Screening of Conditions that Facilitate Crystallization of Oligopeptidase B from Serratia Proteamaculans by Differential Scanning Fluorimetry

Petrenko, D. E.; Лазаренко, В. А.; Dorovatovskii, Pavel V.; Тимофеев, В. И.; Vlaskina, Anna V.; Korzhenevskiy, D.A.; Mikhailova, A. G.; Rakitina, Tatiana V.

doi:10.1134/s1063774520020170

Cited by 5 publications

(6 citation statements)

References 12 publications

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“…Previously, using differential scanning fluorimetry (Thermofluor), we found that lowmolecular weight polyamines, e.g., spermine (Sp), could stabilize PSP in solution [34]. Here, we evaluated the Sp influence on thermal denaturation and catalytic activity of PSP and PSPmod.…”

Section: Comparative Analysis Of Physicochemical Features and Enzymatic Activity Of Pspmodmentioning

confidence: 99%

“…Crystallization of oligopeptidase B from S. proteomaculans with modified hinge region and its E125A and S532A mutants are described in [34,35]. Diffraction data from the crystals were collected at the Kurchatov synchrotron (beamline "BELOK") and processed as described in [34,35]. The structures were solved by the molecular replacement method using BALBES program [36].…”

Section: Protein Crystallization Data Collection Processing Structure Refinement and Analysismentioning

confidence: 99%

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

Petrenko

Тимофеев

Britikov

et al. 2021

Biology

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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.

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Section: Comparative Analysis Of Physicochemical Features and Enzymatic Activity Of Pspmodmentioning

confidence: 99%

Section: Protein Crystallization Data Collection Processing Structure Refinement and Analysismentioning

confidence: 99%

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

Petrenko

Тимофеев

Britikov

et al. 2021

Biology

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“…Protein concentrations were over 20.0 mg/mL. The starting crystallization condition was described in [28], and crystallization screening in [29]. Briefly, the crystallization was conducted at 4 • C, the final growth conditions were 0.2 M C 3 H 2 O 4 Na 2 , pH 7.0, 20-24% PEG 3350 (SpOPBmod); 0.2 M Li 2 SO 4 , 0.1 M Bis-Tris, pH 5.5, 27% PEG 3350 (SpOPBS532A).…”

Section: Crystallography X-ray and Structural Analysismentioning

confidence: 99%

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

et al. 2022

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Oligopeptidase B (OPB) is the least studied group from the prolyl oligopeptidase family. OPBs are found in bacteria and parasitic protozoa and represent pathogenesis factors of the corresponding infections. OPBs consist of two domains connected by a hinge region and have the characteristics of conformational dynamics, which include two types of movements: the bridging/separation of α/β-hydrolase catalytic and β-propeller-regulatory domains and the movement of a loop carrying catalytic histidine, which regulates an assembly/disassembly of the catalytic triad. In this work, an elucidation of the interdomain dynamics of OPB from Serratia proteamaculans (SpOPB) with and without modification of the hinge region was performed using a combination of X-ray diffraction analysis and small-angle X-ray scattering, which was complemented with an essential dynamics sampling (EDS) simulation. The first crystal structure of catalytically deficient SpOPB (SpOPBS532A) with an intact hinge sequence is reported. Similarly to SpOPB with modified hinges, SpOPBS532A was crystallized in the presence of spermine and adopted an intermediate conformation in the crystal lattice. Despite the similarity of the crystal structures, a difference in the catalytic triad residue arrangement was detected, which explained the inhibitory effect of the hinge modification. The SpOPBS532A structure reconstituted to the wild-type form was used as a starting point to the classical MD followed by EDS simulation, which allowed us to simulate the domain separation and the transition of the enzyme from the intermediate to open conformation. The obtained open state model was in good agreement with the experimental SAXS data.

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“…Crystallization of PSPmod-TCK complex were carried out in the presence of 5 mM spermine as described in [26,27]. Crystals were grown at 4 • C in 0.2 M Li 2 SO 4 , 0.1 M TrisHCl, pH 8.5, 30% PEG 4000.…”

Section: Crystallography X-ray and Structural Analysismentioning

confidence: 99%

“…Paraton was used for cryoprotection. Diffraction data were collected at the Kurchatov synchrotron (beamline "BELOK") as described in [26,27]. The structure was solved by the molecular replacement method using BALBES program [28].…”

Section: Crystallography X-ray and Structural Analysismentioning

confidence: 99%

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

et al. 2021

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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.

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Screening of Conditions that Facilitate Crystallization of Oligopeptidase B from Serratia Proteamaculans by Differential Scanning Fluorimetry

Cited by 5 publications

References 12 publications

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

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

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

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