Single amino acid substitutions in the ATP-binding domain of ACVRL1, a key receptor in the bone morphogenetic protein (BMP) signaling pathway, are frequently classified as variants of uncertain significance (VUS), complicating molecular diagnosis for pulmonary arterial hypertension (PAH) and Hereditary Hemorrhagic Telangiectasia (HHT). Since aberrant ATP binding disrupts downstream SMAD1/5/8 phosphorylation, we employed molecular dynamics (MD) simulations to quantitatively assess the functional impact of these variants. We first validated our approach on 20 known pathogenic/likely pathogenic variants within 5Å of the ATP-binding site, finding that 18 (90%) caused significant alterations in binding affinity (|d| ≥ 0.8, p < 0.001). We then applied this protocol to all known VUS, conflicting, and unclassified variants within the same region, reclassifying 20 of 32 (63%) as likely pathogenic. Comprehensive in silico mutagenesis of all possible substitutions at ATP-binding pocket positions, combined with InterVar classification under HHT phenotype, enabled reclassification of 9 of 12 (75%) VUS as likely pathogenic. Finally, we demonstrated the applicability of this approach in two PAH patients with HHT carrying ACVRL1 VUS. This work establishes MD simulation of ATP-binding affinity as an effective and scalable tool for the functional interpretation of kinase variants, with broad potential for application across other disease-associated kinases.

Background Analysis of existing mutations of Angiotensin-I-Converting Enzyme (ACE) led us to hypothesize that the carriers of damaging ACE mutations (accompanied by low ACE levels) could be at risk for the development of late-onset Alzheimer’s disease (AD). Methodology/Principal findings We quantified blood ACE levels in EDTA-containing plasma from 15 patients with 11 different heterozygous ACE mutations and estimated the effects of these mutations on ACE phenotypes, using a set of mAbs to ACE and two ACE substrates. We confirmed prior observations that the relatively frequent Y215C mutation in the N domain of ACE (present in ~1% of the population) is associated with both Alzheimer’s disease (AD) and reduced plasma levels of ACE (~50% of controls), indicating that it likely results in a transport-deficient protein. In addition, we identified another 4 mutations in both ACE domains (M118T, C734Y, V992M and V997M) which are also associated with decreased ACE levels in the blood, and, thus, could be putative risk factors for late-onset AD. One of these mutations, C734Y, is likely transport-deficient, while the other mutations appear to influence ACE catalytic properties. The precipitation of mutant M118T by mAb 2D1 and ACE mutant C734Y by mAb 3F10 increased 2-3-fold compared to native ACE, and therefore, these mAbs could be markers of these mutations. Also, we identified a mutation I989T, which is associated with increased ACE levels in the blood. Conclusions/Significance Conducting a systematic analysis of blood ACE levels in patients with ACE mutations holds promise for identifying individuals with low blood ACE levels. Such individuals may be at increased risk for late-onset AD. The patients with transport-deficient ACE mutations may benefit from therapeutic treatment with a combination of chemical and pharmacological chaperones and proteasome inhibitors, as was demonstrated previously using a cell model of the transport-deficient ACE mutation, Q1069R [Danilov et al, PLoS One, 2010].

The resistance of bacteria to β-lactam antibiotics is primarily caused by the production of β-lactamases. Here, novel crystal structures of the native β-lactamase TEM-171 and two complexes with the widely used inhibitor tazobactam are presented, alongside complementary data from UV spectroscopy and fluorescence quenching. The six chemically identical β-lactamase molecules in the crystallographic asymmetric unit displayed different degrees of disorder. The tazobactam intermediate was covalently bound to the catalytic Ser70 in the trans-enamine configuration. While the conformation of tazobactam in the first complex resembled that in published β-lactamase-tazobactam structures, in the second complex, which was obtained after longer soaking of the native crystals in the inhibitor solution, a new and previously unreported tazobactam conformation was observed. It is proposed that the two complexes correspond to different stages along the deacylation path of the acyl-enzyme intermediate. The results provide a novel structural basis for the rational design of new β-lactamase inhibitors.

Enzymes conjugated to magnetic nanoparticles (MNPs) undergo changes in the catalytic activity of the nonheating low-frequency magnetic field (LFMF). We apply in silico simulations by molecular dynamics (MD) and in vitro spectroscopic analysis of the enzyme kinetics and secondary structure to study alpha-chymotrypsin (CT) conjugated to gold-coated iron oxide MNPs. The latter are functionalized by either carboxylic or amino group moieties to vary the points of enzyme attachment. The MD simulation suggests that application of the stretching force to the CT globule by its amino or carboxylic groups causes shrinkage of the substrate-binding site but little if any changes in the catalytic triad. Consistent with this, in CT conjugated to MNPs by either amino or carboxylic groups, LFMF alters the Michaelis-Menten constant but not the apparent catalytic constant kcat (= Vmax/[E]o). Irrespective of the point of conjugation to MNPs, the CT secondary structure was affected with nearly complete loss of alpha-helices and increase in the random structures in LFMF, as shown by attenuated total reflection Fourier transformed infrared spectroscopy. Both the catalytic activity and the protein structure of MNPCT conjugates restored 3 h after the field exposure. We believe that such remotely actuated systems can find applications in advanced manufacturing, nanomedicine, and other areas.

Background: GH74 xyloglucanases are composed of two separate domains connected by two unstructured peptides. Previously, a hypothesis was made that the movement of domains may affect the enzyme mechanism of catalysis. Methods: The molecular dynamics (MD) simulations of endo-processive xyloglucanases from Paenibacillus odorifer (PoGH74(cat)) and Myceliophthora thermophila (MtXeg74A) were carried out. Results: MD simulations for both enzymes in complex with XXLG and XGXXLG oligosaccharides confirmed the possibility of domain movement. In the case of MtXeg74A, changes in the distances between C-alpha atoms of aromatic residues involved in xyloglucan binding in -3 and +3 subsites of the active site cleft and those of selected residues on the opposite side of the cleft reached values up to 10-12 angstrom. For PoGH74(cat) the conformational changes were less pronounced. In MtXeg74A variants, the deletion of loop 1, which partially closes the entrance to the cleft, and the additional double mutation of two Trp residues in +3 and +5 subsites caused the enhanced mobility of the XGXXLG and also induced changes in topography of the cleft. Conclusions: These findings demonstrate the possibility of existence of GH74 xyloglucanases in a more open and more closed enzyme conformation. The enzyme in an open conformation may more easily accommodate the branched polysaccharide, while its transition to the closed conformation, together with loop 1 function, should aid processivity. General significance: Our results provide an insight into a mechanism of action of GH74 xyloglucanases and may be useful for discussing the catalytic mechanisms of glycoside hydrolases from other families.

NAD(P)(+)-dependent formate dehydrogenase (FDH, EC 1.2.1.2.) is actively used in processes of chiral synthesis by oxidoreductases with systems of reduced cofactor regeneration. The efficient use of FDH in such systems requires simple and fast enzyme purification. Metal-chelate affinity chromatography is widely used for such purposes. The method requires the presence of at least six His residues at N- or C-terminus of protein. The addition of extra His residues can affect enzyme properties. The computer modeling of the structure of FDH from bacteriumPseudomonassp. 101 with different positions of His(6)sequence showed that the optimal case is His-tag at N-terminus. Three types of PseFDH with His(6)were prepared: wild-type NAD(+)-dependent enzyme and two mutant NADP(+)-specific forms. New PseFDHs were obtained as homogeneous preparations through a one-step purification procedure. The comparison of PseFDHs with and without His-tag showed that they have similar kinetic properties.

Bacterial resistance to β-lactams, the most commonly used class of antibiotics, poses a global challenge. This resistance is caused by the production of bacterial enzymes that are termed β-lactamases (βLs). The evolution of serine-class A β-lactamases from penicillin-binding proteins (PBPs) is related to the formation of the Ω-loop at the entrance to the enzyme’s active site. In this loop, the Glu166 residue plays a key role in the two-step catalytic cycle of hydrolysis. This residue in TEM–type β-lactamases, together with Asn170, is involved in the formation of a hydrogen bonding network with a water molecule, leading to the deacylation of the acyl–enzyme complex and the hydrolysis of the β-lactam ring of the antibiotic. The activity exhibited by the Ω-loop is attributed to the positioning of its N-terminal residues near the catalytically important residues of the active site. The structure of the Ω-loop of TEM-type β-lactamases is characterized by low mutability, a stable topology, and structural flexibility. All of the revealed features of the Ω-loop, as well as the mechanisms related to its involvement in catalysis, make it a potential target for novel allosteric inhibitors of β-lactamases.

Magnetomechanical modulation of biochemical processes is a promising instrument for bioengineering and nanomedicine. This work demonstrates two approaches to control activity of an enzyme, α-chymotrypsin immobilized on the surface of gold-coated magnetite magnetic nanoparticles (GM-MNPs) using a nonheating low-frequency magnetic field (LF MF). The measurement of the enzyme reaction rate was carried out in situ during exposure to the magnetic field. The first approach involves α-chymotrypsin-GM-MNPs conjugates, in which the enzyme undergoes mechanical deformations with the reorientation of the MNPs under LF MF (16-410 Hz frequency, 88 mT flux density). Such mechanical deformations result in conformational changes in α-chymotrypsin structure, as confirmed by infrared spectroscopy and molecular modeling, and lead to a 63% decrease of enzyme initial activity. The second approach involves an α-chymotrypsin-GM-MNPs/trypsin inhibitor-GM-MNPs complex, in which the activity of the enzyme is partially inhibited. In this case the reorientation of MNPs in the field leads to disruption of the enzyme-inhibitor complex and an almost 2-fold increase of enzyme activity. The results further demonstrate the utility of magnetomechanical actuation at the nanoscale for the remote modulation of biochemical reactions.

Beta-lactamases (EC 3.5.2.6) represent a superfamily containing more than 2000 members: it includes genetically and functionally different bacterial enzymes capable to degrade the beta-lactam antibiotics. Beta-lactamases of molecular class A with serine residue in the active center are the most common ones. In the context of studies of the mechanisms underlying of evolution of the resistance, TEM type beta-lactamases are of particular interest due to their broad polymorphism. To date, more than 200 sequences of TEM type beta-lactamases have been described and more than 60 structures of different mutant forms of these enzymes have been presented in the Protein Data Bank. We have considered here the main structural features of the enzymes of this type with particular attention to the analysis of key mutations determining drug resistance and the secondary mutations, their location relative to the active center and the surface of the protein globule. We have developed a BlaSIDB database (www.blasidb.org) which is an open information resource combining available data on 3D structures, amino acid sequences and nomenclature of the TEM type beta-lactamases.

The complex formation of TEM-1 β-lactamase and its three mutant forms TEM-32, TEM-37, and TEM-39 with substrates cephalothin and CENTA and serine beta-lactamase inhibitors sulbactam, tazobactam, and clavulanic acid is studied using the methods of molecular dynamics. It is found that the stability of the complexes is caused by the electrostatic attraction between the deprotonated carboxyl group of the β-lactam ring of the substrate (inhibitor) and the positively charged amino groups of the lysine 234 and 73 residues, located in the active site of the enzymes. The formation of a hydrogen bond between this substrate group or its carbonyl oxygen with the hydroxyl group of the catalytic serine 70 residue and also between the negatively charged substituent groups and the positive charge region formed by the arginine 244 guanidine group and the asparagine 276 amino group is observed for some complexes. The binding energy of CENTA with TEM-1 β-lactamase is below the analogous binding energy of cephalothin, which is confirmed by the values of the Michaelis constants, determined experimentally. It is also found that the inhibitors bind to the mutant forms of β-lactamases related to the inhibitor-resistant phenotype, with higher affinity than TEM-1 β-lactamase.

Highly mutable β‐lactamases are responsible for the ability of Gram‐negative bacteria to resist β‐lactam antibiotics. Using site‐directed mutagenesis technique, we have produced in vitro a number of recombinant analogs of naturally occurring TEM‐type β‐lactamases, bearing the secondary substitution Q39K and key mutations related to the extended‐spectrum (E104K, R164S) and inhibitor‐resistant (M69V) β‐lactamases. The mutation Q39K alone was found to be neutral and hardly affected the catalytic properties of β‐lactamases. However, in combination with the key mutations, this substitution resulted in decreased KM values towards hydrolysis of a chromogenic substrate, CENTA. The ability of enzymes to restore catalytic activity after exposure to elevated temperature has been examined. All double and triple mutants of β‐lactamase TEM‐1 bearing the Q39K substitution showed lower thermal stability compared with the enzyme with Q39 intact. A sharp decrease in the stability was observed when Q39K was combined with E104K and M69V. The key R164S substitution demonstrated unusual ability to resist thermal inactivation. Computer analysis of the structure and molecular dynamics of β‐lactamase TEM‐1 revealed a network of hydrogen bonds from the residues Q39 and K32, related to the N‐terminal α‐helix, towards the residues R244 and G236, located in the vicinity of the enzyme's catalytic site. Replacement of Q39 by lysine in combination with the key drug resistance mutations may be responsible for loss of protein thermal stability and elevated mobility of its secondary structure elements. This effect on the activity of β‐lactamases can be used as a new potential target for inhibiting the enzyme.

β-Сyclodextrin (CD) is a perspective class of excipients used in pharmaceutical formulations to enhance solubility, bioavailability, and pharmacokinetics of various poorly soluble drugs, forming a non-covalent guest–host complex. However, the development of such formulations is usually a very laborious and time-consuming process due to lack of appropriate analytical tools to directly track and study the detailed molecular mechanism of such complex formation. Here, using guest–host complexes of fluoroquinolones (FQ) with CDs, as an example, we demonstrate the utility of ATR-FTIR to determine the thermodynamic stability, as well as structural features associated with complex formation, including involvement of certain functional groups. Furthermore, varying the CD’s side groups, we were able to tailor the CD’s geometry and binding surface to make FQ–CD interactions strong enough to potentially affect its pharmacokinetics and justify development of a new sustained-release drug formulation (dissociation constant decreased from 5 * 10−3 M to 10−5 M). 3D molecular modeling with energy optimization supports the findings and conclusions made on the basis of ATR-FTIR data analysis and explains the observed difference in dissociation constants.

The physicochemical properties and structure of moxifloxacin‒methyl-β-cyclodextrin complex have been studied by UV spectroscopy, FTIR spectroscopy, and computer simulation. The optimal conditions for the formation of the complex have been determined, and the dissociation constant of the complex in acidic media (K dis = (5.0 ± 0.3) × 10–5 М) has been obtained. It has been found that complexation significantly slows down the release of the drug in acidic media. Experimental results are in good agreement with computer simulation data. The following mechanism of complex formation has been proposed: the incorporation of the aromatic fragment of moxifloxacin into the cavity of methyl-β-cyclodextrin is followed by additional stabilization of the complex via multiple hydrophobic interactions and hydrogen bonding.

In order to investigate factors affecting the thermostability of GH10 xylanase A from Penicillium canescens (PcXylA) and to obtain its more stable variant, the wild-type (wt) enzyme and its mutant forms, carrying single amino acid substitutions, were cloned and expressed in Penicillium verruculosum B1-537 (niaD-) auxotrophic strain under the control of the cbh1 gene promoter. The recombinant PcXylA-wt and I6V, I6L, L18F, N77D, Y125R, H191R, S246P, A293P mutants were successfully expressed and purified for characterization. The mutations did not affect the enzyme specific activity against xylan from wheat as well as its pH-optimum of activity. One mutant (L18F) displayed a higher thermostability relative to the wild-type enzyme; its half-life time at 50–60°C was 2–2.5–fold longer than that for the PcXylA-wt, and the melting temperature was 60.0 and 56.1°C, respectively. Most of other mutations led to decrease in the enzyme thermostability. This study, together with data of other researchers, suggests that multiple mutations should be introduced into GH10 xylanases in order to dramatically improve their stability.

Synthesis of b-lactamases is one of the common mechanisms of bacterial resistance to b-lactam antibiotics including penicillins and cephalosporins. The widespread use of antibiotics results in appearance of numerous extended-spectrum b-lactamase variants or resistance to inhibitors. Mutations of 92 residues of TEM type were found. Several mutations are the key mutations that determine the extension of spectrum of substrates. However, roles of the most associated mutations, located far from active site, remain unknown. We have investigated the role of associated mutations in structure of b-lactamase TEM-72, which contain two key mutation (G238S, E240K) and two associated mutations (Q39K, M182T) by means of simulation of molecular dynamics. The key mutation lead to destabilization of the protein globule, characterized by increased mobility of amino acid residues at high temperature of modelling. Mutation M182T lead to stabilization protein, whereas mutation Q39K is destabilizing mutation. It seems that the last mutation serves for optimization of conformational mobility of b-lactamase and may influence on enzyme activity.

Lytic transglycosylases are abundant peptidoglycan lysing enzymes that degrade the heteropolymers of bacterial cell walls in metabolic processes or in the course of a bacteriophage infection. The conventional catalytic mechanism of transglycosylases involves only the Glu or Asp residue. Endolysin gp144 of Pseudomonas aeruginosa bacteriophage phiKZ belongs to the family of Gram-negative transglycosylases with a modular composition and C-terminal location of the catalytic domain. Glu115 of gp144 performs the predicted role of a catalytic residue. However, replacement of this residue does not completely eliminate the activity of the mutant protein. Site-directed mutagenesis has revealed the participation of Tyr197 in the catalytic mechanism, as well as the presence of a second active site involving Glu178 and Tyr147. The existence of the dual active site was supported by computer modeling and monitoring of the molecular dynamics of the changes in the conformation and surface charge distribution as a consequence of point mutations.

The dipole interaction model, an implementation of classical electromagnetic theory, is assembled in a computer package: DInaMo/CDCALC. This package is very successful in predicting the far-UV CD π-π∗ transition of proteins, replicating the peak positions, intensity and morphology. The major limitation of the method has been the neglect of the n-π∗ transition. Herein, initial parameters, including the n-π∗ transition, are incorporated with DInaMo/CDCAL. Furthermore, new mean polarizability parameters are developed for the CH3, CH2, and CH groups. Protein structures obtained from the Protein Data Bank (PBD) are energy minimized with NAMD. The energy minimized structures are further modified in six different ways: (1) The methyl (CH3) hydrogens are deleted prior to calculation. (2) Both the methyl (CH3) and methylene (CH2) hydrogens are deleted. (3) All the CH3, CH2, and CH hydrogens are deleted leaving only amide hydrogens. (4) New mean polarizability parameters are implemented only for CH3. (5) New mean polarizability parameters are implemented for both CH3 and CH2 groups. (6) New mean polarizability parameters are implemented for CH3, CH2, and CH groups. There seems to be a correlation between the protein secondary structureal and the kind of modification used for the CD prediction. The β-sheets give best results with including only amide hydrogens while the α-helical proteins give better results ignoring CH3 and/or CH2 groups. Calculations with using new mean polarizability parameters remove the need for different π-π∗ transition parameters and improve the CD results in lower RMSDs and better spectra morphology. In addition, the new parameters for the n-π∗ transition yield normal modes in the correct region and sign for this transition.