental changes. It is also present in archaeons, single-celled eukaryotes, and plants. The kinds of behaviors controlled by two-component signal transduction range from the expression of single genes for nutrient acquisition to well-orchestrated developmental programs that totally redefine a cell’s physiology and sometimes its anatomy. One group of two-component response regulators is formed by proteins known as σ54-dependent transcriptional activators, or bacterial enhancer binding proteins. Each of these contain an AAA+ ATPase domain that is used to couple ATP hydrolysis with conformational changes in the σ54-form of RNA polymerase. The conformational change permits the polymerase to isomerize into productive open complexes at promoters. We are studying one such protein, the dicarboxylate transport protein D (DctD) of Sinorhizobium meliloti. These bacteria reduce nitrogen to ammonia only when living in symbiosis with their legume host, alfalfa. DctD regulates the expression of a dicarboxylate transport protein encoded by dctA, which enables the bacteria to use the food that the host provides to fuel symbiotic nitrogen fixation [1]. DctD, like about 50% of the 600 known σ54dependent activators, has three functional domains. An N-terminal “receiver” domain regulates the functional state of a central AAA+ ATPase output domain, and these are guided to the right promoter region by a C-terminal DNA-binding domain. Analytical ultracentrifugation studies indicated that the DctD receiver domain exists in dimeric but distinct states in both its apo (inactive) and phosphorylated (active) forms. Crystallography revealed a novel dimeric structure for the inactive receiver domain [2, 3]. Substitution E121K destabilized the dimer about 20-fold, and partially activated the ATPase domain. Beryllium fluoride mimicked phosphorylation of the receiver domain, also partially activating the ATPase domain [4]. Together, beryllium fluoride and the substitution E121K sufficiently stabilized the active form of the receiver domain to obtain a crystal structure [5]. Unfortunately, it was unclear which of the four possible dimers present in the crystal lattice represented the solution state [5, 6]. Small-angle x-ray scattering (SAXS) and wide-angle x-ray scattering (WAXS) experiments were explored during a workshop held in the spring of 2002. The purpose was to learn if such data would distinguish between the possible dimers of the DctD receiver domain in its active state and to determine which form exists in solution. Although the studies were only begun during this workshop, they were found to be feasible and were completed and expanded upon in 2003. This report describes the initial studies that were conducted during the workshop in 2002 and relates them to subsequent studies that grew from the workshop experience.