Structural Analysis of Papain-Like NlpC/P60 Superfamily Enzymes with a Circularly Permuted Topology Reveals Potential Lipid Binding Sites

NlpC/P60 superfamily papain-like enzymes play important roles in all kingdoms of life. Two members of this superfamily, LRAT-like and YaeF/YiiX-like families, were predicted to contain a catalytic domain that is circularly permuted such that the catalytic cysteine is located near the C-terminus, instead of at the N-terminus. These permuted enzymes are widespread in virus, pathogenic bacteria, and eukaryotes. We determined the crystal structure of a member of the YaeF/YiiX-like family from Bacillus cereus in complex with lysine. The structure, which adopts a ligand-induced, “closed” conformation, confirms the circular permutation of catalytic residues. A comparative analysis of other related protein structures within the NlpC/P60 superfamily is presented. Permutated NlpC/P60 enzymes contain a similar conserved core and arrangement of catalytic residues, including a Cys/His-containing triad and an additional conserved tyrosine. More surprisingly, permuted enzymes have a hydrophobic S1 binding pocket that is distinct from previously characterized enzymes in the family, indicative of novel substrate specificity. Further analysis of a structural homolog, YiiX (PDB 2if6) identified a fatty acid in the conserved hydrophobic pocket, thus providing additional insights into possible function of these novel enzymes

A conserved fold for fimbrial components revealed by the crystal structure of a putative fimbrial assembly protein (BT1062) from Bacteroides thetaiotaomicron at 2.2 Å resolution

The crystal structure of BT1062 from Bacteroides thetaiotaomicron revealed a conserved fold that is widely adopted by fimbrial components

Structures of the first representatives of Pfam family PF06684 (DUF1185) reveal a novel variant of the Bacillus chorismate mutase fold and suggest a role in amino-acid metabolism

Structures of the first representatives of PF06684 (DUF1185) reveal a Bacillus chorismate mutase-like fold with a potential role in amino-acid synthesis

Structure and functional characterization of a bile acid 7α dehydratase BaiE in secondary bile acid synthesis

Conversion of the primary bile acids cholic acid (CA) and chenodeoxycholic acid (CDCA) to the secondary bile acids deoxycholic acid (DCA) and lithocholic acid (LCA) is performed by a few species of intestinal bacteria in the genus Clostridium through a multistep biochemical pathway that removes a 7α-hydroxyl group. The rate-determining enzyme in this pathway is bile acid 7α-dehydratase (baiE). In this study, crystal structures of apo-BaiE and its putative product-bound [3-oxo-Δ4,6-lithocholyl-Coenzyme A (CoA)] complex are reported. BaiE is a trimer with a twisted α+β barrel fold with similarity to the Nuclear Transport Factor 2 (NTF2) superfamily. Tyr30, Asp35, and His83 form a catalytic triad that is conserved across this family. Site-directed mutagenesis of BaiE from Clostridium scindens VPI 12708 confirm that these residues are essential for catalysis and also the importance of other conserved residues, Tyr54 and Arg146, which are involved in substrate binding and affect catalytic turnover. Steady-state kinetic studies reveal that the BaiE homologs are able to turn over 3-oxo-Δ4-bile acid and CoA-conjugated 3-oxo-Δ4-bile acid substrates with comparable efficiency questioning the role of CoA-conjugation in the bile acid metabolism pathway. Proteins 2016; 84:316-331

Data collection, phasing and refinement statistics for BcPPNE (PDB 3kw0).

a<p>Highest resolution shell in parentheses. The high resolution cutoff was chosen such that the mean I/σ(I) in the highest resolution shell is around 2. These statistics were calculated assuming the equivalence of Friedel pairs.</p><p>ESU = Estimated Standard Uncertainty in atomic coordinates.</p><p>R_merge = Σ_hkl Σ_i|I_i(hkl)−|/Σ_hklΣ_iI_i(hkl), R_meas (redundancy-independent R_merge) = Σ_hkl[N_hkl/(N_hkl−1)]^1/2 Σ_i|I_i(hkl)−|/Σ_hkl Σ_iI_i(hkl), and R_pim (precision-indicating R_merge) = Σ_hkl[1/(N_hkl−1)]^1/2 Σ_i|I_i(hkl)−|/Σ_hkl Σ_iI_i(hkl).</p><p>R_cryst = Σ_hkl||F_obs|−|F_calc||/Σ_hkl|F_obs| where F_calc and F_obs are the calculated and observed structure factor amplitudes, respectively.</p><p>R_free = as for R_cryst, but for 5.0% of the total reflections chosen at random and omitted from refinement.</p

PPNEs have hydrophobic S1 binding pockets.

<p>The S1 binding pockets of YiiX, BcPPNE and PPPDE1 are shown as semi-transparent spheres overlaying ribbon representations, with hydrophobic and aromatic residues colored as gray, hydrophilic residues green and the catalytic cysteines in red.</p

Common core of NlpC/P60 superfamily.

<p>(a) Structure comparison between BcPPNE (left) and CPNE BcYkfC (right, PDB 3h41). The N-terminal subdomains of both proteins are colored grey, and the C-terminal subdomains in violet. The catalytic residues are shown as ball-and-sticks. The sites of the circular permutation are indicated by residue numbers. (b) Core conserved secondary structures within the NlpC/P60 superfamily. C_α traces for the conserved cores of BcPPNE (green), YiiX (cyan), BcYkfC (yellow), PPPDE1 (magenta) the conserved residues are superposed. Secondary structures of BcPPNE (green) and catalytic residues of YiiX (sticks) are highlighted. (c) Sequence alignment between PPNEs (BcPPNE, YiiX, human PPPDE1, vaccinia virus G6R, human LRAT), and CPNE BcYkfC. Sequence numbering and secondary structures of BcPPNE are shown at the top. The 3₁₀ helices are shown in red and other secondary structures are labeled as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022013#pone-0022013-g001" target="_blank">Fig. 1</a>. Sequences of the two subdomains of BcYkfC are swapped in comparison to the others and the respective residue ranges are shown near the ends of the permuted segments.</p

Crystal structure of BcPPNE.

<p>Stereoview of ribbon representation of BcPPNE is color coded from blue (N-terminus) to red (C-terminus). The α-helices are labeled A to G, and β-strands 1 to 6. The bound lysine and active site catalytic residues are shown as sticks. The loop that unites the circular permuted sub-domains (residues 103–110) is colored grey. The 3₁₀ helices are not labeled.</p