First Crystal Structure of Bacterial Oligopeptidase B in an Intermediate State: The Roles of the Hinge Region Modification and Spermine

Simple Summary Oligopeptidase B is a two-domain, trypsin-like peptidase from parasitic protozoa and bacteria which belongs to the least studied group of prolyloligopeptidases. In this study, we describe for the first time a crystal structure of bacterial oligopeptidase B and compare it with those of protozoan oligopeptidases B and related prolyloligopeptidases. The enzyme was crystallized in the presence of spermine and contained a modified sequence of the interdomain linker. Both factors were key for crystallization. The structure showed an uncommon intermediate conformation with a domain arrangement intermediate between open and closed conformations found in the crystals of ligand-free and inhibitor-bound prolyloligopeptidases, respectively. To evaluate the impact of the modification and spermine in the obtained conformation, small-angle X-ray scattering was applied, which showed that in solution wild-type enzymes adopt the open conformation and spermine causes a transition to the intermediate state, while the modification is associated with a partial transition. We suggest that spermine-dependent conformational transition replicates the behavior of the enzyme in bacterial cells and the intermediate state, which is rarely detected in vitro, and might be widely distributed in vivo, and so should be considered during computational studies, including those aimed wanting to develop the small molecule inhibitors targeting prolyloligopeptidases. Oligopeptidase B (OpB) is a two-domain, trypsin-like serine peptidase belonging to the S9 prolyloligopeptidase (POP) family. Two domains are linked by a hinge region that participates in the transition of the enzyme between two major states-closed and open-in which domains and residues of the catalytic triad are located close to each other and separated, respectively. In this study, we described, for the first time, a structure of OpB from bacteria obtained for an enzyme from Serratia proteomaculans with a modified hinge region (PSPmod). PSPmod was crystallized in a conformation characterized by a disruption of the catalytic triad together with a domain arrangement intermediate between open and closed states found in crystals of ligand-free and inhibitor-bound POP, respectively. Two additional derivatives of PSPmod were crystallized in the same conformation. Neither wild-type PSP nor its corresponding mutated variants were susceptible to crystallization, indicating that the hinge region modification was key in the crystallization process. The second key factor was suggested to be polyamine spermine since all crystals were grown in its presence. The influences of the hinge region modification and spermine on the conformational state of PSP in solution were evaluated by small-angle X-ray scattering. SAXS showed that, in solution, wild-type PSP adopted the open state, spermine caused the conformational transition to the intermediate state, and spermine-free PSPmod contained molecules in the open and intermediate conformations in dynamic equilibrium.