A Structural Genomics Approach to the Study of Quorum Sensing: Crystal Structures of Three LuxS Orthologs Speaker: 簡湘誼 Date: 2002/10/08 Structure, vol. 9, , June, 2001

Outline Introduction Quorum sensing, structural genomics, LuxS protein Results and discussion Biological implications

What is Quorum sensing? Quorum sensing is the mechanism by which bacteria control gene expression in response to cell density. It is very important fot pathogenic bacteria during infection of a host (e.g. humans, other animals or plants) to co-ordinate their virulence in order to escape the immune response of the host in order to establish a successful infection. Helicobacter pylori... Two major quorum sensing system: system 1 and system 2

System 1 Signaling molecule: autoinducer-1 (AI-1, N-acyl-L-homoserine lactones) Gram-negative bacteria System 2_more widespread Signaling molecule: autoinducer-2 (AI-2) Gram-positive and gram-negative bacteria AI-2 production depends on the proper function of LuxS gene. Two major quorum sensing system ：

Signaling system 1 Signaling system 2 AI-1AI-2 AI-1 AI-2 Signal 1Sensor 1Signal 2Sensor 2 lux M lux N lux P lux Slux CDABEGH Model for genetic control of luminescence in V. harveyi. (Bassler, Molecular Microbiology (1993) 9(4), )

The advantage of Structural Genomics An emerging field of postgenomic discovery This information will aid the assignment of biochemical function to the large fraction of genomes of unknown function.

In this paper, the authors describe the structure of one component of the AI-2 biosynthesis pathway, LuxS, from three different bacteria, Helicobacter pylori, Deinococcus radiodurans, and Haemophilus influenzae.

Results

* HP, Helicobacter pylori (J99_jsh0097); DR, Deinococcus radiodurans; HI, Haemophilus influenzae; CJ, Campylobacter jejuni; BB, Borrelia burgdorferi. Back

Structure Determination The average, pairwise identity among these five sequences is 38%. * HP, Helicobacter pylori; DR, Deinococcus radiodurans; HI, Haemophilus influenzae; CJ, Campylobacter jejuni; BB, Borrelia burgdorferi. A strong structural similarity between these proteins.

H. pyloriD. radioduransH. influenzaeC. jejuniB. burgdorferi Protein construct C-terminal His-tagged proteins in pET26b-derived expression vectors Induction with IPTG Sample condition 150mM NaCl, 10mM methionine, 1mM β-mercaptoethanol, 10mM HEPES (pH 7.5) Crystallization condition 32%PEG1000, 200mM ammonium sulfate, 100mM MES (pH 7.5), 30mM β-mercaptoethanol (BME), 20°C, 5 mg/ml protein concentration 26% PEG MME 5000, 100mM MES (pH 6.5), 30mM BME, 4°C, 19 mg/ml protein concentration 21% PEG MME 5000, 100mM Bis-Tris (pH 6.25), 30mM BME, 12°C, 10 mg/ml protein concentration Space groupP P2 1 C2 P

Structure of the LuxS Monomer The overall folds observed for LuxS are similar and consist of four-stranded antiparallel β sheet in contact with four α helices. H1-S1-S2-H2-S3-S4-H3-H4 Many of the conserved residues in the LuxS protein are hydrophobic.

Figure2. Stereo Ribbon Diagrams of One Monomer in the Asymmetric Unit of the LuxS Proteins Examined in This Paper Beta strands are displayed in cyan, α helices are displayed in red, and 3 10 helices are displayed in blue. (a) LuxS from H. pylori. (b) LuxS from H. influenza. (c) LuxS from D. radiodurans.

Homodimer The metal and substrate binding sites lie at the dimer interface. Dynamic light-scattering studies showed a dimer interaction in solutions of the LuxS proteins.

Figure 3. Stereo Ribbon Diagram of the LuxS Homodimer The same color coding as that shown in Figure 2 is used. (a) The dimer observed in the asymmetric unit of Hp_LuxS. (b) Same as in (a), except rotated 90 degree out of the plane of the page.

Metal Binding Pocket Go to… Go to…Analysis of the LuxS data revealed electron density in a binding pocket, composed of His57, His61, and Cys131 in coordination distance with a metal atom and with His 137 nearby in supporting role. Go to… Go to… Go to “ substrate binding site ”… Go to “ substrate binding site ”…This is suggestive of an enzymatic role for the metal, as many zinc enzymes have a similarly coordinated zinc ion. Go to “ substrate binding site ”… Go to “ substrate binding site ”…

Figure 4. Experimental Map of Dr_LuxS Displaying Electron Density About the Metal binding Site Back

Figure 5. Ball and Stick and Ribbon Diagrams of the Metal Binding Site (a) The metal binding pocket of Hp_LuxS with His61, His57, His137, and Cys131 side chains shown as a ball and stick.

Figure 5. Ball and Stick and Ribbon Diagrams of the Metal Binding Site (b) The putative metal binding pocket from threonyl-tRNA synthetase (yellow) is superimposed on the LuxS zinc binding pocket (cyan) shown in (a). Go to “ Metal binding pocket “… Go to “ Metal binding pocket “…

Substrate Binding Site An interesting patch of residual density near the metal binding site was observed. Glu60, Arg68, and Asp80 are highly conserved in the LuxS proteins. The extra density was consistent with a bound methionine molecule. Go to Figure 4… Go to Figure 4…Methionine? There is no evidence that methionine palys an in vivo role as a substrate for LuxS. Indeed, the metheionine side chain is too short to reach the metal site. Go to Figure 4… Go to Figure 4…

Figure 6. Ball and Stick and Ribbon Diagram of the Substrate and Metal Biding Sites

Summary LuxS is a homodimer in solution. LuxS has zinc binding site comprised of two histidines and a cysteine, suggesting that the protein is a zinc metalloenzyme. Observations of a bound mehtionine supports arguments that the LuxS substrate is an amino acid derivative.

Discussion

Evidence for the importance of the homodimer in the function of LuxS is that the methionine ligand binding occurs at the dimer interface. Additionally, channels in the protein that lead to and from the substrate binding site, providing access for substrate and egress for product, are visible. One channel leads through one monomer to the binding site of the other.

Figure 7. Molecular Surface Representations of LuxS Homodimer

The methionine backbone interactions with highly conserved residues in LuxS indicate that the physiological substrate is an amino acid or a derivative thereof. Many signals used by bacteria for intercellular communication are amino acid based. The LuxS substrate is S-ribosylhomocysteine. (Bassler, Molecular Microbiology (2001) 41(2), 463–476)

Figure 8. Modleing of S-ribosylhomocysteine into the Ligand Binding Pocket of One Monomer of Dr_LuxS Back

Biological Implications From genetic studies, LuxS is known to be requires for AI-2 generation. The natural substrate for LuxS is an amino acid derivative and the chemical mechanism for AI-2 synthesis involves cleavage of the ribosyl ring of SRH by zinc.SRHzinc Go to... Go to...These provide the groundwork for mutagenesis experiments to confirm the proposed catalytic action of LuxS. Also, drug candidates can now be explored that will interfere with AI-2 production and act as a multispecies antibiotic. Go to... Go to...

Figure 9. SRH is converted to homocysteine and 4,5-dihydroxy-2,3-pentanedione (AI-2) by LuxS protein. (Bassler, Molecular Microbiology (2001) 41(2), 463–476) Back

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