The binding selectivity of the C-terminal SH3 domain of Grb2, but not its folding pathway, is dictated by its contiguous SH2 domain.

The adaptor protein Grb2, or growth factor receptor-bound protein 2, possesses a pivotal role in the transmission of fundamental molecular signals in the cell. Despite lacking enzymatic activity, Grb2 functions as a dynamic assembly platform, orchestrating intracellular signals through its modular structure. This study delves into the energetic communication of Grb2 domains, focusing on the folding and binding properties of the C-SH3 domain linked to its neighboring SH2 domain. Surprisingly, while the folding and stability of C-SH3 remain robust and unaffected by SH2 presence, significant differences emerge in the binding properties when considered within the tandem context compared with isolated C-SH3. Through a double mutant cycle analysis, we highlighted a subset of residues, located at the interface with the SH2 domain and far from the binding site, finely regulating the binding of a peptide mimicking a physiological ligand of the C-SH3 domain. Our results have mechanistic implications about the mechanisms of specificity of the C-SH3 domain, indicating that the presence of the SH2 domain optimizes binding to its physiological target, and emphasizing the general importance of considering supramodular multidomain protein structures to understand the functional intricacies of protein-protein interaction domains.

Learning from Protein Engineering by Deconvolution of Multi-Mutational Variants.

This review analyzes a development in biochemistry, enzymology and biotechnology that originally came as a surprise. Following the establishment of directed evolution of stereoselective enzymes in organic chemistry, the concept of partial or complete deconvolution of selective multi-mutational variants was introduced. Early deconvolution experiments of stereoselective variants led to the finding that mutations can interact cooperatively or antagonistically with one another, not just additively. During the past decade, this phenomenon was shown to be general. In some studies, molecular dynamics (MD) and quantum mechanics/molecular mechanics (QM/MM) computations were performed in order to shed light on the origin of non-additivity at all stages of an evolutionary upward climb. Data of complete deconvolution can be used to construct unique multi-dimensional rugged fitness pathway landscapes, which provide mechanistic insights different from traditional fitness landscapes. Along a related line, biochemists have long tested the result of introducing two point mutations in an enzyme for mechanistic reasons, followed by a comparison of the respective double mutant in so-called double mutant cycles, which originally showed only additive effects, but more recently also uncovered cooperative and antagonistic non-additive effects. We conclude with suggestions for future work, and call for a unified overall picture of non-additivity and epistasis.

Supertertiary protein structure affects an allosteric network.

The notion that protein function is allosterically regulated by structural or dynamic changes in proteins has been extensively investigated in several protein domains in isolation. In particular, PDZ domains have represented a paradigm for these studies, despite providing conflicting results. Furthermore, it is still unknown how the association between protein domains in supramodules, consitituting so-called supertertiary structures, affects allosteric networks. Here, we experimentally mapped the allosteric network in a PDZ:ligand complex, both in isolation and in the context of a supramodular structure, and show that allosteric networks in a PDZ domain are highly dependent on the supertertiary structure in which they are present. This striking sensitivity of allosteric networks to the presence of adjacent protein domains is likely a common property of supertertiary structures in proteins. Our findings have general implications for prediction of allosteric networks from primary and tertiary structures and for quantitative descriptions of allostery.

Impact of E484Q and L452R Mutations on Structure and Binding Behavior of SARS-CoV-2 B.1.617.1 Using Deep Learning AlphaFold2, Molecular Docking and Dynamics Simulation.

During the outbreak of COVID-19, many SARS-CoV-2 variants presented key amino acid mutations that influenced their binding abilities with angiotensin-converting enzyme 2 (hACE2) and neutralizing antibodies. For the B.1.617 lineage, there had been fears that two key mutations, i.e., L452R and E484Q, would have additive effects on the evasion of neutralizing antibodies. In this paper, we systematically investigated the impact of the L452R and E484Q mutations on the structure and binding behavior of B.1.617.1 using deep learning AlphaFold2, molecular docking and dynamics simulation. We firstly predicted and verified the structure of the S protein containing L452R and E484Q mutations via the AlphaFold2-calculated pLDDT value and compared it with the experimental structure. Next, a molecular simulation was performed to reveal the structural and interaction stabilities of the S protein of the double mutant variant with hACE2. We found that the double mutations, L452R and E484Q, could lead to a decrease in hydrogen bonds and higher interaction energy between the S protein and hACE2, demonstrating the lower structural stability and the worse binding affinity in the long dynamic evolutional process, even though the molecular docking showed the lower binding energy score of the S1 RBD of the double mutant variant with hACE2 than that of the wild type (WT) with hACE2. In addition, docking to three approved neutralizing monoclonal antibodies (mAbs) showed a reduced binding affinity of the double mutant variant, suggesting a lower neutralization ability of the mAbs against the double mutant variant. Our study helps lay the foundation for further SARS-CoV-2 studies and provides bioinformatics and computational insights into how the double mutations lead to immune evasion, which could offer guidance for subsequent biomedical studies.

Building 2D classification models and 3D CoMSIA models on small-molecule inhibitors of both wild-type and T790M/L858R double-mutant EGFR.

Epidermal growth factor receptor (EGFR) has received widespread attention because it is an important target for anticancer drug design. Mutations in the EGFR, especially the T790M/L858R double mutation, have made cancer treatment more difficult. We herein built the structure-activity relationship models of small-molecule inhibitors on wild-type and T790M/L858R double-mutant EGFR with a whole dataset of 379 compounds. For 2D classification models, we used ECFP4 fingerprints to build support vector machine and random forest models and used SMILES to build self-attention recurrent neural network models. Each of all six models resulted in an accuracy of above 0.87 and the Matthews correlation coefficient value of above 0.76 on the test set, respectively. We concluded that inhibitors containing anilinoquinoline and methoxy or fluoro phenyl are highly active against wild EGFR. Substructures such as anilinopyrimidine, acrylamide, amino phenyl, methoxy phenyl, and thienopyrimidinyl amide appeared more in highly active inhibitors against double-mutant EGFR. We also used self-organizing map to cluster the inhibitors into six subsets based on ECFP4 fingerprints and analyzed the activity characteristics of different scaffolds in each subset. Among them, three datasets, which are based on pteridin, anilinopyrimidine, and anilinoquinoline scaffold, were selected to build 3D comparative molecular similarity analysis models individually. Models with the leave-one-out coefficient of determination (q2) above 0.65 were selected, and five descriptor types (steric, electrostatic, hydrophobic, donor, and acceptor) were used to study the effects of side chains of inhibitors on the activity against wild-type and mutant-type EGFR.

Biophysical Characterization of Novel DNA Aptamers against K103N/Y181C Double Mutant HIV-1 Reverse Transcriptase.

The human immunodeficiency virus type-1 Reverse Transcriptase (HIV-1 RT) plays a pivotal role in essential viral replication and is the main target for antiviral therapy. The anti-HIV-1 RT drugs address resistance-associated mutations. This research focused on isolating the potential specific DNA aptamers against K103N/Y181C double mutant HIV-1 RT. Five DNA aptamers showed low IC50 values against both the KY-mutant HIV-1 RT and wildtype (WT) HIV-1 RT. The kinetic binding affinity forms surface plasmon resonance of both KY-mutant and WT HIV-1 RTs in the range of 0.06-2 µM and 0.15-2 µM, respectively. Among these aptamers, the KY44 aptamer was chosen to study the interaction of HIV-1 RTs-DNA aptamer complex by NMR experiments. The NMR results indicate that the aptamer could interact with both WT and KY-mutant HIV-1 RT at the NNRTI drug binding pocket by inducing a chemical shift at methionine residues. Furthermore, KY44 could inhibit pseudo-HIV particle infection in HEK293 cells with nearly 80% inhibition and showed low cytotoxicity on HEK293 cells. These together indicated that the KY44 aptamer could be a potential inhibitor of both WT and KY-mutant HIV-RT.

Structural modifications on indole and pyrimidine rings of osimertinib lead to high selectivity towards L858R/T790M double mutant enzyme and potent antitumor activity.

EGFR inhibitors represent a significant milestone for treatment of non-small cell lung cancer, however, they suffer from the acquired drug resistance. Utilizing osimertinib as the lead compound, this work has explored the structural modifications on the indole and pyrimidine rings of osimertinib to generate novel osimertinib derivatives. The in vitro enzymatic and cellular studies showed that the derivatives possessed high selectivity towards double mutant EGFR and potent antitumor activity. Particularly, compound 6b-1, the most active compound, exhibited excellent inhibitory activity against double mutant EGFR (IC50 = 0.18 nM) and wild-type EGFR (IC50 = 2.89 nM) as well as H1975 cells (IC50 = 1.44 nM). Western blot analysis showed that 6b-1 completely inhibited double mutant EGFR and Erk phosphorylation. In vivo test using xenograft model indicated that compound 6b-1 had better antitumor efficacy than osimertinib. More importantly, 6b-1 displayed many advantages in the pharmacokinetic study, including better oral bioavailability and metabolism character.

Discovery of Taroxaz-104: The first potent antidote of SARS-CoV-2 VOC-202012/01 strain.

Polyhydroxyphenols and nitrogenous heterocyclics are two of the most powerful active species of molecules in pharmaceutical chemistry, as each of them is renowned for its various bioactivities for humans. One of their outstanding actions is the antiviral activities, which clearly appear if the principal functional entities of both classes meet into one compound. The recent COVID-19 pandemic pushed us to computationally sift and assess our small library of synthetic 2-(3,4,5-trihydroxyphenyl)-1,3,4-oxadiazoles against the main coronaviral protein/enzymatic targets. Surprisingly, few ligands exhibited interesting low binding energies (strong inhibitory affinities) with some SARS-CoV-2 proteins, mainly the pivotal enzyme RNA-dependent RNA polymerase (nCoV-RdRp). One of these compounds was Taroxaz-104 (5,5'-{5,5'-[(1R,2R)-1,2-dihydroxyethane-1,2-diyl]bis(1,3,4-oxadiazole-5,2-diyl)}dibenzene-1,2,3-triol), which presented lower binding free energies of about -10.60 and -9.10 kcal/mol (as compared to the reference agent, GS-443902, which presented about -9.20 and -7.90 kcal/mol) with nCoV-RdRp-RNA and nCoV-RdRp alone, respectively. Extensive molecular modeling examination disclosed the potent Taroxaz-104 inhibition of one of the possible active/allosteric sites of nCoV-RdRp, since Taroxaz-104 molecule interacts with at least seven main amino acids of the presumed pocket/cavity of this nCoV-RdRp active site. The effective repurposing of Taroxaz-104 molecule was attained after the satisfactorily interesting results of the anti-COVID-19 bioassay were secured, since these data demonstrated that Taroxaz-104 showed very efficient anti-COVID-19 actions (anti-SARS-CoV-2 EC50 = 0.42 µM) with specific promising efficacy against the new SARS-CoV-2 strains. Additional research studies for the progress of Taroxaz-104 and other related polyphenolic 2,5-disubstituted-1,3,4-oxadiazole analogs as successful anti-SARS-CoV-2 medications, via, e.g., preclinical/clinical trials, are pressingly required.

Intracerebral Administration of a Ligand-ASO Conjugate Selectively Reduces α-Synuclein Accumulation in Monoamine Neurons of Double Mutant Human A30P*A53T*α-Synuclein Transgenic Mice.

α-Synuclein (α-Syn) protein is involved in the pathogenesis of Parkinson's disease (PD). Point mutations and multiplications of the α-Syn, which encodes the SNCA gene, are correlated with early-onset PD, therefore the reduction in a-Syn synthesis could be a potential therapy for PD if delivered to the key affected neurons. Several experimental strategies for PD have been developed in recent years using oligonucleotide therapeutics. However, some of them have failed or even caused neuronal toxicity. One limiting step in the success of oligonucleotide-based therapeutics is their delivery to the brain compartment, and once there, to selected neuronal populations. Previously, we developed an indatraline-conjugated antisense oligonucleotide (IND-1233-ASO), that selectively reduces α-Syn synthesis in midbrain monoamine neurons of mice, and nonhuman primates. Here, we extended these observations using a transgenic male mouse strain carrying both A30P and A53T mutant human α-Syn (A30P*A53T*α-Syn). We found that A30P*A53T*α-Syn mice at 4-5 months of age showed 3.5-fold increases in human α-Syn expression in dopamine (DA) and norepinephrine (NE) neurons of the substantia nigra pars compacta (SNc) and locus coeruleus (LC), respectively, compared with mouse α-Syn levels. In parallel, transgenic mice exhibited altered nigrostriatal DA neurotransmission, motor alterations, and an anxiety-like phenotype. Intracerebroventricular IND-1233-ASO administration (100 µg/day, 28 days) prevented the α-Syn synthesis and accumulation in the SNc and LC, and recovered DA neurotransmission, although it did not reverse the behavioral phenotype. Therefore, the present therapeutic strategy based on a conjugated ASO could be used for the selective inhibition of α-Syn expression in PD-vulnerable monoamine neurons, showing the benefit of the optimization of ASO molecules as a disease modifying therapy for PD and related α-synucleinopathies.

AtKPNB1, an Arabidopsis importin-ß protein, is downstream of the RING E3 ubiquitin ligase AtAIRP1 in the ABA-mediated drought stress response.

MAIN CONCLUSION: AtKPNB1, an Arabidopsis importin-ß protein, was regulated by AtAIRP1 E3 ubiquitin ligase, which intensified the ABA-mediated drought stress response. As an early step in the abscisic acid (ABA)-mediated drought response, the ABA signal is transduced into the nucleus, and thus the nuclear transport system is crucially involved in the drought stress response. AtKPNB1, an importin-ß protein, which is a core component of nuclear transport, was previously reported to be a negative factor in the ABA-mediated drought stress response (Luo et al. Luo et al., Plant J 75:377-389, 2013). Here, we report that AtAIPR1, an Arabidopsis RING-type E3 ubiquitin (Ub) ligase, interacted with and ubiquitinated AtKPNB1. A null mutation of AtKPNB1 suppressed the ABA-insensitive germination phenotype of atairp1 mutant seedlings as compared to that of the wild-type plants. Furthermore, the ABA-insensitive stomatal closure and drought-susceptible phenotypes of atairp1 were rescued in atairp1atkpnb1 double mutant progeny, indicating that AtKPNB1 functions downstream of AtAIRP1. These data suggest that AtAIRP1 regulates the ABA-mediated drought response in Arabidopsis via ubiquitination of AtKPNB1.

Biochemical and functional characterization of OsCSD3, a novel CuZn superoxide dismutase from rice.

Superoxide dismutases (SODs, EC 1.15.1.1) belong to an important group of antioxidant metalloenzymes. Multiple SODs exist for scavenging of reactive oxygen species (ROS) in different cellular compartments to maintain an intricate ROS balance. The present study deals with molecular and biochemical characterization of CuZn SOD encoded by LOC_Os03g11960 (referred to as OsCSD3), which is the least studied among the four rice isozymes. The OsCSD3 showed higher similarity to peroxisomal SODs in plants. The OsCSD3 transcript was up-regulated in response to salinity, drought, and oxidative stress. Full-length cDNA encoding OsCSD3 was cloned and expressed in Escherichia coli and analyzed for spectral characteristics. UV (ultraviolet)-visible spectroscopic analysis showed evidences of d-d transitions, while circular dichroism analysis indicated high ß-sheet content in the protein. The OsCSD3 existed as homodimer (∼36 kDa) with both Cu2+ and Zn2+ metal cofactors and was substantially active over a wide pH range (7.0-10.8), with optimum pH of 9.0. The enzyme was sensitive to diethyldithiocarbamate but insensitive to sodium azide, which are the characteristics features of CuZn SODs. The enzyme also exhibited bicarbonate-dependent peroxidase activity. Unlike several other known CuZn SODs, OsCSD3 showed higher tolerance to hydrogen peroxide and thermal inactivation. Heterologous overexpression of OsCSD3 enhanced tolerance of E. coli sod double-knockout (ΔsodA ΔsodB) mutant and wild-type strain against methyl viologen-induced oxidative stress, indicating the in vivo function of this enzyme. The results show that the locus LOC_Os03g11960 of rice encodes a functional CuZn SOD with biochemical characteristics similar to the peroxisomal isozymes.

The impact of Arabidopsis thaliana SNF1-related-kinase 1 (SnRK1)-activating kinase 1 (SnAK1) and SnAK2 on SnRK1 phosphorylation status: characterization of a SnAK double mutant.

Arabidopsis thaliana SNF1-related-kinase 1 (SnRK1)-activating kinase 1 (AtSnAK1) and AtSnAK2 have been shown to phosphorylate in vitro and activate the energy signalling integrator, SnRK1. To clarify this signalling cascade in planta, a genetic- and molecular-based approach was developed. Homozygous single AtSnAK1 and AtSnAK2 T-DNA insertional mutants did not display an apparent phenotype. Crossing of the single mutants did not allow the isolation of double-mutant plants, whereas self-pollinating the S1-/- S2+/- sesquimutant specifically gave approximatively 22% individuals in their offspring that, when rescued on sugar-supplemented media in vitro, were shown to be AtSnAK1 AtSnAK2 double mutants. Interestingly, this was not obtained in the case of the other sesquimutant, S1+/- S2-/-. Although reduced in size, the double mutant had the capacity to produce flowers, but not seeds. Immunological characterization established the T-loop of the SnRK1 catalytic subunit to be non-phosphorylated in the absence of both SnAKs. When the double mutant was complemented with a DNA construct containing an AtSnAK2 open reading frame driven by its own promoter, a normal phenotype was restored. Therefore, wild-type plant growth and development is dependent on the presence of SnAK in vivo, and this is correlated with SnRK1 phosphorylation. These data show that both SnAKs are kinases phosphorylating SnRK1, and thereby they contribute to energy signalling in planta.

Genetic inactivation of mGlu5 receptor improves motor coordination in the Grm1crv4 mouse model of SCAR13 ataxia.

Deleterious mutations in the glutamate receptor metabotropic 1 gene (GRM1) cause a recessive form of cerebellar ataxia, SCAR13. GRM1 and GRM5 code for the metabotropic glutamate type 1 (mGlu1) and type 5 (mGlu5) receptors, respectively. Their different expression profiles suggest they could have distinct functional roles. In a previous study, homozygous mice lacking mGlu1 receptors (Grm1crv4/crv4) and exhibiting ataxia presented cerebellar overexpression of mGlu5 receptors, that was proposed to contribute to the mouse phenotype. To test this hypothesis, we here crossed Grm1crv4 and Grm5ko mice to generate double mutants (Grm1crv4/crv4Grm5ko/ko) lacking both mGlu1 and mGlu5 receptors. Double mutants and control mice were analyzed for spontaneous behavior and for motor activity by rotarod and footprint analyses. In the same mice, the release of glutamate from cerebellar nerve endings (synaptosomes) elicited by 12mM KCl or by α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) was also evaluated. Motor coordination resulted improved in double mutants when compared to Grm1crv4/crv4 mice. Furthermore, in in vitro studies, glutamate release elicited by both KCl depolarization and activation of AMPA autoreceptors resulted reduced in Grm1crv4/crv4 mice compared to wild type mice, while it presented normal levels in double mutants. Moreover, we found that Grm1crv4/crv4 mice showed reduced expression of GluA2/3 AMPA receptor subunits in cerebellar synaptosomes, while it resulted restored to wild type level in double mutants. To conclude, blocking of mGlu5 receptor reduced the dysregulation of glutamate transmission and improved motor coordination in the Grm1crv4 mouse model of SCAR13, thus suggesting the possible usefulness of pharmacological therapies based on modulation of mGlu5 receptor activity for the treatment of this type of ataxia.

Unraveling structural requirements of amino-pyrimidine T790M/L858R double mutant EGFR inhibitors: 2D and 3D QSAR study.

EGFR is an important drug target in cancer. However, the ineffectiveness of first generation inhibitors due to the occurrence of a secondary mutation (T790M) results in the relapse of the disease. Identification of reversible inhibitors against T790M/L858R double mutants (TMLR) thus is a foremost requirement. In this study, various 2 D and 3 D Quantitative Structure-Activity Relationship models were built for amino-pyrimidine compounds with their known biological activity against TMLR mutants. The model developed using multiple linear regression statistical method via stepwise forward-backward variable selection technique showed the best results in terms of internal and external predictivity. The 2D-QSAR model indicated that the presence of electronegative atom, H-bond donors, moderate slogp, count of number of N atoms separated from O (T_N_O_4), 4pathClusterCount and number of S atom connected with two single bonds (SssSE-index), is required for increasing the inhibitory potential of compounds. Also, the 3D-QSAR model suggested that electronegative group at certain positions along with the presence of bulky groups is beneficial for good inhibition activity of the compounds. Thus, the QSAR models developed in the present work can be used for predicting the TMLR bioactivity of a new series of amino-pyrimidine derivatives. To the best of the author's knowledge, this is the first study which deals with the development of 2 D and 3D-QSAR models for double mutant TMLR inhibitors.

Activation of the Integrated Stress Response and Metabolic Dysfunction in a Murine Model of Sleep Apnea.

Intermittent hypoxia (IH) induces activation of the integrated stress response (ISR), but its role in IH-induced visceral white adipose tissue (vWAT) insulin resistance is unknown. CHOP is activated by chronic ISR, whereas GADD34 dephosphorylates the subunit of translation initiation factor 2 (eIF2α), leading to termination of the ISR. We hypothesized that CHOP/Gadd34 null mice would not manifest evidence of insulin resistance after IH exposures. Eight-week-old CHOP/GADD34-/- (double mutant [DM]) and wild-type (WT) littermates were randomly assigned to IH or room air (RA) exposures for 6 weeks. Glucose and insulin tolerance tests were performed, and regulatory T cells (Tregs) and macrophages in vWAT were assessed. Phosphorylated eIF2α:total eIF2α, ATF4, XBP1 expression, and insulin-induced pAKT/AKT expression changes were examined in vWATs. Single GADD34-/- and PERK+/- mice were also evaluated. Body weight and vWAT mass were reduced in DM and WT mice after IH. M1/M2 macrophages and inflammatory macrophages (Ly-6chigh) were significantly increased in WT vWAT but remained unchanged in DM mice. Tregs were significantly decreased in WT vWAT but not in DM mice. Systemic insulin and glucose tolerance tests revealed insulin resistance in IH-WT but not in IH-DM mice. Similarly, decreased pAKT/AKT responses to exogenous insulin emerged in IH-WT compared with RA-WT mice, whereas no significant differences emerged in IH-DM compared with DM-RA. Chronic ISR activation appears to contribute to the insulin resistance and vWAT inflammation that characteristically emerge after long-term IH exposures in a murine model of obstructive sleep apnea.

Allostery Wiring Map for Kinesin Energy Transduction and Its Evolution.

How signals between the kinesin active and cytoskeletal binding sites are transmitted is an open question and an allosteric question. By extracting correlated evolutionary changes within 700+ sequences, we built a model of residues that are energetically coupled and that define molecular routes for signal transmission. Typically, these coupled residues are located at multiple distal sites and thus are predicted to form a complex, non-linear network that wires together different functional sites in the protein. Of note, our model connected the site for ATP hydrolysis with sites that ultimately utilize its free energy, such as the microtubule-binding site, drug-binding loop 5, and necklinker. To confirm the calculated energetic connectivity between non-adjacent residues, double-mutant cycle analysis was conducted with 22 kinesin mutants. There was a direct correlation between thermodynamic coupling in experiment and evolutionarily derived energetic coupling. We conclude that energy transduction is coordinated by multiple distal sites in the protein rather than only being relayed through adjacent residues. Moreover, this allosteric map forecasts how energetic orchestration gives rise to different nanomotor behaviors within the superfamily.

A double-mutant collection targeting MAP kinase related genes in Arabidopsis for studying genetic interactions.

Mitogen-activated protein kinase cascades are conserved in all eukaryotes. In Arabidopsis thaliana there are approximately 80 genes encoding MAP kinase kinase kinases (MAP3K), 10 genes encoding MAP kinase kinases (MAP2K), and 20 genes encoding MAP kinases (MAPK). Reverse genetic analysis has failed to reveal abnormal phenotypes for a majority of these genes. One strategy for uncovering gene function when single-mutant lines do not produce an informative phenotype is to perform a systematic genetic interaction screen whereby double-mutants are created from a large library of single-mutant lines. Here we describe a new collection of 275 double-mutant lines derived from a library of single-mutants targeting genes related to MAP kinase signaling. To facilitate this study, we developed a high-throughput double-mutant generating pipeline using a system for growing Arabidopsis seedlings in 96-well plates. A quantitative root growth assay was used to screen for evidence of genetic interactions in this double-mutant collection. Our screen revealed four genetic interactions, all of which caused synthetic enhancement of the root growth defects observed in a MAP kinase 4 (MPK4) single-mutant line. Seeds for this double-mutant collection are publicly available through the Arabidopsis Biological Resource Center. Scientists interested in diverse biological processes can now screen this double-mutant collection under a wide range of growth conditions in order to search for additional genetic interactions that may provide new insights into MAP kinase signaling.

Double Mutants in DNA Gyrase Lead to Ofloxacin Resistance in Mycobacterium tuberculosis.

Fluoroquinolones are among the most important classes of highly effective antibacterial drugs, exhibiting wide range of activity to cure infectious diseases. Ofloxacin is second generation fluoroquinolone approved by FDA for the treatment of tuberculosis by selectively inhibiting DNA gyrase. However, the emergence of drug resistance owing to mutations in DNA gyrase poses intimidating challenge for the effective therapy of this drug. The double mutants GyrAA90V GyrBD500N and GyrAA90V GyrBT539N are reported to be implicated in conferring higher levels of OFX resistance. The present study was designed to unravel the molecular principles behind development of resistance by the bug against fluoroquinolones. Our results highlighted that polar interactions play critical role in the development of drug resistance and highlight the significant correlation between the free energy calculations predicted by MM-PBSA and stability of the ligand-bound complexes. Modifications at the OFX binding pocket due to amino acid substitution leads to fewer hydrogen bonds in mutants DNA gyrase-OFX complex, which determined the low susceptibility of the ligand in inhibiting the mutant protein. This study provides a structural rationale to the mutation-based resistance to ofloxacin and will pave way for development potent fluoroquinolone-based resistant-defiant drugs. J. Cell. Biochem. 118: 2950-2957, 2017. © 2017 Wiley Periodicals, Inc.

Engineering of Harobin for enhanced fibrinolytic activity obtained by random and site-directed mutagenesis.

We have previously published a report on the cloning and characterization of Harobin, a fibrinolytic serine protease. However, the broad application of this fibrinolytic enzyme is limited by its low expression level that was achieved in Pichia pastoris. To counteract this shortcoming, random and site-directed mutagenesis have been combined in order to improve functional expression and activity of Harobin. By screening 400 clones from random mutant libraries for enhanced fibrinolytic activity, two mutants were obtained: N111R, R230G. By performing site-directed mutagenesis, a Harobin double mutant, N111R/R230G, was constructed and can be functionally expressed at higher level than the wild type enzyme. In addition, it possessed much higher fibrinolytic and amidolytic activity than the wild type enzyme and other single mutants. The N111R/R230G expressed in basal salts medium was purified by a three step purification procedure. At pH of 6.0-9.0, and the temperature range of 40-90 °C, N111R/R230G was more active and more heat resistant. The fibrinolytic activities of Harobin mutants were completely inhibited by PMSF and SBTI, but not by EDTA, EGTA, DTT, indicating that Harobin is a serine protease. N111R/R230G showed much better anti-thrombosis effect than wild type Harobin and single mutants, and could significantly increase bleeding and clotting time. Intravenous injection of N111R/R230G in spontaneous hypertensive rats (SHR) led to a significant reduction in systolic blood pressure (SBP), diastolic blood pressure (DBP) and mean arterial pressure (MAP) (p < 0.01), while heart rate (HR) was not affected. The in vitro and in vivo results of the present study revealed that Harobin double mutant N111R/R230G is an appropriate candidate for biotechnological applications due to its high expression level and high activity in area of thrombosis and hypertension.

Energetically significant networks of coupled interactions within an unfolded protein.

Unfolded and partially unfolded proteins participate in a wide range of biological processes from pathological aggregation to the regulation of normal cellular activity. Unfolded states can be populated under strongly denaturing conditions, but the ensemble which is relevant for folding, stability, and aggregation is that populated under physiological conditions. Characterization of nonnative states is critical for the understanding of these processes, yet comparatively little is known about their energetics and their structural propensities under native conditions. The standard view is that energetically significant coupled interactions involving multiple residues are generally not present in the denatured state ensemble (DSE) or in intrinsically disordered proteins. Using the N-terminal domain of the ribosomal protein L9, a small α-ß protein, as an experimental model system, we demonstrate that networks of energetically significant, coupled interactions can form in the DSE of globular proteins, and can involve residues that are distant in sequence and spatially well separated in the native structure. X-ray crystallography, NMR, dynamics studies, native state pKa measurements, and thermodynamic analysis of more than 25 mutants demonstrate that residues are energetically coupled in the DSE. Altering these interactions by mutation affects the stability of the domain. Mutations that alter the energetics of the DSE can impact the analysis of cooperativity and folding, and may play a role in determining the propensity to aggregate.