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

Abstract

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.

The Crystal Structure of Nα-p-tosyl-lysyl Chloromethylketone-Bound Oligopeptidase B from Serratia Proteamaculans Revealed a New Type of Inhibitor Binding

“…The rate constant of the alkylation step (k2) for PSP is an order of magnitude higher than for PSPmod. These data correspond to the lower hydrolytic activity of PSPmod [11]. For comparison, when trypsin-like enzymes bind one molecule of specific chloromethylketone, the following kinetic parameters of inhibition were obtained: k 2 = 0.78 min −1 , K i = 1.2 µM for trypsin inhibition by D-Val-Phe-Lys-CH 2 C1 [24]; k 2 = 0.78 min −1 , K i = 1.0 µM for the light chain of enterokinase inhibited by Val-(Asp) 4 -Lys-CH 2 C1 [25].…”

“…The rate constant of the alkylation step (k 2 ) for PSP is an order of magnitude higher than for PSPmod. These data correspond to the lower hydrolytic activity of PSPmod [11]. The kinetic parameters of PSP and PSPmod inhibition by TCK determined from the graphs (Figure 2A,B) are presented in Table 2.…”

“…PSPmod-expressing plasmid was obtained as described in [11]. The expression of both recombinant proteins of wild-type PSP and PSPmod were carried out in E. coli BL21(DE3)RIPL (Novagen, Madison, WI, USA).…”

“…The kinetic parameters of the reaction between the enzyme (E) and inhibitor (I) were determined under pseudo-first order conditions ([I] >> [E]) according to Kitz and Wilson [23][24][25]. Recently, we have solved the 3D structures of OpB from bacteria Serratia proteamaculans (PSP) with the hinge region modification (PSPmod) and its derivatives PSPmodS532A and PSPE125A at 2.00, 1.88 and 2.72 Å resolutions, respectively [11]. The hinge region modification caused an inhibitory effect on the PSP catalytic activity but did not affect the basic physicochemical features of the enzyme.…”

“…PSPmod is OpB from S. Proteomaculans with hinge region modification, in which the sequence IPQQEH substituted with TEV protease recognition sequence ENLYFQ [11]. The modification inhibited enzymatic activity but promoted crystallization of the enzyme.…”