The X‐ray crystal structure of PA3566 from Pseudomonas aureginosa at 1.8 Å resolution

Introduction. The structure of the PA3566 protein from Pseudomonas aureginosa was determined at 1.78 Å resolution using the Se-Met multiwavelength anomalous diffraction (MAD) technique. The PA3566 gene encodes a small, conserved protein of unknown function, with a predicted molecular weight of 11.2 kDa (104 amino acids). The protein belongs to a family of conserved proteins of unknown function [COG 1359, Fig. 1(a), top six sequences], but has low sequence identity to any known structures. There is an annotational link to antibiotic biosynthesis monooxygenases (pfam 03992). The PA3566 protein crystallizes as a trimer, although the functional unit is most likely a dimer, as are other members of this structural superfamily. A single monomer assumes a two-layer sandwich fold ( 1 1 2 3 2 3 4) shared by a large number of proteins (CATH number 3.30.70). Helices 2 and 3 form one long helix, with a 74° kink in the middle. The two motifs form a ferredoxin-like split -fold. The four -strands form an antiparallel -sheet with the 1 strand lying between the 3 and 4 strands [Fig. 1(b)] and the three -helices cover one side of the molecule. The monomer is elongated with dimensions of 43 37 25 Å [Fig. 1(b)]. The 310 helix from residues 81–84 is not present in all monomers. The loop from 75–83 in monomer C is highly disordered, resulting in the disordering of part of a3 and the short 310 helix found in monomers A and B. This region contains none of the conserved residues [Fig. 1(a)], but is involved in the contacts between monomers A and B. This suggests that this loop is likely flexible in solution, but stabilized in one position through the crystal contacts. It is possible that the 310 helix is a crystallographic artefact caused by the contacts between the two monomers. There are four turns located in the protein, as defined by DSSP. The turns incorporating residues 14–17 and 35–38 are classical type II turns, while the turn incorporating residues 49–52 is a type I turn and the one at residues 78–81 is a type I turn, based on the turns defined by Hutchinson and Thornton. The monomers B and C form a likely biologically relevant dimer in the asymmetric unit [Fig. 1(c)], while monomer A forms a dimer with a related A subunit through a crystallographic twofold axis. Gel filtration results confirmed that the PA3566 protein is a dimer in solution (see Methods). The dimer buries 1146.2 Å surface area of each monomer and includes 17 hydrogen bonds and two salt bridges. The dimer is predominantly stabilized by contacts along the edge of the -sheets (contacts with all strands, but longest stretches with 2 and 4), plus contacts between the C-terminus and helix 1. The highly conserved residues Arg33 and His44 are buried in the dimer interface. The conserved His44 is noteworthy because previous work shows that this residue is critical for proper folding and thus activity of structurally similar proteins. Arg33 forms a water-mediated hydrogen bond network across the dimer interface, composed of Arg33, two waters, and the carbonyl of Leu96 from the other monomer. Arg33 is also involved in crystal contacts through bridging water molecules. Phe53, an absolutely conserved residue, forms part of a small hydrophobic pocket with the highly conserved residues Ile11, Leu21, and Leu43. This pocket docks the conserved hydrophobic residue Leu96 (also found as Ile or Val) [Fig. 1(a)] of the other monomer, burying 137 Å of the residue and accounting for 12% of the interface. Salt bridges are formed between Arg47 and Asp62 and Asp42 and Arg95 , although neither of these salt bridges appears to be highly conserved across this family of proteins, as all four residues are highly variable [Fig. 1(a)]. A search of the Protein Data Bank using the Dali server yields eight structures with a Z-score over 7, and two of these with a Z-score over 8. The best matches (with Z-scores of 11.5 and 10.3, respectively) are protein yjcs (PDB code: 1Q8B), of unknown function from Bacillus subtilis and ActVA-Orf6 (PDB code: 1LQ9), a monooxygenase involved in actinorhodin biosynthesis from Streptomyces coelicolor [Fig. 1(c)]. The sequence identity between