Mapping the growth effect of previously hidden ubiquitin alleles using an overexpression based mutational scan

AbstractDeep mutational scanning has emerged as a powerful, high throughput approach to determine the growth effect of thousands of alleles at once in bulk competition. However, to date only the growth effect of mutant alleles in isolation has been determined. Building off previous work, we have created a library of all possible single point mutations in ubiquitin and determined the growth effect of mutants overexpressed in the presence of a wild type allele. Using this scan, we explained over half of the previously missing mutants in the single allele scan by showing that they exhibit deleterious effects when co-expressed with wild type ubiquitin. Additionally, unlike the single allele growth effect, these overexpression growth effects were distributed across the entire protein. This overexpression scan methodology can identify likely dominant mutant effects in any essential gene and is highly complementary with traditional deep mutational scanning approaches.

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Structural elements within the methylation loop (residues 112–117) and EF hands III and IV of calmodulin are required for Lys115 trimethylation

Biochemical Journal ◽

10.1042/bj3400417 ◽

1999 ◽

Vol 340 (2) ◽

pp. 417-424 ◽

Cited By ~ 2

Author(s):

Jennifer A. COBB ◽

Chang-Hoon HAN ◽

David M. WILLS ◽

Daniel M. ROBERTS

Keyword(s):

Single Point ◽

Point Mutations ◽

Site Directed Mutagenesis ◽

Structural Elements ◽

Nad Kinase ◽

Wild Type ◽

Loop Sequence ◽

Ef Hand ◽

Single Point Mutations ◽

Ef Hands

Calmodulin is trimethylated by a specific methyltransferase on Lys115, a residue located in a six amino acid loop (LGEKLT) between EF hands III and IV. To investigate the structural requirements for methylation, domain exchange mutants as well as single point mutations of conserved methylation loop residues (E114A, Glu114 → Ala; L116T, Leu116 → Thr) were generated. E114A and L116T activated cyclic nucleotide phosphodiesterase (PDE) and NAD+ kinase (NADK) similar to wild-type calmodulin, but lost their ability to be methylated. Domain exchange mutants in which EF hand III or IV was replaced by EF hand I or II respectively (CaM1214 and CaM1232 respectively) showed a modest effect on PDE and NADK activation (50 to 100% of wild-type), but calmodulin methylation was abolished. A third domain exchange mutant, CaMEKL, has the methylation loop sequence placed at a symmetrical position between EF hands I and II in the N-terminal lobe [residues QNP(41-43) replaced by EKL]. CaMEKL activated PDE normally, but did not activate NADK. However, CaMEKL retained the ability to bind to NADK and inhibited activation by wild-type calmodulin. Site-directed mutagenesis of single residues showed that Gln41 and Pro43 substitutions had the strongest effect on NADK activation. Additionally, CaMEKL was not methylated, suggesting that the introduction of the methylation loop between EF hands I and II is not adequate for methyltransferase recognition. Overall the data indicate that residues in the methylation loop are essential but not sufficient for methyltransferase recognition, and that additional residues unique to EF hands III and IV are required. Secondly, the QNP sequence in the loop between EF hands I and II is necessary for NADK activation.

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Modulation of the Mechanisms Driving Transthyretin Amyloidosis

Frontiers in Molecular Neuroscience ◽

10.3389/fnmol.2020.592644 ◽

2020 ◽

Vol 13 ◽

Author(s):

Filipa Bezerra ◽

Maria João Saraiva ◽

Maria Rosário Almeida

Keyword(s):

Amyloid Fibrils ◽

Single Point ◽

Point Mutations ◽

Disease Process ◽

Wild Type ◽

Transthyretin Amyloidosis ◽

Natural Ligand ◽

Single Point Mutations ◽

Pharmacologic Agents ◽

Plasma Stabilization

Transthyretin (TTR) amyloidoses are systemic diseases associated with TTR aggregation and extracellular deposition in tissues as amyloid. The most frequent and severe forms of the disease are hereditary and associated with amino acid substitutions in the protein due to single point mutations in the TTR gene (ATTRv amyloidosis). However, the wild type TTR (TTR wt) has an intrinsic amyloidogenic potential that, in particular altered physiologic conditions and aging, leads to TTR aggregation in people over 80 years old being responsible for the non-hereditary ATTRwt amyloidosis. In normal physiologic conditions TTR wt occurs as a tetramer of identical subunits forming a central hydrophobic channel where small molecules can bind as is the case of the natural ligand thyroxine (T4). However, the TTR amyloidogenic variants present decreased stability, and in particular conditions, dissociate into partially misfolded monomers that aggregate and polymerize as amyloid fibrils. Therefore, therapeutic strategies for these amyloidoses may target different steps in the disease process such as decrease of variant TTR (TTRv) in plasma, stabilization of TTR, inhibition of TTR aggregation and polymerization or disruption of the preformed fibrils. While strategies aiming decrease of the mutated TTR involve mainly genetic approaches, either by liver transplant or the more recent technologies using specific oligonucleotides or silencing RNA, the other steps of the amyloidogenic cascade might be impaired by pharmacologic compounds, namely, TTR stabilizers, inhibitors of aggregation and amyloid disruptors. Modulation of different steps involved in the mechanism of ATTR amyloidosis and compounds proposed as pharmacologic agents to treat TTR amyloidosis will be reviewed and discussed.

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HIV recombination: what is the impact on antiretroviral therapy?

Journal of The Royal Society Interface ◽

10.1098/rsif.2005.0064 ◽

2005 ◽

Vol 2 (5) ◽

pp. 489-503 ◽

Cited By ~ 50

Author(s):

Christophe Fraser

Keyword(s):

Antiretroviral Therapy ◽

Single Point ◽

Point Mutations ◽

Wild Type Virus ◽

Wild Type ◽

Genetic Barrier ◽

Resistant Strains ◽

Single Point Mutations ◽

The Impact ◽

Retroviral Recombination

Retroviral recombination is a potential mechanism for the development of multiply drug resistant viral strains but the impact on the clinical outcomes of antiretroviral therapy in HIV-infected patients is unclear. Recombination can favour resistance by combining single-point mutations into a multiply resistant genome but can also hinder resistance by breaking up associations between mutations. Previous analyses, based on population genetic models, have suggested that whether recombination is favoured or hindered depends on the fitness interactions between loci, or epistasis. In this paper, a mathematical model is developed that includes viral dynamics during therapy and shows that population dynamics interact non-trivially with population genetics. The outcome of therapy depends critically on the changes to the frequency of cell co-infection and I review the evidence available. Where recombination does have an effect on therapy, it is always to slow or even halt the emergence of multiply resistant strains. I also find that for patients newly infected with multiply resistant strains, recombination can act to prevent reversion to wild-type virus. The analysis suggests that treatment targeted at multiple parts of the viral life-cycle may be less prone to drug resistance due to the genetic barrier caused by recombination but that, once selected, mutants resistant to such regimens may be better able to persist in the population.

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Dinitroanilines Bind α-Tubulin to Disrupt Microtubules

Molecular Biology of the Cell ◽

10.1091/mbc.e03-07-0530 ◽

2004 ◽

Vol 15 (4) ◽

pp. 1960-1968 ◽

Cited By ~ 101

Author(s):

Naomi S. Morrissette ◽

Arpita Mitra ◽

David Sept ◽

L. David Sibley

Keyword(s):

Bos Taurus ◽

Single Point ◽

Point Mutations ◽

Docking Studies ◽

Chemical Mutagenesis ◽

Wild Type ◽

Protozoan Parasites ◽

Resistant Lines ◽

Single Point Mutations ◽

A Site

Protozoan parasites are remarkably sensitive to dinitroanilines such as oryzalin, which disrupt plant but not animal microtubules. To explore the basis of dinitroaniline action, we isolated 49 independent resistant Toxoplasma gondii lines after chemical mutagenesis. All 23 of the lines that we examined harbored single point mutations in α-tubulin. These point mutations were sufficient to confer resistance when transfected into wild-type parasites. Several mutations were in the M or N loops, which coordinate protofilament interactions in the microtubule, but most of the mutations were in the core of α-tubulin. Docking studies predict that oryzalin binds with an average affinity of 23 nM to a site located beneath the N loop of Toxoplasma α-tubulin. This binding site included residues that were mutated in several resistant lines. Moreover, parallel analysis of Bos taurus α-tubulin indicated that oryzalin did not interact with this site and had a significantly decreased, nonspecific affinity for vertebrate α-tubulin. We propose that the dinitroanilines act through a novel mechanism, by disrupting M-N loop contacts. These compounds also represent the first class of drugs that act on α-tubulin function.

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gyrA Mutations in Ciprofloxacin-Resistant Bartonella bacilliformis Strains Obtained In Vitro

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.47.1.383-386.2003 ◽

2003 ◽

Vol 47 (1) ◽

pp. 383-386 ◽

Cited By ~ 20

Author(s):

Michael F. Minnick ◽

Zachary R. Wilson ◽

Laura S. Smitherman ◽

D. Scott Samuels

Keyword(s):

Single Point ◽

Point Mutations ◽

Dna Gyrase ◽

Wild Type ◽

Bartonella Bacilliformis ◽

E Coli ◽

A Subunit ◽

Resistant Strains ◽

Single Point Mutations

ABSTRACT We isolated and characterized mutants of Bartonella bacilliformis that are resistant to the fluoroquinolone antibiotic ciprofloxacin, which targets the A subunit of DNA gyrase. Mutants had single point mutations in the gyrA gene that changed either Asp-90 to Gly or Asp-95 to Asn and had 3- or 16-fold higher resistance, respectively, to ciprofloxacin than did wild-type B. bacilliformis. Asp-95 is homologous to Asp-87 of Escherichia coli GyrA and is a common residue mutated in fluoroquinolone-resistant strains of other bacteria. This is the first report of a mutation at an Asp-90 homologue, which corresponds to Asp-82 in E. coli GyrA.

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

10.26434/chemrxiv.11481834.v1 ◽

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.

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Single-Point Mutations in Qβ Virus-like Particles Change Binding to Cells

Biomacromolecules ◽

10.1021/acs.biomac.1c00443 ◽

2021 ◽

Author(s):

Marisa L. Martino ◽

Stephen N. Crooke ◽

Marianne Manchester ◽

M.G. Finn

Keyword(s):

Single Point ◽

Point Mutations ◽

Virus Like Particles ◽

Single Point Mutations

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MinD directly interacting with FtsZ at the H10 helix suggests a model for robust activation of MinC to destabilize FtsZ polymers

Biochemical Journal ◽

10.1042/bcj20170357 ◽

2017 ◽

Vol 474 (18) ◽

pp. 3189-3205 ◽

Cited By ~ 6

Author(s):

Ashoka Chary Taviti ◽

Tushar Kant Beuria

Keyword(s):

Cell Division ◽

Single Point ◽

Point Mutations ◽

Direct Interaction ◽

Ring Formation ◽

Z Ring ◽

Single Point Mutations ◽

Biochemical Analyses ◽

Ftsz Assembly ◽

The Mind

Cell division in bacteria is a highly controlled and regulated process. FtsZ, a bacterial cytoskeletal protein, forms a ring-like structure known as the Z-ring and recruits more than a dozen other cell division proteins. The Min system oscillates between the poles and inhibits the Z-ring formation at the poles by perturbing FtsZ assembly. This leads to an increase in the FtsZ concentration at the mid-cell and helps in Z-ring positioning. MinC, the effector protein, interferes with Z-ring formation through two different mechanisms mediated by its two domains with the help of MinD. However, the mechanism by which MinD triggers MinC activity is not yet known. We showed that MinD directly interacts with FtsZ with an affinity stronger than the reported MinC–FtsZ interaction. We determined the MinD-binding site of FtsZ using computational, mutational and biochemical analyses. Our study showed that MinD binds to the H10 helix of FtsZ. Single-point mutations at the charged residues in the H10 helix resulted in a decrease in the FtsZ affinity towards MinD. Based on our findings, we propose a novel model for MinCD–FtsZ interaction, where MinD through its direct interaction with FtsZ would trigger MinC activity to inhibit FtsZ functions.

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