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

Abstract

A covalent serine protease inhibitor—Na-p-Tosyl-Lysyl Chloromethylketone (TCK) is a modified lysine residue tosylated at the N-terminus and chloromethylated at the C-terminus, one molecule of which is capable of forming two covalent bonds with both Ser and His catalytic residues, was co-crystallized with modified oligopeptidase B (OpB) from Serratia proteomaculans (PSPmod). The kinetics study, which preceded crystallization, shows that the stoichiometry of TCK-dependent inhibition of PSPmod was 1:2 (protein:inhibitor). The crystal structure of the PSPmod-TCK complex, solved at a resolution of 2.3 Å, confirmed a new type of inhibitor binding. Two TCK molecules were bound to one enzyme molecule: one with the catalytic Ser, the other with the catalytic His. Due to this mode of binding, the intermediate state of PSPmod and the disturbed conformation of the catalytic triad were preserved in the PSPmod-TCK complex. Nevertheless, the analysis of the amino acid surroundings of the inhibitor molecule bound to the catalytic Ser and its comparison with that of antipain-bound OpB from Trypanosoma brucei provided an insight in the structure of the PSPmod substrate-binding pocket. Supposedly, the new type of binding is typical for the interaction of chloromethylketone derivatives with two-domain OpBs. In the open conformational state that these enzymes are assumed in solution, the disordered configuration of the catalytic triad prevents simultaneous interaction of one inhibitor molecule with two catalytic residues.

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

“…Previously, crystal structures were obtained for the following mutated forms of SpOpB: catalytically inactive SpOpB_S532A, carrying the alanine substitution of the catalytic residue S532 (PDB ID 7ZJZ), and low-active enzymes SpOpBmod, in which the first hinge peptide (residues 71-77, IPQQEH) was replaced by the tobacco etching virus protease site (ENLYFQ) [8,22,23]. SpOpBmod was crystallized in both the inhibitor-free form (PDB ID 7OB1, 7YWS, and 7YX7) and in the TCK-bound form (PDB ID 7NE7).…”

“…TCK was added to SpOpB dropwise with constant stirring at +4 • C until the final concentration of 1 mM (stoichiometric ratio protein:inhibitor was about 1:4). The loss of the catalytic activity was monitored by hydrolysis of Nα-benzoyl-D,L-arginine-p-nitroanilide (BAPNA) (Sigma-Aldrich, St. Louis, MI, USA) as described in [22]. In brief, we measure the increase in the absorption at 405 nm (0.1 M Tris-HCl, pH 8.0, 2% DMSO, 25 • C), which occurred due to the formation of free p-nitroaniline (∆ε405 = 10,400 M -1 •cm -1 ).…”

“…In addition, bacterial OpBs play an important role in the resistance to non-lytic proline-rich antimicrobial peptides, which are usually enriched with basic amino acid residues [21]. Nevertheless, the restricted distribution of OpBs caused a delay in their structural studies: spatial structures were solved only for two protozoan enzymes (TbOpB and LmOpB) [7,9] and for modified (mutated) derivatives of SpOpB in the intermediate state [8,22,23]. At the same time, numerous structures of both inhibitor-bound and free POPs from nearly a dozen species, including those from mammals, bacteria, archaea, fungi, and mollusks, were solved [4][5][6]10,11,15,24,25].…”

Elucidation of the Conformational Transition of Oligopeptidase B by an Integrative Approach Based on the Combination of X-ray, SAXS, and Essential Dynamics Sampling Simulation

“…The propeller domain performs a regulatory function, blocking access to the active center to bulk substrates larger than 30 amino acids. Such an architecture allows the enzymes to adopt different conformations, which were discovered by X-ray diffraction (XRD) studies [8][9][10][11][12][13][14][15][16] (Figure 1B).…”

“…Three conformations of POPs include a closed (catalytically active) state, in which the domains and residues of the catalytic triad are brought together [8][9][10][11][12][13], an open (inactive), in which the domains and residues of the catalytic triad are spatially separated [8][9][10][11][12], and an intermediate state, which combines the closeness of the domains with the disassembly of the catalytic triad [14][15][16]. In the absence of ligands, the bacterial PEPs and protozoan OPBs were captured in the open conformations [8,9,12], while the binding of inhibitors caused a transition to the closed states [8,9,12,13].…”