The results of investigation of DNA-protein complexes by two independent structural methods, namely, molecular dynamics (MD) and small-angle X-ray scattering (SAXS), were compared. Molecular dynamics is a computational method enabling the visualization of the behavior of macromolecules in real media, which is based on the laws of physics, but it is limited by numerous simplifications. Small-angle X-ray scattering is an X-ray method, which allows the reconstruction of the three-dimensional structure of a system in solution from a one-dimensional small-angle scattering profile, but it faces the problem of ambiguity in solving inverse problems. The use of structural characteristics of the complexes determined by SAXS for the validation of 3D structural models generated by MD simulations made it possible to significantly reduce the ambivalence of theoretical predictions and demonstrated the efficiency of a combination of MD simulations and SAXS for solving problems of structural biology.

Myelin basic protein (MBP) is the second most abundant protein in the central nervous system and is responsible for structural maintenance of the myelin sheath covering axons. Previously, we showed that MBP has a more proactive role in the oligodendrocyte homeostasis, interacting with membrane-associated proteins, including integral membrane protein 2B (ITM2B or Bri2) that is associated with familial dementias. Here, we report that the molecular dynamics of the in silico-generated MBP-Bri2 complex revealed that MBP covers a significant portion of the Bri2 ectodomain, assumingly trapping the furin cleavage site, while the surface of the BRICHOS domain, which is responsible for the multimerization and activation of the Bri2 high-molecular-weight oligomer chaperone function, remains unmasked. These observations were supported by the co-expression of MBP with Bri2, its mature form, and disease-associated mutants, which showed that in mammalian cells, MBP indeed modulates the post-translational processing of Bri2 by restriction of the furin-catalyzed release of its C-terminal peptide. Moreover, we showed that the co-expression of MBP and Bri2 also leads to an altered cellular localization of Bri2, restricting its membrane trafficking independently of the MBP-mediated suppression of the Bri2 C-terminal peptide release. Further investigations should elucidate if these observations have physiological meaning in terms of Bri2 as a MBP chaperone activated by the MBP-dependent postponement of Bri2 membrane trafficking.

This study aims to perform a comparative analysis of the interfaces between monomers in the dimers of bacterial histone-like DNA-binding proteins (HU proteins) and investigate the correlation between the thermal stability and the binding free energy of monomers in the dimers. The interfaces were analyzed using the PDBePISA server, and the binding free energy of monomers in the dimers (Δ G dim ) was calculated by the MM-GBSA method based on a combination of molecular mechanics and the generalized Born equation. It was demonstrated that the increase in Δ G dim of the HU protein from the mycoplasma Spiroplasma melliferum (HUSpM) correlates well with the increase in the thermal stability. It was found that there is a correlation between the decrease in the thermal stability of mutant variants of HUSpM, which bear alanine substitutions at the interface between monomers in the dimer, and the calculated Δ G dim values for the corresponding mutants. The results of this study confirm the applicability of the MM-GBSA method to the prediction of the thermal stability of histone-like proteins and the effect of single amino acid substitutions at the interface between monomers on the thermal stability of mutant proteins.

In order to elucidate the effect of modification of the hinge region on structural polymorphism associated with conformational transitions, structural studies of hinge-modified oligopeptidase B from Serratia proteamaculans (SpOpBmod) in the crystalline state and solution were carried out. A new crystal structure of SpOpBmod in the intermediate conformation was obtained, and a molecular model of SpOpBmod in the open conformation was created using a combination of small-angle X-ray scattering with MD simulations. The improved electron density of the mobile H-loop carrying the catalytic H652 distinguished the obtained crystal structure from that which was previously reported. Good electron density in this region was previously found only in the inhibitor-bound SpOpBmod structure, in which one of the inhibitor molecules was covalently bound to H652. Comparison of the above structures of free and inhibitor-bound enzymes showed that both tertiary folds are the result of the internal conformational dynamics of SpOpBmod, which were captured by inhibitor binding. Comparison of the SpOpBmod structures with the structures of the same enzyme with a native hinge peptide made it possible to establish the influence of hinge modification on the rearrangement of the interdomain interface during conformational transitions. The above analysis also used models of native and hinge-modified enzymes in open conformations. We found that the interdomain interface observed in the crystal structures of hinge-modified enzymes could be considered an extreme version of the H-loop arrangement, in which closure of the domains does not lead to the assembly of the catalytic triad, whereas the intermediate conformation observed in the structure of the enzyme with the native hinge sequence illustrates a productive transition to the catalytically active closed conformation.

Nucleoid-associated proteins (NAPs) control the structure and functions of bacterial nucleoid. Histone-like HU proteins are most abundant NAPs in dividing bacterial cells. Previously, structural ensembles of conformations of HU proteins from pathogenic mycoplasmas Spiroplasma melliferum and Mycoplasma gallisepticum were obtained using NMR spectroscopy. A structural study of these mycoplasma proteins is performed by small-angle X-ray scattering (SAXS). The occurrence of individual conformations from the ensemble, obtained by NMR, is estimated from the scattering data on HU protein solutions. In particular, an approach based on characterization of equilibrium mixtures in terms of volume fractions of their components was applied. The general shape of the proteins and their oligomeric state are independently confirmed using ab initio bead modelling. The flexibility of DNA-binding protein domains is analyzed by the ensemble optimization method, which is based on comparison of the structural characteristics of conformations fitting the SAXS data to the distribution of these characteristics in a randomly generated set. The results obtained give a new insight on the variability of the structure of HU proteins, which is necessary for their functioning.

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 β-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 β-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.

The determination of the three-dimensional structures of viral proteins is a necessary step both for understanding the mechanisms of virus pathogenicity and for developing methods to combat viral infections. This study aimed to explore the folding and oligomeric state of the major component of the virion surface of the tick-borne encephalitis virus (TBEV), the ectodomain of the envelope E protein (ectoE), which was expressed in E. coli in a soluble form and purified from inclusion bodies as a mixture of dimeric and monomeric forms. The time-dependent assembly of monomers into dimers was detected using size-exclusion chromatography. An X-ray diffraction study of the ectoE crystals grown at pH 4.5 confirmed the dimeric folding of the recombinant protein typical for ectoE. The ability of ectoE dimers to self-assemble into tetramers was detected via small-angle X-ray scattering (SAXS) in combination with molecular dynamics. Such self-assembly occurred at protein concentrations above 4 mg/mL and depended on the pH of the solution. In contrast to stable, specific dimers, we observed that tetramers were stabilized with weak intermolecular contacts and were sensitive to environmental conditions. We discovered the ability of ectoE tetramers to change conformation under crystallization conditions. These results are important for understanding the crystallization process of viral proteins and may be of interest for the development of virus-like particles.

Genetically encoded monomeric blue-to-red fluorescent timers (mFTs) change their fluorescent color over time. mCherry-derived mFTs were used for the tracking of the protein age, visualization of the protein trafficking, and labeling of engram cells. However, the brightness of the blue and red forms of mFTs are 2–3- and 5–7-fold dimmer compared to the brightness of the enhanced green fluorescent protein (EGFP). To address this limitation, we developed a blue-to-red fluorescent timer, named mRubyFT, derived from the bright mRuby2 red fluorescent protein. The blue form of mRubyFT reached its maximum at 5.7 h and completely transformed into the red form that had a maturation half-time of 15 h. Blue and red forms of purified mRubyFT were 4.1-fold brighter and 1.3-fold dimmer than the respective forms of the mCherry-derived Fast-FT timer in vitro. When expressed in mammalian cells, both forms of mRubyFT were 1.3-fold brighter than the respective forms of Fast-FT. The violet light-induced blue-to-red photoconversion was 4.2-fold less efficient in the case of mRubyFT timer compared to the same photoconversion of the Fast-FT timer. The timer behavior of mRubyFT was confirmed in mammalian cells. The monomeric properties of mRubyFT allowed the labeling and confocal imaging of cytoskeleton proteins in live mammalian cells. The X-ray structure of the red form of mRubyFT at 1.5 Å resolution was obtained and analyzed. The role of the residues from the chromophore surrounding was studied using site-directed mutagenesis.

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.

This study is devoted to the modeling and thermodynamic analysis of enzyme–substrate complexes. The complexes consist of wild-type oligopeptidase B (ОрВ) from the bacterium Serratia proteamaculans (PSP) or its mutants with E125A and D649A substitutions, resulting in an increase in the efficiency of the catalysis and a change in the secondary substrate specificity, respectively, and the peptide substrates GRRG and GKRG containing arginine at the P1 position and arginine or lysine at the Р2 positions, respectively. The binding free energies of the peptide substrates in the active sites of the enzymes were calculated by the molecular mechanics with generalized Born surface area (MM-GBSA) method and compared with the experimental hydrolysis efficiencies (kcat/Km) for the corresponding enzyme–substrate pairs. It was shown that the binding free energy for the PSP/GRRG complex is almost two times higher compared to that for the PSP/GKRG complex, which correlates well with the 2.7-fold higher hydrolysis efficiency. The amino-acid substitutions E125A and D649A led to an increase in the binding free energy for both types of complexes, which corresponds to a higher catalytic efficiency of PSP-E125A for both substrates and of PSP-D649A for the lysine-containing substrate compared to wild-type PSP. It was demonstrated that the MM-GBSA method allows the prediction of the specificity of bacterial ОрВs for peptide substrates and also of the effect of amino-acid substitutions on the hydrolysis efficiency. Therefore, this method can be used to study the mechanisms of catalytic activation and to model low-molecular-weight inhibitors of bacterial ОрВs.

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.

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.