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Membrane proteins (MPs) encompass a large family of proteins with distinct cellular functions, and although representing over 50% of existing pharmaceutical drug targets, their structural and functional information is still very scarce. Over the last years, in silico analysis and algorithm development were essential to characterize MPs and overcome some limitations of experimental approaches. The optimization and improvement of these methods remain an ongoing process, with key advances in MPs' structure, folding, and interface prediction being continuously tackled. Herein, we discuss the latest trends in computational methods toward a deeper understanding of the atomistic and mechanistic details of MPs.

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Microbial fibrinolytic proteases are therapeutic enzymes responsible to ameliorate thrombosis, a fatal cardiac-disorder which effectuates due to excessive fibrin accumulation in blood vessels. Inadequacies such as low fibrin specificity, lethal after-effects and short life-span of available fibrinolytic enzymes stimulates an intensive hunt for novel, efficient and safe substitutes. Therefore, we herewith suggest a novel and potent fibrinolytic enzyme RFEA1 from Bacillus cereus RSA1 (MK288105). Although, attributes such as in-vitro purification, characterization and thrombolytic potential of RFEA1 were successfully accomplished in our previous study. However, it is known that structure-function traits and mode of action significantly aid to commercialization of an enzyme. Also, predicting structural model of a protein from its amino acid sequence is challenging in computational biology owing to intricacy of energy functions and inspection of vast conformational space. Our present study thus reports In-silico structural-functional analysis of RFEA1. Sequence based modelling approaches such as-Iterative threading ASSEmbly Refinement (I-TASSER), SWISS-MODEL, RaptorX and Protein Homology/analogY Recognition Engine V 2.0 (Phyre2) were employed to model three-dimensional structure of RFEA1 and the modelled RFEA1 was validated by structural analysis and verification server (SAVES v6.0). The modelled crystal structure revealed the presence of high affinity Ca1 binding site, associated with hydrogen bonds at Asp147, Leu181, Ile185 and Val187residues. RFEA1 is structurally analogous to Subtilisin E from Bacillus subtilis 168. Molecular docking analysis using PATCH DOCK and FIRE DOCK servers was performed to understand the interaction of RFEA1 with substrate fibrin. Strong RFEA1-fibrin interaction was observed with high binding affinity (-21.36 kcal/mol), indicating significant fibrinolytic activity and specificity of enzyme RFEA1. Overall, the computational research suggests that RFEA1 is a subtilisin-like serine endopeptidase with proteolytic potential, involved in thrombus hydrolysis.

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Diacylglycerol acyltransferases (DGAT) catalyze the final committed step of de novo biosynthesis of triacylglycerol (TAG) in plant seeds. This study was to functionally characterize DGAT3 genes in Camelina sativa, an important oil crops accumulating high levels of unsaturated fatty acids (UFAs) in seeds. Three camelina DGAT3 genes (CsDGAT3-1, CsDGAT3-2 and CsDGAT3-3) were identified, and the encoded proteins were predicted to be cytosolic-soluble proteins present as a homodimer containing the 2Fe-2S domain. They had divergent expression patterns in various tissues, suggesting that they may function in tissue-specific manner with CsDGAT3-1 in roots, CsDGAT3-2 in flowers and young seedlings, and CsDGAT3-3 in developing seeds. Functional complementation assay in yeast demonstrated that CsDGAT3-3 restored TAG synthesis. TAG content and UFAs, particularly eicosenoic acid (EA, 20:1n-9) were largely increased by adding exogenous UFAs in the yeast medium. Further heterogeneously transient expression in N. benthamiana leaves and seed-specific expression in tobacco seeds indicated that CsDGAT3-3 significantly enhanced oil and UFA accumulation with much higher level of EA. Overall, CsDGAT3-3 exhibited a strong abilty catalyzing TAG synthesis and high substrate preference for UFAs, especially for 20:1n-9. The present data provide new insights for further understanding oil biosynthesis mechanism in camelina seeds, indicating that CsDGAT3-3 may have practical applications for increasing both oil yield and quality.

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BACKGROUND: Eqolisins are rare acid proteases found in archaea, bacteria and fungi. Certain fungi secrete acids as part of their lifestyle and interestingly these also have many eqolisin paralogs, up to nine paralogs have been recorded. This suggests a process of functional redundancy and diversification has occurred, which was the subject of the research we performed and describe here. RESULTS: We identified eqolisin homologs by means of iterative HMMER analysis of the NR database. The identified sequences were scrutinized for which new hallmarks were identified by molecular dynamics simulations of mutants in highly conserved positions, using the structure of an eqolisin that was crystallized in the presence of a transition state inhibitor. Four conserved glycines were shown to be important for functionality. A substitution of W67F is shown to be accompanied by the L105W substitution. Molecular dynamics shows that the W67 binds to the substrate via a π-π stacking and a salt bridge, the latter being stronger in a virtual W67F/L105W double mutant of the resolved structure of Scytalido-carboxyl peptidase-B (PDB ID: 2IFW). Additional problematic mutations are discussed. Upon sequence scrutiny we obtained a set of 233 sequences that was used to reconstruct a Bayesian phylogenetic tree. We identified 14 putative specificity determining positions (SDPs) of which four are explained by mere structural explanations and nine seem to correspond to functional diversification related with substrate binding and specificity. A first sub-network of SDPs is related to substrate specificity whereas the second sub-network seems to affect the dynamics of three loops that are involved in substrate binding. CONCLUSION: The eqolisins form a small superfamily of acid proteases with nevertheless many paralogs in acidic fungi. Functional redundancy has resulted in diversification related to substrate specificity and substrate binding.

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BACKGROUND: The microtubule associated protein Tau (MAPT) promotes assembly and interaction of microtubules with the cytoskeleton, impinging on axonal transport and synaptic plasticity. Its neuronal expression and intrinsic disorder implicate it in some 30 tauopathies such as Alzheimer's disease and frontotemporal dementia. These pathophysiological studies have yet to be complemented by computational analyses of its molecular evolution and structural models of all its functional domains to explain the molecular basis for its conservation profile, its site-specific interactions and the propensity to conformational disorder and aggregate formation. RESULTS: We systematically annotated public sequence data to reconstruct unspliced MAPT, MAP2 and MAP4 transcripts spanning all represented genomes. Bayesian and maximum likelihood phylogenetic analyses, genetic linkage maps and domain architectures distinguished a nonvertebrate outgroup from the emergence of MAP4 and its subsequent ancestral duplication to MAP2 and MAPT. These events were coupled to other linked genes such as KANSL1L and KANSL and may thus be consequent to large-scale chromosomal duplications originating in the extant vertebrate genomes of hagfish and lamprey. Profile hidden Markov models (pHMMs), clustered subalignments and 3D structural predictions defined potential interaction motifs and specificity determining sites to reveal distinct signatures between the four homologous microtubule binding domains and independent divergence of the amino terminus. CONCLUSION: These analyses clarified ambiguities of MAPT nomenclature, defined the order, timing and pattern of its molecular evolution and identified key residues and motifs relevant to its protein interaction properties and pathogenic role. Additional unexpected findings included the expansion of cysteine-containing, microtubule binding domains of MAPT in cold adapted Antarctic icefish and the emergence of a novel multiexonic saitohin (STH) gene from repetitive elements in MAPT intron 11 of certain primate genomes.

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The A1 family of eukaryotic aspartic proteinases (APs) forms one of the 16 AP families. Although one of the best characterized families, the recent increase in genome sequence data has revealed many fungal AP homologs with novel sequence characteristics. This study was performed to explore the fungal AP sequence space and to obtain an in-depth understanding of fungal AP evolution. Using a comprehensive phylogeny of approximately 700 AP sequences from the complete proteomes of 87 fungi and 20 nonfungal eukaryotes, 11 major clades of APs were defined of which clade I largely corresponds to the A1A subfamily of pepsin-archetype APs. Clade II largely corresponds to the A1B subfamily of nepenthesin-archetype APs. Remarkably, the nine other clades contain only fungal APs, thus indicating that fungal APs have undergone a large sequence diversification. The topology of the tree indicates that fungal APs have been subject to both "birth and death" evolution and "functional redundancy and diversification." This is substantiated by coclustering of certain functional sequence characteristics. A meta-analysis toward the identification of Cluster Determining Positions (CDPs) was performed in order to investigate the structural and biochemical basis for diversification. Seven CDPs contribute to the secondary structure of the enzyme. Three other CDPs are found in the vicinity of the substrate binding cleft. Tree topology, the large sequence variation among fungal APs, and the apparent functional diversification suggest that an amendment to update the current A1 AP classification based on a comprehensive phylogenetic clustering might contribute to refinement of the classification in the MEROPS peptidase database.

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