Structure Guided Design of Protein Biosensors for Phenolic Pollutants

Phenolic aromatic compounds are a major source of environmental pollution. Currently there are no in situ methods for specifically and selectively detecting these pollutants. Here, we exploit the nature’s biosensory machinery by employing Acinetobacter calcoaceticus NCIB8250 protein, MopR, as a model system to develop biosensors for selective detection of a spectrum of these pollutants. The X-ray structure of the sensor domain of MopR was used as a scaffold for logic-based tunable biosensor design. By employing a combination of in silico structure guided approaches, mutagenesis and isothermal calorimetric studies, we were able to generate biosensor templates, that can selectively and specifically sense harmful compounds like chlorophenols, cresols, catechol, and xylenols. Furthermore, the ability of native protein to selectively sense phenol as the primary ligand was also enhanced. Overall, this methodology can be extended as a suitable framework for development of a series of exclusive biosensors for accurate and selective detection of aromatic pollutants from real time environmental samples

Structural Basis of Selective Aromatic Pollutant Sensing by the Effector Binding Domain of MopR, an NtrC Family Transcriptional Regulator

Phenol and its derivatives are common pollutants that are present in industrial discharge and are major xenobiotics that lead to water pollution. To monitor as well as improve water quality, attempts have been made in the past to engineer bacterial <i>in vivo</i> biosensors. However, due to the paucity of structural information, there is insufficiency in gauging the factors that lead to high sensitivity and selectivity, thereby impeding development. Here, we present the crystal structure of the sensor domain of MopR (MopR^AB) from <i>Acinetobacter calcoaceticus</i> in complex with phenol and its derivatives to a maximum resolution of 2.5 Å. The structure reveals that the N-terminal residues 21–47 possess a unique fold, which are involved in stabilization of the biological dimer, and the central ligand binding domain belongs to the “nitric oxide signaling and golgi transport” fold, commonly present in eukaryotic proteins that bind long-chain fatty acids. In addition, MopR^AB nests a zinc atom within a novel zinc binding motif, crucial for maintaining structural integrity. We propose that this motif is crucial for orchestrated motions associated with the formation of the effector binding pocket. Our studies reveal that residues W134 and H106 play an important role in ligand binding and are the key selectivity determinants. Furthermore, comparative analysis of MopR with XylR and DmpR sensor domains enabled the design of a MopR binding pocket that is competent in binding DmpR-specific ligands. Collectively, these findings pave way towards development of specific/broad based biosensors, which can act as useful tools for detection of this class of pollutants

Steps involved in the ‘context-specific’ bioinformatics study.

<p>The chart is organized in the consecutive major steps labelled as 1 to 7, and it contains four columns; the first column shows the number of protein sequences before and the last column that of after the execution of each step (No seq INPUT and No seq OUTPUT), respectively. The second column shows the description of the steps, the third column the references to the steps, respectively. For details see ‘<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089313#s4" target="_blank">Materials and Methods</a>.’ To carry out these steps, we have written a few Python-routines for the steps 1 through 3 and employed several open access programs (steps in light grey).</p

Crystal Structure of F209W mutant.

<p>(A) mFo-DFc electron density map for Trp209 at 3.0 σ is shown in black mesh (B) Xe3 cavity region of F209W structure is shown in magenta cartoons and StPurL-Xenon complex is shown color coded with FGAM synthetase domain in blue and linker domain in yellow color. Location of xenon atom is depicted as an orange sphere. Select residues with rmsd of more than 1 Å are shown in sticks.</p

Data processing and refinement statistics.

a<p>values for the highest-resolution shell are given in parentheses.</p

Crystallographic statistics.

a<p>Numbers given in brackets are from the last resolution shell.</p>b<p><i>R_sym</i> = (Σ_hklΣ_i|I_i(hkl)-)/Σ_hklΣI_i(hkl), where I_i(hkl) is the intensity of the <i>i</i>th measurement of reflection (hkl) and is the average intensity.</p>c<p><i>R_meas</i> = (Σ_hkl (sqrt(N_hkl/(N_hkl-1))Σ_i|I_i(hkl)-)/Σ_hklΣI_i(hkl), where I_i(hkl) is the intensity of the <i>i</i>th measurement of reflection (hkl) and is the average intensity.</p>d<p><i>R_work</i> = (Σ_hkl|Fo-Fc|/Σ_hklFo where Fo and Fc are the observed and calculated structure factors.</p>e<p><i>R_free</i> is calculated as for <i>R_work</i> but from a randomly selected subset of the data (5%) which were excluded from the refinement <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089313#pone.0089313-Brunger1" target="_blank">[48]</a>.</p>f<p>Ramachandran et al., 1963 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089313#pone.0089313-Ramachandran1" target="_blank">[49]</a>.</p>g<p><i>I</i> is the integrated intensity and σ(<i>I</i>) is the estimated standard deviation of that intensity.</p

Alignments of the WXG100 subfamilies reveal conserved subfamily specific residues and generally conserved C-terminal residues pattern.

<p>(A) The position of helices, according to the structures of ESAT-6, CFP-10, and <i>sag</i>Esx-A, are shown below the alignments of each subfamily. The four-helix bundle requires mostly hydrophobic residues at the position of ‘a’ and ‘d’ of a helix turn consisting of the heptad helix repeat (a-b-c-d-e-f-g), shown as grey shading on the aligned residues. The key features of ‘ESAT-6-like’ subfamily (top panel): Shown are three highly conserved residues besides the almost invariant WXG motif (marked with red triangles), boxed in K/P38 (yellow), Y51 (green) and Q55 (red). Numbering of residues followed those of ESAT-6 (Rv3875). In the ‘CFP-10-like’ subfamily (middle panel), there are almost no conserved features, except for the C-terminal sequence conservation (marked with asterisks, filled with black for hydrophobic residues and unfilled for acidic residues), shared by all WXG100 superfamily members. In the ‘<i>sag</i>Esx-like’ subfamily (bottom panel), all residues involved in the inter-dimer interactions are hydrophobic except two residues, boxed in cyan and black. The gene IDs of the WXG targets are shown on each line. The numbers correspond to the locus of each genes depicted here. The bacterial species out of the phylum “Actinobacteria” are abbreviated as: Mmcs0071: <i>Mycobacterium sp</i>. MCS, Mvan: <i>M. vanbaalenii</i>, ML: <i>M. leprae</i>, jk: <i>Corynebacterium (C.) jeikeium</i>, cur: <i>C. urealyticum</i>, Ncgl: <i>C. glutamicum</i>, DIP: <i>C. diphtheria</i>, Mflv: <i>M.gilvum</i>, Sare: <i>Salinispora (Sa.) arenicola</i>, Strop: <i>Sa. tropica</i>, SACE: <i>Saccharopolyspora erythraea</i>, and those from the phylum “Firmicutes” as: CAC: <i>Clostridium acetobutylicum</i>, BPUM: <i>Bacillus pumilus</i>, GTNG: <i>Geobacillus thermodenitrificans</i>, ABC: <i>alkaliphilic Bacillus clausii,</i> BC: <i>Bacillus cereus</i>, Sez: <i>Streptococcus equi,</i> Lmo: <i>Listeria monocytogenes serovar</i>, SAV: <i>Staphylococcus aureus</i>, SAG: <i>Streptococcus agalactiae</i>, BH: <i>Bacillus halodurans</i>, Cthe: <i>Clostridium thermocellum</i>, respectively. (B) The C-terminal consensus sequence <i>HxxxD/ExxhxxxH</i> is shown as a sequence logo diagram. The residue at the eighth position is marked with ‘<i>h</i>’ indicating lower conservation on hydrophobic residues (see panel A). (C) Structural superposition of CFP-10 (blue), ESAT-6 (cyan), <i>sag</i>EsxA (orange) and <i>sauEsx</i>A (violet): Only the C-terminal helices along with the adjacent WXG loops facing towards helices are shown. For better visibility only the side chains of <i>sag</i>EsxA are shown. (D) The side chains of the conserved C-terminal residues decorate the same side of the C-terminal helix as observed in the structures of the WXG100 proteins, shown is that of <i>sag</i>EsxA (see text), marked with asterisks in panel A. To emphasize the structural feature, the C-terminal helix is shown in a surface representation, where the consensus hydrophobic residues are in grey and the acid residue is in red. The remaining residues (x) are shown in light grey.</p

Structures of <i>sag</i>EsxA and CFP-10/ESAT-6 complexes, and comparisons of the loop conformation, as observed in the three WXG100 proteins.

<p>(A) The four-helix bundle structures of the homodimeric <i>sag</i>EsxA and heterodimeric CFP-10/ESAT-6 complexes are shown. (B) The WXG motif-containing loops of ESAT-6 showing an extended hydrogen-bonding network as indicated by dashed lines and labelled with their hydrogen bond donor-acceptor distances. (C) Comparisons of the loops of CFP-10 and ESAT-6. The asymmetric unit (AU) of CFP-10/ESAT-6 crystal contains two copies of the heterodimer. The view shows down towards the central long axis of the dimer. The relation of top to bottom panel views are 180° rotation around central short axis of the dimer, showing the WXG containing loop of ESAT-6 (top) and that of CFP-10 (bottom). Superimpositions of the structures of the AU content show that the WXG containing loops of ESAT-6 exhibit lower B-values and overlap better than that of CFP-10. (D) A hydrogen bond interaction formed by Y18 and Q38 at the inter-dimer interface of <i>sag</i>EsxA is shown.</p

Xenon binding in StPurL.

<p>(A) StPurL structure is depicted in cartoon with N-terminal domain in green, linker domain in yellow, glutaminase domain in red, and FGAM synthetase domain with structural sub-domains 1 and 2 shown in blue and cyan colors respectively. The two active sites and the auxillary ADP are shown in sticks and Xenon atoms are depicted as orange spheres. In the side panel (B, C and D), mFo-DFc densitiy maps of the three Xenon atoms at 5.0 σ are shown in black mesh and residues forming the cavities around them are shown in sticks.</p

Loop regions around cavity 2.

<p>(A) Glutaminase domain is shown in surface representation in purple color with active site residues, helix α27 and α30 highlighted in cartoon and stick representations. A2 domain of the FGAM synthetase is shown in pink cartoons with the long and short loops in green color. The structurally conserved long loop region of TmPurL is shown in yellow cartoon. ADP is shown in magenta sticks. The β-sheet regions of A1 domain involved in making the β-barrel core of the FGAM synthetase domain are shown in marine blue cartoon representation. Locations of the xenon atoms trapped in the structure are depicted as orange spheres. (B) View of the interface between the FGAM synthetase loop regions and the glutaminase domain showing various hydrogen bonding and van der waals interactions.</p