scholarly journals The Iron-containing Domain Is Essential in Rad3 Helicases for Coupling of ATP Hydrolysis to DNA Translocation and for Targeting the Helicase to the Single-stranded DNA-Double-stranded DNA Junction

2007 ◽  
Vol 283 (3) ◽  
pp. 1732-1743 ◽  
Author(s):  
Robert A. Pugh ◽  
Masayoshi Honda ◽  
Haley Leesley ◽  
Alvin Thomas ◽  
Yuyen Lin ◽  
...  
Keyword(s):  
Atp Hydrolysis ◽  
Point Mutations ◽  
Structural Features ◽  
Fine Tuning ◽  
Dna Interactions ◽  
Double Stranded Dna ◽  
Distinct Disease ◽  
Relevant Point ◽  
Conserved Core ◽  
Xpd Helicase

Helicases often achieve functional specificity through utilization of unique structural features incorporated into an otherwise conserved core. The archaeal Rad3 (xeroderma pigmentosum group D protein (XPD)) helicase is a prototypical member of the Rad3 family, distinct from other related (superfamily II) SF2 enzymes because of a unique insertion containing an iron-sulfur (FeS) cluster. This insertion may represent an auxiliary domain responsible for modifying helicase activity or for conferring specificity for selected DNA repair intermediates. The importance of the FeS cluster for the fine-tuning of Rad3-DNA interactions is illustrated by several clinically relevant point mutations in the FeS domain of human Bach1 (FancJ) and XPD helicases that result in distinct disease phenotypes. Here we analyzed the substrate specificity of the Rad3 (XPD) helicase from Ferroplasma acidarmanus (FacRad3) and probed the importance of the FeS cluster for Rad3-DNA interactions. We found that the FeS cluster stabilizes secondary structure of the auxiliary domain important for coupling of single-stranded (ss) DNA-dependent ATP hydrolysis to ssDNA translocation. Additionally, we observed specific quenching of the Cy5 fluorescent dye when the FeS cluster of a bound helicase is positioned in close proximity to a Cy5 fluorophore incorporated into the DNA molecule. Taking advantage of this Cy5 quenching, we developed an equilibrium assay for analysis of the Rad3 interactions with various DNA substrates. We determined that the FeS cluster-containing domain recognizes the ssDNA-double-stranded DNA junction and positions the helicase in an orientation consistent with duplex unwinding. Although it interacts specifically with the junction, the enzyme binds tightly to ssDNA, and the single-stranded regions of the substrate are the major contributors to the energetics of FacRad3-substrate interactions.

scholarly journals Myosinome: A Database of Myosins from Select Eukaryotic Genomes to Facilitate Analysis of Sequence-Structure-Function Relationships

2012 ◽  
Vol 6 ◽  
pp. BBI.S9902 ◽  
Author(s):  
Divya P. Syamaladevi ◽  
Margaret S Sunitha ◽  
S. Kalaimathy ◽  
Chandrashekar C. Reddy ◽  
Mohammed Iftekhar ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Atp Hydrolysis ◽  
Homo Sapiens ◽  
Relevant Literature ◽  
Myosin Ii ◽  
Coiled Coil ◽  
Structural Features ◽  
Model Organisms ◽  
Congenital Diseases ◽  
C Elegans

Myosins are one of the largest protein superfamilies with 24 classes. They have conserved structural features and catalytic domains yet show huge variation at different domains resulting in a variety of functions. Myosins are molecules driving various kinds of cellular processes and motility until the level of organisms. These are ATPases that utilize the chemical energy released by ATP hydrolysis to bring about conformational changes leading to a motor function. Myosins are important as they are involved in almost all cellular activities ranging from cell division to transcriptional regulation. They are crucial due to their involvement in many congenital diseases symptomatized by muscular malfunctions, cardiac diseases, deafness, neural and immunological dysfunction, and so on, many of which lead to death at an early age. We present Myosinome, a database of selected myosin classes (myosin II, V, and VI) from five model organisms. This knowledge base provides the sequences, phylogenetic clustering, domain architectures of myosins and molecular models, structural analyses, and relevant literature of their coiled-coil domains. In the current version of Myosinome, information about 71 myosin sequences belonging to three myosin classes (myosin II, V, and VI) in five model organisms ( Homo Sapiens, Mus musculus, D. melanogaster, C. elegans and S. cereviseae) identified using bioinformatics surveys are presented, and several of them are yet to be functionally characterized. As these proteins are involved in congenital diseases, such a database would be useful in short-listing candidates for gene therapy and drug development. The database can be accessed from http://caps.ncbs.res.in/myosinome .

scholarly journals Extending the Coding Potential of Viral Genomes with Overlapping Antisense ORFs: A Case for the De Novo Creation of the Gene Encoding the Antisense Protein ASP of HIV-1

Viruses ◽  
2022 ◽  
Vol 14 (1) ◽  
pp. 146
Author(s):  
Angelo Pavesi ◽  
Fabio Romerio
Keyword(s):  
De Novo ◽  
Point Mutations ◽  
Selective Advantage ◽  
Genomic Region ◽  
Fine Tuning ◽  
Sequence Evolution ◽  
Viral Genomes ◽  
Stop Codons ◽  
Hiv 1

Gene overprinting occurs when point mutations within a genomic region with an existing coding sequence create a new one in another reading frame. This process is quite frequent in viral genomes either to maximize the amount of information that they encode or in response to strong selective pressure. The most frequent scenario involves two different reading frames in the same DNA strand (sense overlap). Much less frequent are cases of overlapping genes that are encoded on opposite DNA strands (antisense overlap). One such example is the antisense ORF, asp in the minus strand of the HIV-1 genome overlapping the env gene. The asp gene is highly conserved in pandemic HIV-1 strains of group M, and it is absent in non-pandemic HIV-1 groups, HIV-2, and lentiviruses infecting non-human primates, suggesting that the ~190-amino acid protein that is expressed from this gene (ASP) may play a role in virus spread. While the function of ASP in the virus life cycle remains to be elucidated, mounting evidence from several research groups indicates that ASP is expressed in vivo. There are two alternative hypotheses that could be envisioned to explain the origin of the asp ORF. On one hand, asp may have originally been present in the ancestor of contemporary lentiviruses, and subsequently lost in all descendants except for most HIV-1 strains of group M due to selective advantage. Alternatively, the asp ORF may have originated very recently with the emergence of group M HIV-1 strains from SIVcpz. Here, we used a combination of computational and statistical approaches to study the genomic region of env in primate lentiviruses to shed light on the origin, structure, and sequence evolution of the asp ORF. The results emerging from our studies support the hypothesis of a recent de novo addition of the antisense ORF to the HIV-1 genome through a process that entailed progressive removal of existing internal stop codons from SIV strains to HIV-1 strains of group M, and fine tuning of the codon sequence in env that reduced the chances of new stop codons occurring in asp. Altogether, the study supports the notion that the HIV-1 asp gene encodes an accessory protein, providing a selective advantage to the virus.

scholarly journals Inductive Transfer Learning for Molecular Activity Prediction: Next-Gen QSAR Models with MolPMoFiT

2020 ◽  
Author(s):  
Xinhao Li ◽  
Denis Fourches
Keyword(s):  
Transfer Learning ◽  
High Throughput Screening ◽  
Structure Prediction ◽  
Large Scale ◽  
High Reliability ◽  
Structural Features ◽  
Fine Tuning ◽  
Qsar Modeling ◽  
Chemical Structures ◽  
Effective Transfer

<p>Deep neural networks can directly learn from chemical structures without extensive, user-driven selection of descriptors in order to predict molecular properties/activities with high reliability. But these approaches typically require large training sets to learn the endpoint-specific structural features and ensure reasonable prediction accuracy. Even though large datasets are becoming the new normal in drug discovery, especially when it comes to high-throughput screening or metabolomics datasets, one should also consider smaller datasets with challenging endpoints to model and forecast. Thus, it would be highly relevant to better utilize the tremendous compendium of unlabeled compounds from publicly-available datasets for improving the model performances for the user’s particular series of compounds. In this study, we propose the <b>Mol</b>ecular <b>P</b>rediction <b>Mo</b>del <b>Fi</b>ne-<b>T</b>uning (<b>MolPMoFiT</b>) approach, an effective transfer learning method based on self-supervised pre-training + task-specific fine-tuning for QSPR/QSAR modeling. A large-scale molecular structure prediction model is pre-trained using one million unlabeled molecules from ChEMBL in a self-supervised learning manner, and can then be fine-tuned on various QSPR/QSAR tasks for smaller chemical datasets with specific endpoints. Herein, the method is evaluated on four benchmark datasets (lipophilicity, FreeSolv, HIV, and blood-brain barrier penetration). The results showed the method can achieve strong performances for all four datasets compared to other state-of-the-art machine learning modeling techniques reported in the literature so far. <br></p>

scholarly journals Structures of the ATP-fueled ClpXP proteolytic machine bound to protein substrate

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Xue Fei ◽  
Tristan A Bell ◽  
Simon Jenni ◽  
Benjamin M Stinson ◽  
Tania A Baker ◽  
...  
Keyword(s):  
Single Molecule ◽  
Atp Hydrolysis ◽  
Functional Results ◽  
Structural Features ◽  
Specific Protein ◽  
Power Stroke ◽  
E Coli ◽  
Protein Substrates ◽  
Biophysical Studies ◽  
Sequential Models

ClpXP is an ATP-dependent protease in which the ClpX AAA+ motor binds, unfolds, and translocates specific protein substrates into the degradation chamber of ClpP. We present cryo-EM studies of the E. coli enzyme that show how asymmetric hexameric rings of ClpX bind symmetric heptameric rings of ClpP and interact with protein substrates. Subunits in the ClpX hexamer assume a spiral conformation and interact with two-residue segments of substrate in the axial channel, as observed for other AAA+ proteases and protein-remodeling machines. Strictly sequential models of ATP hydrolysis and a power stroke that moves two residues of the substrate per translocation step have been inferred from these structural features for other AAA+ unfoldases, but biochemical and single-molecule biophysical studies indicate that ClpXP operates by a probabilistic mechanism in which five to eight residues are translocated for each ATP hydrolyzed. We propose structure-based models that could account for the functional results.

Inhibition of Hill Reaction by 2-Azido-s-triazine Derivatives: QSAR Study with Molecular Connectivity Indices

1989 ◽  
Vol 44 (3-4) ◽  
pp. 255-261 ◽  
Author(s):  
Milan Š Šoškić ◽  
Aleksandar Sabljić
Keyword(s):  
Structural Features ◽  
Second Order ◽  
Fine Tuning ◽  
Superior Performance ◽  
Hill Reaction ◽  
Retention Data ◽  
Molecular Connectivity ◽  
Inhibitory Potency ◽  
Molecular Connectivity Indices ◽  
Connectivity Indices

Abstract 2-A zido-s-T riazines, Connectivity Indices. Inhibition of Hill Reaction. Photosystem II, OSAR Modelling This study was undertaken to find a simple and accurate structural parameters for the quantita­tive description of inhibitory potency of 2-azido-s-triazines in Hill reaction and to gain more information about the mechanism of inhibition on molecular level. A very good correlation (r = 0.946) was obtained between the pl50 values (the negative logarithm of the molar concentra­tion that causes 50% inhibition) and the valence zero-order and the difference between the second-order and the valence second-order molecular connectivity indices. This model, when com pared with the empirical models based on the 1-octano/water partition coefficients and the chromatographic retention data, shows superior performance in accuracy and range of applicabili­ty. In addition, the direct correspondence between molecular structure and above connectivity indices makes it possible to locate structural features responsible for the inhibitory potency of 2-azido-s-triazines in Hill reaction. From our OSAR analysis, the interaction between the chloro­plast receptor site and 2-azido-s-triazines, which causes inhibition of Hill reaction, is primarily influenced by the size of alkylamino substituents and it accounts for the most variation in the pl50 data. The structural features of secondary importance that control the magnitude of pl50’s are the polarity of alkylamino chains and the degree of branching on alpha carbon atom of R:alkylamino substituent. Com pared with the main factor, the size of alkylamino substituents, they can be viewed as a fine tuning elements for the inhibitory potency of 2-azido-s-triazines.

A method to predict the impact of point mutations on local structural features

2000 ◽  
Vol 28 (3) ◽  
pp. A78-A78
Author(s):  
S.N. Ruzheinikov ◽  
M.E. Popov ◽  
I.V. Kashparov
Keyword(s):  
Point Mutations ◽  
Structural Features ◽  
The Impact

scholarly journals Analysis of the structural features of the C-terminus of GLUT1 that are required for transport catalytic activity

1995 ◽  
Vol 311 (2) ◽  
pp. 699-704 ◽  
Author(s):  
A Muraoka ◽  
M Hashiramoto ◽  
A E Clark ◽  
L C Edwards ◽  
H Sakura ◽  
...  
Keyword(s):  
Amino Acids ◽  
Glucose Transport ◽  
Cytochalasin B ◽  
Point Mutations ◽  
Chinese Hamster ◽  
Structural Features ◽  
Transport Activity ◽  
Wild Type ◽  
C Terminus ◽  
Wild Type Clone

C-terminally truncated and mutated forms of GLUT1 have been constructed to determine the minimum structure at the C-terminus required for glucose transport activity and ligand binding at the outer and inner binding sites. Four truncated mutants have been constructed (CTD24 to CTD27) in which 24 to 27 amino acids are deleted. In addition, point substitutions of R468-->L, F467-->L and G466-->E have been produced. Chinese hamster ovary clones which were transfected with these mutant GLUT1s were shown, by Western blotting and cell-surface carbohydrate labelling, to have expression levels which were comparable with the wild-type clone. Wild-type levels of 2-deoxy-D-glucose transport activity were retained only in the clone transfected with the construct in which 24 amino acids were deleted (CTD24). The CTD25, CTD26 and CTD27 clones showed markedly reduced transport activity. From a kinetic comparison of the CTD24 and CTD26 clones it was found that the reduced transport was mainly associated with a reduced Vmax. value for 2-deoxy-D-glucose uptake but with a slight lowering of the Km. These data establish that the 24 amino acids at the C-terminus of GLUT1 are not required for the transport catalysis. However, the point mutations of F467L and G466E (26 and 27 residues from the C-terminus) did not significantly perturb the kinetics of 2-deoxy-D-glucose transport. The substitution of R468L produced a slight, but significant, lowering of the Km. The ability of the truncated GLUt1s to bind the exofacial ligand, 2-N-4-(1-zai-2,2,2-trifluoroethyl)benzoyl-1,3-bis-(D-mannos- 4-yl-oxy) -2-propylamine (ATB-BMPA), and the endofacial ligand, cytochalasin B, were assessed by photolabelling procedures. The ability to bind ATB-BMPA was retained only in the CTD24 truncated mutant and was reduced to levels comparable with those of the non-transfected clone in the other mutant clones. Cytochalasin B labelling was unimpaired in all four mutated GLUT1s. These data establish that a minimum structure at the C-terminus of GLUT1, which is required for the conformational change to expose the exofacial site, includes amino acids at positions Phe-467 and Arg-468; however, these amino acids are not individually essential.

scholarly journals Nucleosome Remodeling by the Human SWI/SNF Complex Requires Transient Global Disruption of Histone-DNA Interactions

2002 ◽  
Vol 22 (11) ◽  
pp. 3653-3662 ◽  
Author(s):  
Sayura Aoyagi ◽  
Geeta Narlikar ◽  
Chunyang Zheng ◽  
Saïd Sif ◽  
Robert E. Kingston ◽  
...  
Keyword(s):  
Restriction Enzyme ◽  
Atp Hydrolysis ◽  
Dnase I ◽  
Cross Linking ◽  
Dna Interactions ◽  
Nucleosome Remodeling ◽  
Histone Octamer ◽  
Nucleosomal Dna ◽  
Single Cross ◽  
A Site

ABSTRACT We utilized a site-specific cross-linking technique to investigate the mechanism of nucleosome remodeling by hSWI/SNF. We found that a single cross-link between H2B and DNA virtually eliminates the accumulation of stably remodeled species as measured by restriction enzyme accessibility assays. However, cross-linking the histone octamer to nucleosomal DNA does not inhibit remodeling as monitored by DNase I digestion assays. Importantly, we found that the restriction enzyme-accessible species can be efficiently cross-linked after remodeling and that the accessible state does not require continued ATP hydrolysis. These results imply that the generation of stable remodeled states requires at least transient disruption of histone-DNA interactions throughout the nucleosome, while hSWI/SNF-catalyzed disruption of just local histone-DNA interactions yields less-stable remodeled states that still display an altered DNase I cleavage pattern. The implications of these results for models of the mechanism of SWI/SNF-catalyzed nucleosome remodeling are discussed.

scholarly journals Genetic and biochemical analysis of Msh2p-Msh6p: role of ATP hydrolysis and Msh2p-Msh6p subunit interactions in mismatch base pair recognition.

1997 ◽  
Vol 17 (5) ◽  
pp. 2436-2447 ◽  
Author(s):  
E Alani ◽  
T Sokolsky ◽  
B Studamire ◽  
J J Miret ◽  
R S Lahue
Keyword(s):  
Base Pair ◽  
Biochemical Analysis ◽  
Atp Hydrolysis ◽  
Point Mutations ◽  
Binding Domain ◽  
Dominant Negative ◽  
Mutant Protein ◽  
Atp Binding ◽  
Mutant Proteins ◽  
Mismatch Recognition

Recent studies have shown that Saccharomyces cerevisiae Msh2p and Msh6p form a complex that specifically binds to DNA containing base pair mismatches. In this study, we performed a genetic and biochemical analysis of the Msh2p-Msh6p complex by introducing point mutations in the ATP binding and putative helix-turn-helix domains of MSH2. The effects of these mutations were analyzed genetically by measuring mutation frequency and biochemically by measuring the stability, mismatch binding activity, and ATPase activity of msh2p (mutant msh2p)-Msh6p complexes. A mutation in the ATP binding domain of MSH2 did not affect the mismatch binding specificity of the msh2p-Msh6p complex; however, this mutation conferred a dominant negative phenotype when the mutant gene was overexpressed in a wild-type strain, and the mutant protein displayed biochemical defects consistent with defects in mismatch repair downstream of mismatch recognition. Helix-turn-helix domain mutant proteins displayed two different properties. One class of mutant proteins was defective in forming complexes with Msh6p and also failed to recognize base pair mismatches. A second class of mutant proteins displayed properties similar to those observed for the ATP binding domain mutant protein. Taken together, these data suggested that the proposed helix-turn-helix domain of Msh2p was unlikely to be involved in mismatch recognition. We propose that the MSH2 helix-turn-helix domain mediates changes in Msh2p-Msh6p interactions that are induced by ATP hydrolysis; the net result of these changes is a modulation of mismatch recognition.

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