Conformational transitions induced by NADH binding promote reduction half-reaction in 2-hydroxybiphenyl-3-monooxygenase catalytic cycle.

2-Hydroxybiphenyl-3-monoxygenase from Pseudomonas azelaica is an effective catalyst of the regiospecific conversions of various aromatic compounds. A comprehensive understanding of the complete catalytic cycle, including the as yet unclear details of NADH binding, NADH/FAD interaction as well as related conformational changes could facilitate the rational design of improved enzyme variants for practical applications. Induced fit formation of a specific pocket for the nicotinamide ring at NADH binding has been revealed using advanced molecular simulation methods including metadynamics and QM/MM modeling. The resulting triple stacking interaction of the nicotinamide as well as isoalloxazine rings and evolutionarily correlated amino acid residues of the active site greatly contributes to the stabilization of the charge-transfer complex and determines the Pro-S stereospecificity of the hydride transfer and the low energy barrier 11 kcal/mol. Then the resulting FADH- anion undergoes the consequent conformational transition of the FAD isoalloxazine ring from the open out to the closed in position which is followed by the binding of an oxygen molecule what is crucial for the next step of substrate oxidation and the completion of the catalytic cycle.

Soft Tissue Sarcoma Study: Association of Genetic Alterations in the Apoptosis Pathways with Chemoresistance to Doxorubicin.

Soft tissue sarcomas (STS) are heterogeneous cancers with more than 100 histological subtypes, different in molecular alterations, which make its personalized therapy very complex. Gold standard of chemotherapy for advanced STS includes combinations of Doxorubicin and Ifosfamide or Gemcitabine and Docetaxel. Chemotherapy is efficient for less than 50% of patients and it is followed by a fast development of drug resistance. Our study was directed to the search of genetic alterations in cancer cells associated with chemoresistance of undifferentiated pleomorphic and synovial sarcomas to the abovementioned genotoxic drugs. We analyzed chemoresistance of cancer cells in vitro using primary STS cultures and performed genetic analysis for the components of apoptotic signaling. In 27% of tumors, we revealed alterations in TP53, ATM, PIK3CB, PIK3R1, NTRK1, and CSF2RB. Cells from STS specimens with found genetic alterations were resistant to Dox, excluding the only one case when TP53 mutation resulted in the substitution Leu344Arg associated with partial oligomerization loss and did not cause total loss of TP53 function. Significant association between alterations in the components of apoptosis signaling and chemoresistance to Dox was found. Our data are important to elaborate further the therapeutic strategy for STS patients with alterations in apoptotic signaling.

Analysis of Multiple Drug Resistance Mechanism in Different Types of Soft Tissue Sarcomas: Assessment of the Expression of ABC-Transporters, MVP, YB-1, and Analysis of Their Correlation with Chemosensitivity of Cancer Cells.

Chemotherapy of soft tissue sarcomas (STS) is restricted by low chemosensitivity and multiple drug resistance (MDR). The purpose of our study was the analysis of MDR mechanism in different types of STS. We assessed the expression of ABC-transporters, MVP, YB-1, and analyzed their correlation with chemosensitivity of cancer cells. STS specimens were obtained from 70 patients without metastatic disease (2018-2020). Expression level of MDR-associated genes was estimated by qRT-PCR and cytofluorimetry. Mutations in ABC-transporter genes were captured by exome sequencing. Chemosensitivity (SI) of STS to doxorubicin (Dox), ifosfamide (Ifo), gemcitabine (Gem), and docetaxel (Doc) was analyzed in vitro. We found strong correlation in ABCB1, ABCC1, and ABCG2 expression. We demonstrated strong negative correlations in ABCB1 and ABCG2 expression with SI (Doc) and SI (Doc + Gem), and positive correlation of MVP expression with SI (Doc) and SI (Doc + Gem) in undifferentiated pleomorphic sarcoma. Pgp expression was shown in 5 out of 44 STS samples with prevalence of synovial sarcoma relapses and it is strongly correlated with SI (Gem). Mutations in MDR-associated genes were rarely found. Overall, STS demonstrated high heterogeneity in chemosensitivity that makes reasonable in vitro chemosensitivity testing to improve personalized STS therapy, and classic ABC-transporters are not obviously involved in MDR appearance.

Computational design of a pH stable enzyme: understanding molecular mechanism of penicillin acylase's adaptation to alkaline conditions.

Protein stability provides advantageous development of novel properties and can be crucial in affording tolerance to mutations that introduce functionally preferential phenotypes. Consequently, understanding the determining factors for protein stability is important for the study of structure-function relationship and design of novel protein functions. Thermal stability has been extensively studied in connection with practical application of biocatalysts. However, little work has been done to explore the mechanism of pH-dependent inactivation. In this study, bioinformatic analysis of the Ntn-hydrolase superfamily was performed to identify functionally important subfamily-specific positions in protein structures. Furthermore, the involvement of these positions in pH-induced inactivation was studied. The conformational mobility of penicillin acylase in Escherichia coli was analyzed through molecular modeling in neutral and alkaline conditions. Two functionally important subfamily-specific residues, Gluß482 and Aspß484, were found. Ionization of these residues at alkaline pH promoted the collapse of a buried network of stabilizing interactions that consequently disrupted the functional protein conformation. The subfamily-specific position Aspß484 was selected as a hotspot for mutation to engineer enzyme variant tolerant to alkaline medium. The corresponding Dß484N mutant was produced and showed 9-fold increase in stability at alkaline conditions. Bioinformatic analysis of subfamily-specific positions can be further explored to study mechanisms of protein inactivation and to design more stable variants for the engineering of homologous Ntn-hydrolases with improved catalytic properties.

Bioinformatic analysis of protein families for identification of variable amino acid residues responsible for functional diversity.

Proteins within a single family usually share a common function but differ in more specific features and can be divided into subfamilies with different properties. Availability of genomic, structural, and functional information implemented into numerous databases provides new opportunities for bioinformatic analysis of homologous proteins. In this work, new method of bioinformatic analysis has been developed to identify subfamily-specific positions (SSPs)--conserved only within protein subfamilies, but different between subfamilies--that seem to play important role in functional diversity. A novel scoring function is suggested to consider structural information as well as physicochemical and residue conservation in protein subfamilies. Random shuffling is performed to rank results by significance, and Bernoulli statistics is applied to calculate p-values. Algorithm does not require predefined subfamily classification and can propose it automatically by graph-based clustering. This method can be used as a tool to explore SSPs with different structural localization in order to understand their implication to structure-function relationship and protein function. Web interface to the program is available at http://biokinet.belozersky.msu.ru/zebra.

Zebra: a web server for bioinformatic analysis of diverse protein families.

During evolution of proteins from a common ancestor, one functional property can be preserved while others can vary leading to functional diversity. A systematic study of the corresponding adaptive mutations provides a key to one of the most challenging problems of modern structural biology - understanding the impact of amino acid substitutions on protein function. The subfamily-specific positions (SSPs) are conserved within functional subfamilies but are different between them and, therefore, seem to be responsible for functional diversity in protein superfamilies. Consequently, a corresponding method to perform the bioinformatic analysis of sequence and structural data has to be implemented in the common laboratory practice to study the structure-function relationship in proteins and develop novel protein engineering strategies. This paper describes Zebra web server - a powerful remote platform that implements a novel bioinformatic analysis algorithm to study diverse protein families. It is the first application that provides specificity determinants at different levels of functional classification, therefore addressing complex functional diversity of large superfamilies. Statistical analysis is implemented to automatically select a set of highly significant SSPs to be used as hotspots for directed evolution or rational design experiments and analyzed studying the structure-function relationship. Zebra results are provided in two ways - (1) as a single all-in-one parsable text file and (2) as PyMol sessions with structural representation of SSPs. Zebra web server is available at http://biokinet.belozersky.msu.ru/zebra .