scholarly journals Structure-based design and discovery of novel anti-tissue factor antibodies with cooperative double-point mutations, using interaction analysis

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Shuntaro Chiba ◽  
Aki Tanabe ◽  
Makoto Nakakido ◽  
Yasushi Okuno ◽  
Kouhei Tsumoto ◽  
...  
Keyword(s):  
Point Mutation ◽  
Triple Point ◽  
In Silico ◽  
Double Point ◽  
Interaction Analysis ◽  
Single Point ◽  
Point Mutations ◽  
Point Mutant ◽  
Wide Range ◽  
Mutation Strategy

Abstract The generation of a wide range of candidate antibodies is important for the successful development of drugs that simultaneously satisfy multiple requirements. To find cooperative mutations and increase the diversity of mutants, an in silico double-point mutation approach, in which 3D models of all possible double-point mutant/antigen complexes are constructed and evaluated using interaction analysis, was developed. Starting from an antibody with very high affinity, four double-point mutants were designed in silico. Two of the double-point mutants exhibited improved affinity or affinity comparable to that of the starting antibody. The successful identification of two active double-point mutants showed that a cooperative mutation could be found by utilizing information regarding the interactions. The individual single-point mutants of the two active double-point mutants showed decreased affinity or no expression. These results suggested that the two active double-point mutants cannot be obtained through the usual approach i.e. a combination of improved single-point mutants. In addition, a triple-point mutant, which combines the distantly located active double-point mutation and an active single-point mutation collaterally obtained in the process of the double-point mutation strategy, was designed. The triple-point mutant showed improved affinity. This finding suggested that the effects of distantly located mutations are independent and additive. The double-point mutation approach using the interaction analysis of 3D structures expands the design repertoire for mutants, and hopefully paves a way for the identification of cooperative multiple-point mutations.

Cupin Variants as Macromolecular Ligand Library for Stereoselective Michael Addition of Nitroalkanes

2019 ◽  
Author(s):  
Nobutaka Fujieda ◽  
Miho Yuasa ◽  
Yosuke Nishikawa ◽  
Genji Kurisu ◽  
Shinobu Itoh ◽  
...  
Keyword(s):  
Michael Addition ◽  
In Silico ◽  
Addition Reaction ◽  
Single Point ◽  
Point Mutations ◽  
Unsaturated Ketone ◽  
Michael Addition Reaction ◽  
Beta Barrel ◽  
Cupin Superfamily ◽  
Single Point Mutations

Cupin superfamily proteins (TM1459) work as a macromolecular ligand framework with a double-stranded beta-barrel structure ligating to a Cu ion through histidine side chains. Variegating the first coordination sphere of TM1459 revealed that H52A and H54A/H58A mutants effectively catalyzed the diastereo- and enantio-selective Michael addition reaction of nitroalkanes to an α,β-unsaturated ketone. Moreover, in silico substrate docking signified C106N and F104W single-point mutations, which inverted the diastereoselectivity of H52A and further improved the stereoselectivity of H54A/H58A, respectively.

scholarly journals An Intramolecular Salt Bridge in Bacillus thuringiensis Cry4Ba Toxin Is Involved in the Stability of Helix α-3, Which Is Needed for Oligomerization and Insecticidal Activity

2017 ◽  
Vol 83 (20) ◽  
Author(s):  
Sabino Pacheco ◽  
Isabel Gómez ◽  
Jorge Sánchez ◽  
Blanca-Ines García-Gómez ◽  
Mario Soberón ◽  
...  
Keyword(s):  
Bacillus Thuringiensis ◽  
Double Point ◽  
Single Point ◽  
Three Dimensional ◽  
Point Mutations ◽  
Salt Bridge ◽  
Dimensional Structure ◽  
Cry Toxins ◽  
Content Type ◽  
Domain I

ABSTRACT Bacillus thuringiensis three-domain Cry toxins kill insects by forming pores in the apical membrane of larval midgut cells. Oligomerization of the toxin is an important step for pore formation. Domain I helix α-3 participates in toxin oligomerization. Here we identify an intramolecular salt bridge within helix α-3 of Cry4Ba (D111-K115) that is conserved in many members of the family of three-domain Cry toxins. Single point mutations such as D111K or K115D resulted in proteins severely affected in toxicity. These mutants were also altered in oligomerization, and the mutant K115D was more sensitive to protease digestion. The double point mutant with reversed charges, D111K-K115D, recovered both oligomerization and toxicity, suggesting that this salt bridge is highly important for conservation of the structure of helix α-3 and necessary to promote the correct oligomerization of the toxin. IMPORTANCE Domain I has been shown to be involved in oligomerization through helix α-3 in different Cry toxins, and mutations affecting oligomerization also elicit changes in toxicity. The three-dimensional structure of the Cry4Ba toxin reveals an intramolecular salt bridge in helix α-3 of domain I. Mutations that disrupt this salt bridge resulted in changes in Cry4Ba oligomerization and toxicity, while a double point reciprocal mutation that restored the salt bridge resulted in recovery of toxin oligomerization and toxicity. These data highlight the role of oligomer formation as a key step in Cry4Ba toxicity.

scholarly journals Unraveling the stability landscape of mutations in the SARS-CoV-2 receptor-binding domain

2020 ◽  
Author(s):  
Mohamed Raef Smaoui ◽  
Hamdi Yahyaoui
Keyword(s):  
Receptor Binding ◽  
Single Point ◽  
Protein Structures ◽  
Point Mutations ◽  
Binding Domain ◽  
Spike Protein ◽  
Receptor Binding Domain ◽  
Point Mutant ◽  
The Stability ◽  
Spike Proteins

Abstract The interaction between the receptor-binding domain of the SARS-CoV-2 spike glycoprotein and the ACE2 enzyme is believed to be the entry point of the virus into various cells in the body, including the lungs, heart, liver, and kidneys. The current focus of several therapeutic design efforts explore attempts at affecting the binding interaction between the two proteins to limit the activity of the virus and disease progression. In this work, we analyze the stability of the spike protein under all possible single-point mutations in the receptor-binding domain and computationally explore mutations that can affect the binding with the ACE2 enzyme. We unravel the mutation landscape of the receptor region and assess the toxicity potential of single and multi-point mutations, generating insights for future vaccine efforts on potential mutations that might further stabilize the spike protein and increase its infectivity. We developed a tool, called SpikeMutator, to construct full atomic protein structures of the mutant spike proteins and shared a database of 3,800 single-point mutant structures. We analyzed the recent 65,000 reported spike sequences across the globe and observed the emergence of stable multi-point mutant structures. Using the landscape, we searched through 7.5 million possible 2-point mutation combinations and report that the (R355D K424E) mutation produces one of the strongest spike proteins that therapeutic efforts should investigate for the sake of developing an effective vaccine.

scholarly journals The highly conserved U small nuclear RNA 3'-end formation signal is quite tolerant to mutation.

1987 ◽  
Vol 7 (6) ◽  
pp. 2070-2079 ◽  
Author(s):  
R A Ach ◽  
A M Weiner
Keyword(s):  
Point Mutation ◽  
Hela Cells ◽  
Double Point ◽  
Xenopus Oocytes ◽  
Point Mutations ◽  
Small Nuclear Rna ◽  
Conserved Residues ◽  
Conserved Sequence ◽  
Nuclear Rna

Formation of the 3' end of U1 and U2 small nuclear RNA (snRNA) precursors is directed by a conserved sequence called the 3' box located 9 to 28 nucleotides downstream of all metazoan U1 to U4 snRNA genes sequenced so far. Deletion of part or all of the 3' box from human U1 and U2 genes drastically reduces 3'-end formation. To define the essential nucleotides within this box that direct 3'-end formation, we constructed a set of point mutations in the conserved residues of the human U1 3' box. The ability of the various mutations to direct 3'-end formation was tested by microinjection into Xenopus oocytes and transfection into HeLa cells. We found that the point mutations had diverse effects on 3'-end formation, ranging from no effect at all to severe inhibition; however, no single or double point mutation we tested completely eliminated 3'-end formation. We also showed that a rat U3 3' flank can effectively substitute for the human U1 3' flank, indicating that the 3' boxes of the different U snRNA genes are functionally equivalent.

scholarly journals GEDpm-cg: Genome Editing Automated Design Platform for Point Mutation Construction in Corynebacterium glutamicum

2021 ◽  
Vol 9 ◽  
Author(s):  
Yi Yang ◽  
Yufeng Mao ◽  
Ye Liu ◽  
Ruoyu Wang ◽  
Hui Lu ◽  
...  
Keyword(s):  
Genome Editing ◽  
Point Mutation ◽  
In Silico ◽  
Computer Aided Design ◽  
Large Scale ◽  
Point Mutations ◽  
Dna Assembly ◽  
Cell Factories ◽  
Assembly Method ◽  
Counter Selection

Advances in robotic system-assisted genome editing techniques and computer-aided design tools have significantly facilitated the development of microbial cell factories. Although multiple separate software solutions are available for vector DNA assembly, genome editing, and verification, by far there is still a lack of complete tool which can provide a one-stop service for the entire genome modification process. This makes the design of numerous genetic modifications, especially the construction of mutations that require strictly precise genetic manipulation, a laborious, time-consuming and error-prone process. Here, we developed a free online tool called GEDpm-cg for the design of genomic point mutations in C. glutamicum. The suicide plasmid-mediated counter-selection point mutation editing method and the overlap-based DNA assembly method were selected to ensure the editability of any single nucleotide at any locus in the C. glutamicum chromosome. Primers required for both DNA assembly of the vector for genetic modification and sequencing verification were provided as design results to meet all the experimental needs. An in-silico design task of over 10,000 single point mutations can be completed in 5 min. Finally, three independent point mutations were successfully constructed in C. glutamicum guided by GEDpm-cg, which confirms that the in-silico design results could accurately and seamlessly be bridged with in vivo or in vitro experiments. We believe this platform will provide a user-friendly, powerful and flexible tool for large-scale mutation analysis in the industrial workhorse C. glutamicum via robotic/software-assisted systems.

scholarly journals Assessing Protein-Drug Resistance Due to Mutations via a Rigidity Analysis in silico Approach

10.29007/7gnf ◽  
2020 ◽  
Author(s):  
Dylan Carpenter ◽  
Tess Thackray ◽  
Cecilia Kalthoff ◽  
Filip Jagodzinski
Keyword(s):  
Drug Resistance ◽  
Amino Acid ◽  
In Silico ◽  
Single Point ◽  
Point Mutations ◽  
Single Point Mutation ◽  
Protein Drug ◽  
Ligand Complex ◽  
Rigidity Analysis ◽  
The Impact

A mutation to the amino acid sequence of a protein can cause a biomolecule to be resistant to the intended effects of a drug. Assessing the changes of a drug’s efficacy in response to mutations via mutagenesis wet-lab experiments is prohibitively time consuming for even a single point mutation, let alone for all possible mutations. Existing approaches for inferring mutation-induced drug resistance are available, but all of them reason about mutations of residues at or very near the protein-drug interface. However, there are examples of mutations far away from the region where the ligand binds, but which nonetheless render a protein resistant to the effects of the drug. We present a proof-of-concept computational pipeline that generates in silico the set of all possible single point mutations in a protein-ligand complex. We assess drug resistance using a graph theoretic rigidity analysis approach. Unlike existing methods, we are able to assess the impact of mutations far away from the protein-drug interface. We introduce several visualizations for exploring how amino acid substitutions both near and far away from where the ligand interacts with a protein target have a stabilizing or destabilizing effect on the protein-drug complex. We discuss our analytical approach in the context of experimental data from the literature about clinically known protein-drug interactions.

Cupin Variants as Macromolecular Ligand Library for Stereoselective Michael Addition of Nitroalkanes

2019 ◽  
Author(s):  
Nobutaka Fujieda ◽  
Haruna Ichihashi ◽  
Miho Yuasa ◽  
Yosuke Nishikawa ◽  
Genji Kurisu ◽  
...  
Keyword(s):  
Michael Addition ◽  
In Silico ◽  
Addition Reaction ◽  
Single Point ◽  
Point Mutations ◽  
Unsaturated Ketone ◽  
Michael Addition Reaction ◽  
Beta Barrel ◽  
Cupin Superfamily ◽  
Single Point Mutations

Cupin superfamily proteins (TM1459) work as a macromolecular ligand framework with a double-stranded beta-barrel structure ligating to a Cu ion through histidine side chains. Variegating the first coordination sphere of TM1459 revealed that H52A and H54A/H58A mutants effectively catalyzed the diastereo- and enantio-selective Michael addition reaction of nitroalkanes to an α,β-unsaturated ketone. Moreover, in silico substrate docking signified C106N and F104W single-point mutations, which inverted the diastereoselectivity of H52A and further improved the stereoselectivity of H54A/H58A, respectively.

scholarly journals Specific point mutations in Lactobacillus casei ATCC 27139 cause a phenotype switch from Lac− to Lac+

Microbiology ◽  
2009 ◽  
Vol 155 (3) ◽  
pp. 751-760 ◽  
Author(s):  
Yu-Kuo Tsai ◽  
Hung-Wen Chen ◽  
Ta-Chun Lo ◽  
Thy-Hou Lin
Keyword(s):  
Gene Cluster ◽  
Point Mutation ◽  
Lactobacillus Casei ◽  
Stop Codon ◽  
Single Point ◽  
Point Mutations ◽  
Premature Stop Codon ◽  
Gene Clusters ◽  
Single Point Mutation ◽  
Phenotype Switch

Lactose metabolism is a changeable phenotype in strains of Lactobacillus casei. In this study, we found that L. casei ATCC 27139 was unable to utilize lactose. However, when exposed to lactose as the sole carbon source, spontaneous Lac+ clones could be obtained. A gene cluster (lacTEGF–galKETRM) involved in the metabolism of lactose and galactose in L. casei ATCC 27139 (Lac−) and its Lac+ revertant (designated strain R1) was sequenced and characterized. We found that only one nucleotide, located in the lacTEGF promoter (lacTp), of the two lac–gal gene clusters was different. The protein sequence identity between the lac–gal gene cluster and those reported previously for some L. casei (Lac+) strains was high; namely, 96–100 % identity was found and no premature stop codon was identified. A single point mutation located within the lacTp promoter region was also detected for each of the 41 other independently isolated Lac+ revertants of L. casei ATCC 27139. The revertants could be divided into six classes based on the positions of the point mutations detected. Primer extension experiments conducted on transcription from lacTp revealed that the lacTp promoter of these six classes of Lac+ revertants was functional, while that of L. casei ATCC 27139 was not. Northern blotting experiments further confirmed that the lacTEGF operon of strain R1 was induced by lactose but suppressed by glucose, whereas no blotting signal was ever detected for L. casei ATCC 27139. These results suggest that a single point mutation in the lacTp promoter was able to restore the transcription of a fully functional lacTEGF operon and cause a phenotype switch from Lac− to Lac+ for L. casei ATCC 27139.

scholarly journals Carbendazim Resistance of Fusarium graminearum From Henan Wheat

Plant Disease ◽  
2019 ◽  
Vol 103 (10) ◽  
pp. 2536-2540 ◽  
Author(s):  
Shengming Liu ◽  
Liuyuan Fu ◽  
Shuan Wang ◽  
Jinpeng Chen ◽  
Jia Jiang ◽  
...  
Keyword(s):  
Point Mutation ◽  
Fusarium Graminearum ◽  
Single Point ◽  
Point Mutations ◽  
Henan Province ◽  
Resistant Isolate ◽  
Head Blight ◽  
Tubulin Gene ◽  
Predominant Type ◽  
Moderately Resistant

Fusarium head blight, also called scab, is caused by Fusarium graminearum and is one of the most important destructive diseases of wheat. The frequency of carbendazim resistance in 1,132 isolates of F. graminearum recovered from fields in different regions of Henan Province in 2016, 2017, and 2018 was determined. A total of 31 F. graminearum isolates resistant to carbendazim were detected, including 30 moderately resistant isolates and one highly resistant isolate. The frequency of resistance of F. graminearum isolates to carbendazim was 2.7%. The range of effective concentration (EC50) values of 1,101 sensitive isolates and 30 moderately resistant isolates was 0.08 to 0.98 μg ml−1 and 2.73 to 13.28 μg ml−1, respectively. The mean ± SD EC50 value was 0.55 ± 0.13 μg ml−1 and 5.61 ± 2.58 μg ml−1, respectively. The EC50 value of the highly resistant isolate was 21.12 μg ml−1. Point mutation types of the carbendazim-resistant isolates were characterized by cloning the β2-tubulin gene of 31 resistant isolates. Three point mutation types at amino acids F167Y, E198Q, and E198L in the β2-tubulin gene of resistant isolates were identified. Among 31 resistant isolates, the frequency of point mutation types in F167Y, E198Q, and E198L of the β2-tubulin gene was 71.0, 25.8, and 3.2%, respectively. The data indicate that F. graminearum has developed resistance to carbendazim in Henan Province, and single point mutations at amino acid F167Y were the predominant type of mutation detected.

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