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

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 ß-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 ß-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.

Highly Similar Sequences of Mature IgA1 Proteases from Neisseria meningitidis, Neisseria gonorrhoeae and Haemophilus influenzae.

The mature serine-type IgA1 protease from Neisseria meningitidis serogroup B strain H44/76 (IgA1pr1_28-1004) is considered here as the basis for creating a candidate vaccine against meningococcal meningitis. In this work, we examine the primary structure similarity of IgA1 proteases from various strains of a number of Gram-negative bacteria (N. meningitidis, Neisseria gonorrhoeae, Haemophilus influenzae) in order to find a structural groundwork for creating a broad-spectrum vaccine based on fragments of this enzyme. BLAST has shown high similarity between the primary structure of IgA1pr1_28-1004 and hypothetical sequences of mature IgA1 proteases from N. meningitidis (in 1060 out of 1061 examined strains), N. gonorrhoeae (in all 602 examined strains) and H. influenzae (in no less than 137 out of 521 examined strains). For these enzymes, common regions of sequence correspond to IgA1pr1_28-1004 fragments 28-84, 146-193, 253-539, 567-628, 639-795 and 811-1004, with identity of at least 85%. We believe that these fragments can be used in the development of a vaccine to prevent diseases caused by pathogenic strains of N. meningitidis and N. gonorrhoeae as well as a significant number of strains of H. influenzae.

Molecular dynamics complemented by site-directed mutagenesis reveals significant difference between the interdomain salt bridge networks stabilizing oligopeptidases B from bacteria and protozoa in their active conformations.

Oligopeptidases B (OpdBs) are trypsin-like peptidases from protozoa and bacteria that belong to the prolyl oligopeptidase (POP) family. All POPs consist of C-terminal catalytic domain and N-terminal ß-propeller domain and exist in two major conformations: closed (active), where the domains and residues of the catalytic triad are positioned close to each other, and open (non-active), where two domains and residues of the catalytic triad are separated. The interdomain interface, particularly, one of its salt bridges (SB1), plays a role in the transition between these two conformations. However, due to double amino acid substitution (E/R and R/Q), this functionally important SB1 is absent in γ-proteobacterial OpdBs including peptidase from Serratia proteamaculans (PSP). In this study, molecular dynamics was used to analyze inter- and intradomain interactions stabilizing PSP in the closed conformation, in which catalytic H652 is located close to other residues of the catalytic triad. The 3D models of either wild-type PSP or of mutant PSPs carrying activating mutations E125A and D649A in complexes with peptide-substrates were subjected to the analysis. The mechanism that regulates transition of H652 from active to non-active conformation upon domain separation in PSP and other γ-proteobacterial OpdB was proposed. The complex network of polar interactions within H652-loop/C-terminal α-helix and between these areas and ß-propeller domain, established in silico, was in a good agreement with both previously published results on the effects of single-residue mutations and new data on the effects of the activating mutations on each other and on the low active mutant PSP-K655A.Communicated by Ramaswamy H. Sarma.

Activity modulation of the oligopeptidase B from Serratia proteamaculans by site-directed mutagenesis of amino acid residues surrounding catalytic triad histidine.

Oligopeptidase B (OpdB; EC 3.4.21.83) is a trypsin-like peptidase belonging to the family of serine prolyl oligopeptidases; two-domain structure of the enzyme includes C-terminal peptidase catalytic domain and N-terminal seven-bladed ß-propeller domain. Importance of the interface between these domains and particularly of the 5 salt bridges for enzyme activity was established for protozoan OpdBs. However, these salt bridges are not conserved in γ -proteobacterial OpdBs including the peptidase from Serratia proteamaculans (PSP). In this work, using comparative modelling and protozoan OpdBs' crystal structures we created 3D models of PSP in open and closed forms to elucidate the mechanism underlying inactivation of the truncated form of PSP1-655 obtained earlier. Analysis of the models shows that in the closed form of PSP charged amino acid residues of histidine loop, surrounding the catalytic triad His652, participate in formation of the inter-domain contact interface between catalytic and ß-propeller domains, while in the open form of PSP disconnection of the catalytic triad and distortion of these contacts can be observed. Complete destruction of this interface by site-directed mutagenesis causes inactivation of PSP while elimination of the individual contacts leads to differential effects on the enzyme activity and substrate specificity. Thus, we identified structural factors regulating activity of PSP and supposedly of other γ-proteobacterial OpdBs and discovered the possibility of directed modulation of their enzymatic features.

Complement C1q-target proteins recognition is inhibited by electric moment effectors.

Classical complement pathway is an important innate immune mechanism, which is usually triggered by binding of C1q to immunoglobulins, pentraxins and other target molecules. Although the activation of the classical pathway is crucial in the host defence, its undesirable and uncontrolled activation can lead to tissue damage. Thus, understanding the molecular basis of complement activation and its inhibition are of great biomedical importance. Recently, we proposed a mechanism for target recognition and classical pathway activation by C1q, which is likely governed by calcium-controlled reorientation of macromolecular electric moment vectors. Here we sought to define the mechanism of C1q inhibition by low molecular weight disulphate compounds that bind to the globular (gC1q) domain, using experimental, computational docking and theoretical modelling approaches. Our experimental results demonstrate that betulin disulphate (B2S) and 9,9-bis(4'-hydroxyphenyl)fluorene disulphate (F2S) inhibit the interaction of C1q and its recombinant globular modules with target molecules IgG1, C-reactive protein (CRP) and long pentraxin 3 (PTX3). In most C1q-inhibitor docked complexes, there is a reduction of electric moment scalar values and similarly altered direction of electric/dipole moment vectors. This could explain the inhibitory effect by impaired electrostatic steering, lacking optimal target recognition and formation of functional complex. In the presence of the inhibitor, the tilt of gC1q domains is likely to be blocked by the altered direction of the electric moment vector. Thus, the transition from the inactive (closed) towards the active (open) conformation of C1q (i.e. the complement activation signal transmission) will be impaired and the cascade initiation disrupted. These results could serve as a starting point for the exploration of a new form of 'electric moment inhibitors/effectors'.