W0223

Structural Studies of BenM, A Transcriptional Activator With a Synergist Response to Two Effectors. Cory Momany¹, Todd J. Clark², Sandra Haddad², and Ellen L. Neidle², Dept. of Pharmaceutical & Biomedical Sciences¹ and Dept. of Microbiology², Univ. of Georgia, Athens, GA 30602.

BenM is a LysR-type transcriptional regulator of the soil bacterium Acinetobacter sp. ADP1. LysR-type proteins form one of the largest families of prokaryotic transcriptional regulators and control diverse physiological processes including biodegradation, bioremediation, biosynthesis, and bacterial virulence. BenM regulates the expression of more than a dozen genes for the degradation of aromatic compounds serving as carbon and energy sources. BenM responds to two effectors, benzoate and its catabolite cis,cis-muconate. The level of BenM-regulated transcription is significantly higher in response to both compounds than the combined levels due to each alone. To understand this novel synergistic response at an atomic level, the effector-binding domain (EBD) of BenM, BenM-EBD, was crystallized. The structure of the BenM-EBD dimer was solved by SAD phasing using a crystal of selenomethionyl-modified BenM-EBD collected at the selenium peak in conjunction with the (Re)Solve structure determination package. Topologically, each 224-residue BenM-EBD monomer consisted of two major domains, both α/β structures, connected by two β-strands. The N- and C-termini were associated with domain I (residues 81-161 and 268-296), while the second domain II consisted of the intervening residues (162-267). Within the dimer, domain I of one monomer was associated with domain II from the neighboring two-fold (non-crystallographic) related monomer. Two binding pockets, separated by approximately 20 Å, were exposed on the same face of the dimer. One monomer contained a bound sulfate ion, which appeared to mimic an effector-bound structure. Two helices from separate subdomains shifted 1-2 Å to enclose the sulfate ion, with smaller shifts apparent in the surrounding β-strands. Crystals of BenM-EBD with different combinations of the two effectors prepared from various crystallization conditions are being evaluated to characterize the conformational changes associated with synergistic transcriptional activation.