The Crystal Structure of the Escherichia coli Autoinducer-2 Processing Protein LsrF

Citation: Diaz Z, Xavier KB, Miller ST ( The Crystal Structure of the Escherichia coli Autoinducer- 2 Processing Protein LsrF Zamia Diaz 0 Karina B. Xavier 0 Stephen T. Miller 0 Raphael H. Valdivia, Duke University Medical Center, United States of America 0 1 Department of Chemistry and Biochemistry, Swarthmore College, Swarthmore, Pennsylvania, United States of America, 2 Instituto Gulbenkian de Ciencia , Oeiras , Portugal , 3 Instituto de Tecnologia Qu mica e Biolo gica, Estac a o Agrono mica Nacional , Oeiras , Portugal Many bacteria produce and respond to the quorum sensing signal autoinducer-2 (AI-2). Escherichia coli and Salmonella typhimurium are among the species with the lsr operon, an operon containing AI-2 transport and processing genes that are up regulated in response to AI-2. One of the Lsr proteins, LsrF, has been implicated in processing the phosphorylated form of AI-2. Here, we present the structure of LsrF, unliganded and in complex with two phospho-AI-2 analogues, ribose-5phosphate and ribulose-5-phosphate. The crystal structure shows that LsrF is a decamer of (ab)8-barrels that exhibit a previously unseen N-terminal domain swap and have high structural homology with aldolases that process phosphorylated sugars. Ligand binding sites and key catalytic residues are structurally conserved, strongly implicating LsrF as a class I aldolase. - Funding: The work in the laboratory of S.T.M. was supported by grants from the Camille and Henry Dreyfus Foundation (http://www.dreyfus.org/), the Merck/ AAAS Undergraduate Science Research Program (http://www.merckaaasusrp.org/), and Swarthmore College (http://www.swarthmore.edu). The research performed by K.B.X. was supported by Fundacao para a Ciencia e Tecnologia (FCT) grant PTDC/ DG/BIA/82010/2006 (http://alfa.fct.mctes.pt/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Many bacterial species control expression of specific genes thorough the production, release, and detection of small signal molecules called autoinducers. This process, termed quorum sensing, allows bacteria to regulate behavior in a populationdependant manner, effectively coordinating their activity. Behaviors regulated by quorum sensing include bioluminescence, biofilm formation, and production of virulence factors [1]. While autoinducer production and recognition is generally species specific, autoinducer-2 (AI-2) has been shown to be produced and recognized by a variety of bacterial species, both Gram-positive and Gram-negative. First identified as a regulator of bioluminescence in Vibrio harveyi [2,3], AI-2 has been shown to control a wide variety of behaviors in different species, including motility in Helicobacter pylori [4], division, stress response, and biofilm formation in Streptococcus mutans [5,6], virulence and formation of biofilms in Vibrio cholerae [79] and Staphylococcus epidermis [10], social and pluricellular behavior of Bacillus subtilis [11], and virulence in Erwinia carotovora ssp. carotovora [12]. Since many species produce and respond to AI-2, it is believed to facilitate interspecies communication, potentially allowing bacteria to modulate gene expression in response to both the concentration and species composition of bacteria in the local environment; indeed, some species of bacteria have been shown to respond to AI-2 produced by other species in co-culture experiments [13,14]. AI-2 is produced by the highly conserved synthase LuxS, which catalyzes the production of 4,5-dihydroxy-2,3-pentanedione (DPD) from S-ribosylhomocysteine [2]. Crystal structures of AI-2 receptor/ ligand complexes from V. harveyi [15], S. typhimurium [16], and S. meliloti [13] have shown that these species recognize chemically distinct DPD adducts as AI-2: (2S, 4S)-2-methyl-2,3,3,4-tetrahydroxyterahydrofuran-borate in the case of V. harveyi and (2R, 4S)-2-methyl-2,3,3,4tetrahydroxyterahydrofuran in the case of S. typhimurium and S. meliloti. The known forms of AI-2 are able to interconvert spontaneously in solution, suggesting that a mix of DPD-derived molecules exists in environments with LuxS-containing bacteria [16]; however, because the different forms of AI-2 can interconvert, bacteria that recognize chemically distinct forms of AI-2 can nonetheless communicate with each other [14]. While AI-2 has been shown to act as a signaling molecule in many bacterial species [17,18], the molecular details of AI-2 recognition and response have been studied in only a small number of species including E. coli [19,20], S. typhimurium [21,22], Sinorhizobium meliloti [13], V. cholerae [9,2325], and V. harveyi [3,15,26,27]. E. coli and S. typhimurium share an operon, named lsr (for LuxS Regulated), that consists of lsrA, lsrB, LsrC, lsrD, lsrF, and lsrG (and, in the case of S. typhimurium, lsrE) and is responsible for the reco (...truncated)