Protein flexibility and dissociation pathway differentiation can explain onset of resistance mutations in kinases

Understanding how point mutations can render a ligand or a drug ineffective against a given biological target is a problem of immense fundamental and practical relevance. Often the efficacy of such resistance mutations can be explained purely on a thermodynamic basis wherein the mutated system displays a reduced binding affinity for the ligand. However, the more perplexing and harder to explain situation is when two protein sequences have the same binding affinity for a drug. In this work, we demonstrate how all-atom molecular dynamics simulations, specifically using recent developments grounded in statistical mechanics and information theory, can provide a detailed mechanistic rationale for such variances. We establish the dissociation mechanism for the popular anti-cancer drug Imatinib (Gleevec) against wild-type and N387S mutant of Abl kinase. We show how this single point mutation triggers a non-local response in the protein's flexibility and eventually leads to pathway differentiation during dissociation. This pathway differentiation explains why Gleevec has a long residence time in the wild-type Abl, but for the mutant, by opening up a backdoor pathway for ligand exit, an order of magnitude shorter residence time is obtained. We thus believe that this work marks an efficient and scalable approach to pinpoint the molecular determinants of resistance mutations in biomolecular receptors of pharmacological relevance that are hard to explain using a simple structural perspective and require mechanistic and kinetic insights.

Download Full-text

Genetic perturbation enhances functional heterogeneity in alkaline phosphatase

10.1101/2021.11.25.470051 ◽

2021 ◽

Author(s):

Morito Sakuma ◽

Shingo Honda ◽

Hiroshi Ueno ◽

Kentaro Miyazaki ◽

Nobuhiko Tokuriki ◽

...

Keyword(s):

Alkaline Phosphatase ◽

Catalytic Activity ◽

Single Molecule ◽

Single Point ◽

Point Mutations ◽

Clear Correlation ◽

Wild Type ◽

Functional Heterogeneity ◽

Genetic Perturbations ◽

In The Wild

Enzymes inherently exhibit molecule-to-molecule heterogeneity in catalytic activity or function, which underlies the acquisition of new functions in evolutionary processes. However, correlations between the functional heterogeneity of an enzyme and its multi-functionality or promiscuity remain elusive. In addition, the modulation of functional heterogeneity upon genetic perturbation is currently unexplored. Here, we quantitatively analyzed functional heterogeneity in the wild-type and 69 single-point mutants of Escherichia coli alkaline phosphatase (AP) by employing single-molecule assay with a femtoliter reactor array device. Most mutant enzymes exhibited higher functional heterogeneity than the wild-type enzyme, irrespective of catalytic activity. These results indicated that the wild-type AP minimizes functional heterogeneity, and single-point mutations can significantly expand the span of functional heterogeneity in AP. Moreover, we identified a clear correlation between functional heterogeneity and promiscuous activities. These findings suggest that enzymes can acquire greater functional heterogeneity following marginal genetic perturbations that concomitantly promote catalytic promiscuity.

Download Full-text

mmCSM-AB: guiding rational antibody engineering through multiple point mutations

Nucleic Acids Research ◽

10.1093/nar/gkaa389 ◽

2020 ◽

Vol 48 (W1) ◽

pp. W125-W131 ◽

Cited By ~ 2

Author(s):

Yoochan Myung ◽

Douglas E V Pires ◽

David B Ascher

Keyword(s):

Binding Affinity ◽

Single Point ◽

Point Mutations ◽

Affinity Maturation ◽

Antibody Engineering ◽

Multiple Point ◽

Single Point Mutation ◽

Atomic Interaction ◽

Therapeutic Class ◽

Antibody Design

Abstract While antibodies are becoming an increasingly important therapeutic class, especially in personalized medicine, their development and optimization has been largely through experimental exploration. While there have been many efforts to develop computational tools to guide rational antibody engineering, most approaches are of limited accuracy when applied to antibody design, and have largely been limited to analysing a single point mutation at a time. To overcome this gap, we have curated a dataset of 242 experimentally determined changes in binding affinity upon multiple point mutations in antibody-target complexes (89 increasing and 153 decreasing binding affinity). Here, we have shown that by using our graph-based signatures and atomic interaction information, we can accurately analyse the consequence of multi-point mutations on antigen binding affinity. Our approach outperformed other available tools across cross-validation and two independent blind tests, achieving Pearson's correlations of up to 0.95. We have implemented our new approach, mmCSM-AB, as a web-server that can help guide the process of affinity maturation in antibody design. mmCSM-AB is freely available at http://biosig.unimelb.edu.au/mmcsm_ab/.

Download Full-text

An Autism-Associated Neuroligin-3 Mutation Affects Developmental Synapse Elimination in the Cerebellum

Frontiers in Neural Circuits ◽

10.3389/fncir.2021.676891 ◽

2021 ◽

Vol 15 ◽

Author(s):

Esther Suk King Lai ◽

Hisako Nakayama ◽

Taisuke Miyazaki ◽

Takanobu Nakazawa ◽

Katsuhiko Tabuchi ◽

...

Keyword(s):

Cell Adhesion Molecule ◽

Synapse Formation ◽

Single Point ◽

Autism Spectrum ◽

Mutant Mice ◽

Single Point Mutation ◽

Synapse Elimination ◽

Wild Type ◽

Post Mortem Brain ◽

And Function

Neuroligin is a postsynaptic cell-adhesion molecule that is involved in synapse formation and maturation by interacting with presynaptic neurexin. Mutations in neuroligin genes, including the arginine to cystein substitution at the 451st amino acid residue (R451C) of neuroligin-3 (NLGN3), have been identified in patients with autism spectrum disorder (ASD). Functional magnetic resonance imaging and examination of post-mortem brain in ASD patients implicate alteration of cerebellar morphology and Purkinje cell (PC) loss. In the present study, we examined possible association between the R451C mutation in NLGN3 and synaptic development and function in the mouse cerebellum. In NLGN3-R451C mutant mice, the expression of NLGN3 protein in the cerebellum was reduced to about 10% of the level of wild-type mice. Elimination of redundant climbing fiber (CF) to PC synapses was impaired from postnatal day 10–15 (P10–15) in NLGN3-R451C mutant mice, but majority of PCs became mono-innervated as in wild-type mice after P16. In NLGN3-R451C mutant mice, selective strengthening of a single CF relative to the other CFs in each PC was impaired from P16, which persisted into juvenile stage. Furthermore, the inhibition to excitation (I/E) balance of synaptic inputs to PCs was elevated, and calcium transients in the soma induced by strong and weak CF inputs were reduced in NLGN3-R451C mutant mice. These results suggest that a single point mutation in NLGN3 significantly influences the synapse development and refinement in cerebellar circuitry, which might be related to the pathogenesis of ASD.

Download Full-text

Mutational Analysis of Quinolone-Resistant Determining Region gyrA and parC Genes in Quinolone-Resistant ESBL-Producing E. Coli

IIUM Medical Journal Malaysia ◽

10.31436/imjm.v20i3.1825 ◽

2021 ◽

Vol 20 (3) ◽

Author(s):

Hairul Aini Hamzah ◽

Rahmatullah Sirat ◽

Mohammed Imad A. Mustafa Mahmud ◽

Roesnita Baharudin

Keyword(s):

Mutational Analysis ◽

Single Point ◽

Point Mutations ◽

Multidrug Resistant ◽

Quinolone Resistance ◽

Resistant Bacteria ◽

Single Point Mutation ◽

Phenotypic Resistance ◽

Drug Effectiveness ◽

E Coli

Introduction: Co-resistance to quinolones among extended spectrum β[1]lactamase (ESBL)-producing E. coli commonly occurs in clinical settings. Quinolones act on DNA gyrase and DNA topoisomerase enzymes, which are coded by gyrA and parC genes, thus any mutation to the genes may affect the drug effectiveness. The objective of the study was to characterize gyrA and parC genes in quinolone-resistant E. coli isolates and correlated the mutations with their phenotypic resistance. Materials and Methods: Thirty-two quinolone-resistant (QR) and six quinolone-sensitive (QS) ESBL-E. coli isolates were identified by antibiotic susceptibility and minimum inhibitory concentration tests. Bioinformatics analysis were conducted to study any mutations occurred in the genes and generate their codon compositions. Results: All the QR ESBL-E. coli isolates were identified as multidrug-resistant bacteria. A single point mutation in the quinolone resistance-determining region (QRDR) of gyrA, at codon 83, caused the substitution amino acid Ser83Leu. It is associated with a high level of resistance to nalidixic acid. However, double mutations Ser83Leu and Asp87Asn in the same region were significantly linked to higher levels of resistance to ciprofloxacin. Cumulative point mutations in gyrA and/or in parC were also correlated significantly (p<0.05) to increased resistance to ciprofloxacin. Conclusion: Together, the findings showed that the mutations in gyrA and parC genes handled the institution of intrinsic quinolone resistance in the ESBL-E. coli isolates. Thus, vigilant monitoring for emergence of new mutation in resistance genes may give an insight into dissemination of QR ESBL-E. coli in a particular region.

Download Full-text

FtsZ-Dependent Elongation of a Coccoid Bacterium

mBio ◽

10.1128/mbio.00908-16 ◽

2016 ◽

Vol 7 (5) ◽

Cited By ~ 13

Author(s):

Ana R. Pereira ◽

Jen Hsin ◽

Ewa Król ◽

Andreia C. Tavares ◽

Pierre Flores ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Cell Shape ◽

Single Point ◽

Spherical Shape ◽

Single Point Mutation ◽

Wild Type ◽

Dead End ◽

Dynamics Simulations

ABSTRACT A mechanistic understanding of the determination and maintenance of the simplest bacterial cell shape, a sphere, remains elusive compared with that of more complex shapes. Cocci seem to lack a dedicated elongation machinery, and a spherical shape has been considered an evolutionary dead-end morphology, as a transition from a spherical to a rod-like shape has never been observed in bacteria. Here we show that a Staphylococcus aureus mutant (M5) expressing the ftsZ G193D allele exhibits elongated cells. Molecular dynamics simulations and in vitro studies indicate that FtsZ G193D filaments are more twisted and shorter than wild-type filaments. In vivo , M5 cell wall deposition is initiated asymmetrically, only on one side of the cell, and progresses into a helical pattern rather than into a constricting ring as in wild-type cells. This helical pattern of wall insertion leads to elongation, as in rod-shaped cells. Thus, structural flexibility of FtsZ filaments can result in an FtsZ-dependent mechanism for generating elongated cells from cocci. IMPORTANCE The mechanisms by which bacteria generate and maintain even the simplest cell shape remain an elusive but fundamental question in microbiology. In the absence of examples of coccus-to-rod transitions, the spherical shape has been suggested to be an evolutionary dead end in morphogenesis. We describe the first observation of the generation of elongated cells from truly spherical cocci, occurring in a Staphylococcus aureus mutant containing a single point mutation in its genome, in the gene encoding the bacterial tubulin homologue FtsZ. We demonstrate that FtsZ-dependent cell elongation is possible, even in the absence of dedicated elongation machinery.

Download Full-text

An Unusual Response Regulator Influences Sporulation at Early and Late Stages in Streptomyces coelicolor

Journal of Bacteriology ◽

10.1128/jb.01615-06 ◽

2007 ◽

Vol 189 (7) ◽

pp. 2873-2885 ◽

Cited By ~ 24

Author(s):

Yuqing Tian ◽

Kay Fowler ◽

Kim Findlay ◽

Huarong Tan ◽

Keith F. Chater

Keyword(s):

Streptomyces Coelicolor ◽

Point Mutations ◽

Aerial Mycelium ◽

Site Directed Mutagenesis ◽

Null Mutant ◽

Wild Type ◽

New Variant ◽

In The Wild ◽

Pcr Targeting ◽

Spore Maturation

ABSTRACT WhiI, a regulator required for efficient sporulation septation in the aerial mycelium of Streptomyces coelicolor, resembles response regulators of bacterial two-component systems but lacks some conserved features of typical phosphorylation pockets. Four amino acids of the abnormal “phosphorylation pocket” were changed by site-directed mutagenesis. Unlike whiI null mutations, these point mutations did not interfere with sporulation septation but had various effects on spore maturation. Transcriptome analysis was used to compare gene expression in the wild-type strain, a D27A mutant (pale gray spores), a D69E mutant (wild-type spores), and a null mutant (white aerial mycelium, no spores) (a new variant of PCR targeting was used to introduce the point mutations into the chromosomal copy of whiI). The results revealed 45 genes that were affected by the deletion of whiI. Many of these showed increased expression in the wild type at the time when aerial growth and development were taking place. About half of them showed reduced expression in the null mutant, and about half showed increased expression. Some, but not all, of these 45 genes were also affected by the D27A mutation, and a few were affected by the D69E mutation. The results were consistent with a model in which WhiI acts differently at sequential stages of development. Consideration of the functions of whiI-influenced genes provides some insights into the physiology of aerial hyphae. Mutation of seven whiI-influenced genes revealed that three of them play roles in spore maturation.

Download Full-text

Definitive demonstration by synthesis of genome annotation completeness

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1905990116 ◽

2019 ◽

Vol 116 (48) ◽

pp. 24206-24213 ◽

Cited By ~ 7

Author(s):

Paul R. Jaschke ◽

Gabrielle A. Dotson ◽

Kay S. Hung ◽

Diane Liu ◽

Drew Endy

Keyword(s):

Essential Gene ◽

Single Point ◽

Viable Cell ◽

Essential Genes ◽

Single Point Mutation ◽

Evolutionary Adaptation ◽

Wild Type ◽

Bioinformatic Tools ◽

Design And Synthesis ◽

New Genes

We develop a method for completing the genetics of natural living systems by which the absence of expected future discoveries can be established. We demonstrate the method using bacteriophage øX174, the first DNA genome to be sequenced. Like many well-studied natural organisms, closely related genome sequences are available—23 Bullavirinae genomes related to øX174. Using bioinformatic tools, we first identified 315 potential open reading frames (ORFs) within the genome, including the 11 established essential genes and 82 highly conserved ORFs that have no known gene products or assigned functions. Using genome-scale design and synthesis, we made a mutant genome in which all 11 essential genes are simultaneously disrupted, leaving intact only the 82 conserved but cryptic ORFs. The resulting genome is not viable. Cell-free gene expression followed by mass spectrometry revealed only a single peptide expressed from both the cryptic ORF and wild-type genomes, suggesting a potential new gene. A second synthetic genome in which 71 conserved cryptic ORFs were simultaneously disrupted is viable but with ∼50% reduced fitness relative to the wild type. However, rather than finding any new genes, repeated evolutionary adaptation revealed a single point mutation that modulates expression of gene H, a known essential gene, and fully suppresses the fitness defect. Taken together, we conclude that the annotation of currently functional ORFs for the øX174 genome is formally complete. More broadly, we show that sequencing and bioinformatics followed by synthesis-enabled reverse genomics, proteomics, and evolutionary adaptation can definitely establish the sufficiency and completeness of natural genome annotations.

Download Full-text

In Silico Insights into HIV-1 Vpu-Tetherin Interactions and Its Mutational Counterparts

Medical Sciences ◽

10.3390/medsci7060074 ◽

2019 ◽

Vol 7 (6) ◽

pp. 74

Author(s):

Patil Sneha ◽

Urmi Shah ◽

Seetharaman Balaji

Keyword(s):

Cell Surface ◽

Binding Affinity ◽

Hydrophobic Interactions ◽

Single Point ◽

Resistance Mechanism ◽

Point Mutations ◽

Protein Docking ◽

Host Protein ◽

Single Point Mutations ◽

Hiv 1

Tetherin, an interferon-induced host protein encoded by the bone marrow stromal antigen 2 (BST2/CD317/HM1.24) gene, is involved in obstructing the release of many retroviruses and other enveloped viruses by cross-linking the budding virus particles to the cell surface. This activity is antagonized in the case of human immunodeficiency virus (HIV)-1 wherein its accessory protein Viral Protein U (Vpu) interacts with tetherin, causing its downregulation from the cell surface. Vpu and tetherin connect through their transmembrane (TM) domains, culminating into events leading to tetherin degradation by recruitment of β-TrCP2. However, mutations in the TM domains of both proteins are reported to act as a resistance mechanism to Vpu countermeasure impacting tetherin’s sensitivity towards Vpu but retaining its antiviral activity. Our study illustrates the binding aspects of blood-derived, brain-derived, and consensus HIV-1 Vpu with tetherin through protein–protein docking. The analysis of the bound complexes confirms the blood-derived Vpu–tetherin complex to have the best binding affinity as compared to other two. The mutations in tetherin and Vpu are devised computationally and are subjected to protein–protein interactions. The complexes are tested for their binding affinities, residue connections, hydrophobic forces, and, finally, the effect of mutation on their interactions. The single point mutations in tetherin at positions L23Y, L24T, and P40T, and triple mutations at {L22S, F44Y, L37I} and {L23T, L37T, T45I}, while single point mutations in Vpu at positions A19H and W23Y and triplet of mutations at {V10K, A11L, A19T}, {V14T, I18T, I26S}, and {A11T, V14L, A15T} have revealed no polar contacts with minimal hydrophobic interactions between Vpu and tetherin, resulting in reduced binding affinity. Additionally, we have explored the aggregation potential of tetherin and its association with the brain-derived Vpu protein. This work is a possible step toward an understanding of Vpu–tetherin interactions.

Download Full-text

ethA, inhA, and katG Loci of Ethionamide-Resistant Clinical Mycobacterium tuberculosis Isolates

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.47.12.3799-3805.2003 ◽

2003 ◽

Vol 47 (12) ◽

pp. 3799-3805 ◽

Cited By ~ 159

Author(s):

Glenn P. Morlock ◽

Beverly Metchock ◽

David Sikes ◽

Jack T. Crawford ◽

Robert C. Cooksey

Keyword(s):

Mycobacterium Tuberculosis ◽

Structural Gene ◽

Single Point ◽

Resistance Mechanism ◽

Structural Analog ◽

Mycolic Acid ◽

Single Point Mutation ◽

Structural Genes ◽

Resistance Mutations ◽

Catalase Peroxidase

ABSTRACT Ethionamide (ETH) is a structural analog of the antituberculosis drug isoniazid (INH). Both of these drugs target InhA, an enzyme involved in mycolic acid biosynthesis. INH requires catalase-peroxidase (KatG) activation, and mutations in katG are a major INH resistance mechanism. Recently an enzyme (EthA) capable of activating ETH has been identified. We sequenced the entire ethA structural gene of 41 ETH-resistant Mycobacterium tuberculosis isolates. We also sequenced two regions of inhA and all or part of katG. The MICs of ETH and INH were determined in order to associate the mutations identified with a resistance phenotype. Fifteen isolates were found to possess ethA mutations, for all of which the ETH MICs were ≥50 μg/ml. The ethA mutations were all different, previously unreported, and distributed throughout the gene. In eight of the isolates, a missense mutation in the inhA structural gene occurred. The ETH MICs for seven of the InhA mutants were ≥100 μg/ml, and these isolates were also resistant to ≥8 μg of INH per ml. Only a single point mutation in the inhA promoter was identified in 14 isolates. A katG mutation occurred in 15 isolates, for which the INH MICs for all but 1 were ≥32 μg/ml. As expected, we found no association between katG mutation and the level of ETH resistance. Mutations within the ethA and inhA structural genes were associated with relatively high levels of ETH resistance. Approximately 76% of isolates resistant to ≥50 μg of ETH per ml had such mutations.

Download Full-text

C1GALT1C1 (COSMC) Mutations in Four Individuals with Tn+ Phenotype.

Blood ◽

10.1182/blood.v110.11.1727.1727 ◽

2007 ◽

Vol 110 (11) ◽

pp. 1727-1727

Author(s):

Vanja Karamatic Crew ◽

Carole Green ◽

Stephen Parsons ◽

Belinda K. Singleton ◽

Geoff Daniels ◽

...

Keyword(s):

Cell Line ◽

Dna Analysis ◽

Single Point ◽

Jurkat Cells ◽

Erythroid Cell ◽

Single Point Mutation ◽

Wild Type ◽

Membrane Glycoproteins ◽

Hemopoietic Cells ◽

Elevated Expression

Abstract Tn is a cryptantigen located on O-linked oligosaccharides of cell membrane glycoproteins and is composed of N-acetylgalactosamine a -linked to serine or threonine of the protein backbone. Tn is expressed on hemopoietic cells of individuals with the rare idiopathic Tn syndrome, characterized by a variable pattern of Tn expression suggestive of its somatic and clonal origin. Tn is also associated with overexpression in several autoimmune diseases and malignancies. Tn is a bioprecursor of the T cryptantigen and subsequently the disialotetrasaccharide units typical of O-linked oligosaccharides of RBC sialoglycoproteins. Tn results from defective oligosaccharide biosynthesis caused by the malfunction of T-synthase, a b1,3-galactosyltransferase. Recent evidence suggested that the activity of T-synthase is dependent on a molecular chaperone, Cosmc (Ju and Cummings, PNAS2002;99:16613–18). In view of this evidence, we investigated whether Cosmc is required for T-synthase activity and ultimately for Tn phenotype by obtaining material from 4 apparently healthy, unrelated Caucasian individuals with Tn phenotype. Case 1 was 93.9% Tn+ on an EBV-transformed lymphoblastoma cell line. Analysis of the Cosmc gene, C1GALT1C1, showed a homozygous 428C>T, Ala143Val mutation and Case 1 showed a complete lack of expression of C1GALT1C1 cDNA. Case 2 revealed no C1GALT1C1 mutations in DNA extracted from plasma, but in DNA of lymphocyte origin an apparent heterozygous 454G>A, Glu152Lys change was observed, in agreement with Ju and Cummings (Nature2005, 437:1252). When Case 2 hemopoietic progenitor cells were expanded into the erythroid cell line, the mutation appeared homozygous. Tn expression varied from 76.1% in lymphocytes, 90.7% in RBCs to 96.9% in erythroblasts. Case 3 showed 19.4% Tn+ lymphocytes, compared to 97.0% Tn+ RBCs. In Case 3 we found a single point mutation 577T>C, Ser193Pro. Case 4 exhibited 46.5% Tn+ lymphocytes, 96.0% Tn+ RBCs and 90.2% Tn+ erythroblasts. DNA analysis revealed 3G>C, converting the translation-initiating methionine to isoleucine and predicting the loss of first 12 amino acids of the protein, potentially altering its morphology. The mutations found in Cases 2–4 reflected the clonal nature of Tn, appearing heterozygous in DNA of lymphocyte origin and homozygous in DNA of erythroid origin. To confirm that the observed mutations are indeed responsible for Tn phenotype, pBabe puro vector with Tn or wild type C1GALT1C1 inserts was transfected into Jurkat cells. Untransfected cells and cells transfected with bare vector expressed Tn. Jurkat cells transfected with wild-type C1GALT1C1 were Tn-negative while those transfected with C1GALT1C1 from Cases 2–4 expressed Tn. From this evidence we postulate that Cosmc is directly involved in the expression of Tn phenotype. To investigate the involvement of other genes, we performed expression profiling of 3 Tn and 4 control samples hybridized to HG-U133A arrays. A list of 100 up-regulated and 173 down-regulated genes, with 1.5× fold difference in expression, was obtained. Some genes, relating to erythrocyte development/heme biosynthesis were upregulated, while down-regulated genes were related to cholesterol/lipid metabolism. Real-time Q-PCR on six differentially expressed genes of interest, down-regulated FABP5, CYP1B1 and LRP8 and up-regulated AQP1, AQP3 and EPB42, confirmed the microarray results, and elevated expression of AQP3 on Tn + RBCs was detected serologically. The effects of C1GALT1C1 mutations are wider than Tn expression on hemopoietic cells.

Download Full-text