Crystal Structure of Inhibitor-Bound Bacterial Oligopeptidase B in the Closed State: Similarity and Difference between Protozoan and Bacterial Enzymes

Abstract

The crystal structure of bacterial oligopeptidase B from Serratia proteamaculans (SpOpB) in complex with a chloromethyl ketone inhibitor was determined at 2.2 Å resolution. SpOpB was crystallized in a closed (catalytically active) conformation. A single inhibitor molecule bound simultaneously to the catalytic residues S532 and H652 mimicked a tetrahedral intermediate of the catalytic reaction. A comparative analysis of the obtained structure and the structure of OpB from Trypanosoma brucei (TbOpB) in a closed conformation showed that in both enzymes, the stabilization of the D-loop (carrying the catalytic D) in a position favorable for the formation of a tetrahedral complex occurs due to interaction with the neighboring loop from the β-propeller. However, the modes of interdomain interactions were significantly different for bacterial and protozoan OpBs. Instead of a salt bridge (as in TbOpB), in SpOpB, a pair of polar residues following the catalytic D617 and a pair of neighboring arginine residues from the β-propeller domain formed complementary oppositely charged surfaces. Bioinformatics analysis and structural modeling show that all bacterial OpBs can be divided into two large groups according to these two modes of D-loop stabilization in closed conformations.

How the Hinge Region Affects Interactions between the Catalytic and β-Propeller Domains in Oligopeptidase B

“…The distance between the guanidino and carboxyl groups of the corresponding residues was 2.77 Å. This salt bridge was absent in structures of enzymes with intact hinge regions crystallized in either intermediate or closed conformation (PDB ID 7ZJZ and 7YWP) [17,24]. This salt bridge appears to be one of the reasons for the loss of activity observed for the enzyme with a modified hinge region (see Section 3.3 for a detailed explanation).…”

“…As a starting model for the closed conformation of SpOpBmod, we used the crystal structure of SpOpB in a complex with TCK (PDB ID 7YWP, [17]), without TCK, and with the replacement of the first hinge peptide sequence with the sequence of the TEV protease site.…”

“…The polypeptide chain was folded similarly to previously determined structures of oligopeptidases in intermediate conformations, including the structures of free SpOpBmod (PDB ID 7OB1) [15], free SpOpB-S532A (PDB ID 7ZJZ) [24], and TCK-bound SpOpBmod (PDB ID 7NE7) [23]. Comparison of the above structures gave RMSD values for Cα-atoms ranging from 0.3 to 0.8 Å, whereas comparison of the new structure with that of TCK-bound SpOpB in a closed conformation (PDB ID 7YWP) [17] gave an RMSD of 1.8 Å. Five spermine molecules were detected in the interdomain cavity of SpOpBmod, whereas one spermine molecule was located outside the interdomain cavity and participated in the crystal lattice formation (Figure 2A).…”

“…The same crystal structures and structural models that were used for the comparative analysis of the interdomain interface were taken, but the hinge-modified enzyme in the intermediate conformation was presented only by the SpOpBmod structure. The crystal structure of the closed conformation obtained for SpOpB in complex with TCK (PDB ID 7YWP) [17] was added to the comparison.…”

“…A closed conformation, in which the catalytic triad is assembled and the domains are closed, has been found in crystals of inhibitor-bound POPs from bacteria and OpBs from protozoa [12][13][14]16,17]. Mammalian POPs (both free and inhibitor-bound) always crystallize in the closed conformation [11,18], although a number of studies have suggested that mammalian POPs also exhibit conformational transitions similar to those observed in bacterial POPs [19][20][21].…”