1. Field of the Invention
This invention relates to the LuxP protein, and more particularly to a crystalline form of the LuxP protein having sufficient crystal quality to allow crystallographic data to be obtained, as well as to methods of structure-based drug design based on the crystallographic data.
2. Description of the Related Art
Cell-cell communication in bacteria occurs through the exchange of extracellular signaling compounds called autoinducers. This process, termed quorum sensing, allows bacterial populations to coordinate gene expression. Community cooperation likely enhances the effectiveness of processes including bioluminescence, virulence factor expression, antibiotic production, and biofilm development. Unlike other autoinducers, which are specific to particular species of bacteria, a recently discovered autoinducer (AI-2) is produced by a large number of bacterial species. AI-2 has been proposed to serve as a universal signal for inter-species communication.
AI-2 was originally identified in the bioluminescent marine bacterium Vibrio harveyi as one of two autoinducers that regulate light production in response to cell density. The synthase required for AI-2 production, LuxS, is widely conserved among gram-negative and -positive bacteria. Bacteria produce AI-2 from S-adenosylmethionine in several enzymatic steps, as shown in FIG. 1. Consumption of S-adenosylmethionine as a methyl donor produces S-adenosylhomocysteine, which undergoes hydrolysis catalyzed by the nucleosidase Pfs to yield adenine and S-ribosylhomocysteine. Subsequently LuxS catalyzes cleavage of S-ribosylhomocysteine to homocysteine and 4,5-dihydroxy-2,3-pentanedione, which can then cyclize either by itself or in an enzyme-mediated process.
Detection of AI-2 by V. harveyi involves two proteins, LuxP and LuxQ. LuxP belongs to a large family of periplasmic binding proteins whose members bind diverse ligands, while LuxQ is a two-component hybrid sensor kinase embedded in the bacterial inner membrane. It is believed that LuxP is the primary AI-2 receptor, see X. Chen, S. Schauder, N. Potier, A. Van Dorsselaer, I. Pelczer, B. Bassler, and F. Hughson, “Structural Identification of a Bacterial Quorum-Sensing Signal Containing Boron,” Nature, Vol. 415, pp. 545–549 (2002). In quorum sensing, the periplasmic LuxP-AI-2 complex likely interacts with LuxQ to transduce the autoinducer signal. However, heretofore LuxP has not been isolated in crystalline form suitable for structural determination by X-ray crystallography, and thus neither the crystal structure of LuxP nor the LuxP binding site for AI-2 were known prior to the instant invention.
USPTO Backgrounds
USPTO Backgrounds