Chemical RNA digestion enables robust RNA-binding site mapping at single amino acid resolution

The RNA-binding domain of the protein kinase DAI, the double-stranded RNA inhibitor of translation, contains two repeats of a motif that is also found in a number of other RNA-binding proteins. This motif consists of 67 amino acid residues and is predicted to contain a positively charged alpha helix at its C terminus. We have analyzed the effects of equivalent single amino acid changes in three conserved residues distributed over each copy of the motif. Mutants in the C-terminal portion of either repeat were severely defective, indicating that both copies of the motif are essential for RNA binding. Changes in the N-terminal and central parts of the motif were more debilitating if they were made in the first motif than in the second, suggesting that the first motif is the more important for RNA binding and that the second motif is structurally more flexible. When the second motif was replaced by a duplicate of the first motif, the ectopic copy retained its greater sensitivity to mutation, implying that the two motifs have distinct functions with respect to the process of RNA binding. Furthermore, the mutations have the same effect on the binding of double-stranded RNA and VA RNA, consistent with the existence of a single RNA-binding domain for both activating and inhibitory RNAs.

Download Full-text

G- to F-actin modulation by a single amino acid substitution in the actin binding site of actobindin and thymosin beta 4.

The EMBO Journal ◽

10.1002/j.1460-2075.1992.tb05579.x ◽

1992 ◽

Vol 11 (13) ◽

pp. 4739-4746 ◽

Cited By ~ 60

Author(s):

K. Vancompernolle ◽

M. Goethals ◽

C. Huet ◽

D. Louvard ◽

J. Vandekerckhove

Keyword(s):

Amino Acid ◽

Binding Site ◽

Amino Acid Substitution ◽

Single Amino Acid Substitution ◽

Single Amino Acid ◽

Actin Binding ◽

Thymosin Beta 4 ◽

Actin Binding Site

Download Full-text

Mutations in Plasmodium falciparumCytochrome b That Are Associated with Atovaquone Resistance Are Located at a Putative Drug-Binding Site

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.44.8.2100-2108.2000 ◽

2000 ◽

Vol 44 (8) ◽

pp. 2100-2108 ◽

Cited By ~ 232

Author(s):

Michael Korsinczky ◽

Nanhua Chen ◽

Barbara Kotecka ◽

Allan Saul ◽

Karl Rieckmann ◽

...

Keyword(s):

Amino Acid ◽

Binding Site ◽

Point Mutations ◽

Drug Binding ◽

Single Amino Acid ◽

Malaria Parasites ◽

Drug Binding Site ◽

Sole Agent ◽

Amino Acid Mutations

ABSTRACT Atovaquone is the major active component of the new antimalarial drug Malarone. Considerable evidence suggests that malaria parasites become resistant to atovaquone quickly if atovaquone is used as a sole agent. The mechanism by which the parasite develops resistance to atovaquone is not yet fully understood. Atovaquone has been shown to inhibit the cytochrome bc 1 (CYTbc 1) complex of the electron transport chain of malaria parasites. Here we report point mutations in Plasmodium falciparum CYT b that are associated with atovaquone resistance. Single or double amino acid mutations were detected from parasites that originated from a cloned line and survived various concentrations of atovaquone in vitro. A single amino acid mutation was detected in parasites isolated from a recrudescent patient following atovaquone treatment. These mutations are associated with a 25- to 9,354-fold range reduction in parasite susceptibility to atovaquone. Molecular modeling showed that amino acid mutations associated with atovaquone resistance are clustered around a putative atovaquone-binding site. Mutations in these positions are consistent with a reduced binding affinity of atovaquone for malaria parasite CYTb.

Download Full-text

Two Trypanosome-Specific Proteins Are Essential Factors for 5S rRNA Abundance and Ribosomal Assembly in Trypanosoma brucei

Eukaryotic Cell ◽

10.1128/ec.00119-07 ◽

2007 ◽

Vol 6 (10) ◽

pp. 1766-1772 ◽

Cited By ~ 19

Author(s):

Kristina M. Hellman ◽

Martin Ciganda ◽

Silvia V. Brown ◽

Jinlei Li ◽

William Ruyechan ◽

...

Keyword(s):

Amino Acid ◽

Trypanosoma Brucei ◽

Ribosome Biogenesis ◽

Rna Binding ◽

Rna Binding Proteins ◽

5S Rrna ◽

Single Amino Acid ◽

Ribosomal Subunits ◽

Morphological Alterations ◽

Specific Proteins

ABSTRACT We have previously identified and characterized two novel nuclear RNA binding proteins, p34 and p37, which have been shown to bind 5S rRNA in Trypanosoma brucei. These two proteins are nearly identical, with one major difference, an 18-amino-acid insert in the N-terminal region of p37, as well as three minor single-amino-acid differences. Homologues to p34 and p37 have been found only in other trypanosomatids, suggesting that these proteins are unique to this ancient family. We have employed RNA interference (RNAi) studies in order to gain further insight into the interaction between p34 and p37 with 5S rRNA in T. brucei. In our p34/p37 RNAi cells, decreased expression of the p34 and p37 proteins led to morphological alterations, including loss of cell shape and vacuolation, as well as to growth arrest and ultimately to cell death. Disruption of a higher-molecular-weight complex containing 5S rRNA occurs as well as a dramatic decrease in 5S rRNA levels, suggesting that p34 and p37 serve to stabilize 5S rRNA. In addition, an accumulation of 60S ribosomal subunits was observed, accompanied by a significant decrease in overall protein synthesis within p34/p37 RNAi cells. Thus, the loss of the trypanosomatid-specific proteins p34 and p37 correlates with a diminution in 5S rRNA levels as well as a decrease in ribosome activity and an alteration in ribosome biogenesis.

Download Full-text

Loss of Function in Zeaxanthin Epoxidase of Dunaliella tertiolecta Caused by a Single Amino Acid Mutation within the Substrate-Binding Site

Marine Drugs ◽

10.3390/md16110418 ◽

2018 ◽

Vol 16 (11) ◽

pp. 418 ◽

Cited By ~ 2

Author(s):

Minjae Kim ◽

Jisu Kang ◽

Yongsoo Kang ◽

Beom Kang ◽

EonSeon Jin

Keyword(s):

Amino Acid ◽

Binding Site ◽

Conformational Change ◽

Substrate Binding ◽

Single Amino Acid ◽

Wild Type ◽

Amino Acid Mutation ◽

Zeaxanthin Epoxidase ◽

Substrate Binding Site ◽

Single Amino Acid Mutation

The zea1 mutant of marine microalga Dunaliella tertiolecta accumulates zeaxanthin under normal growth conditions, and its phenotype has been speculated to be related to zeaxanthin epoxidase (ZEP). In this study, we isolated the ZEP gene from both wild-type D. tertiolecta and the mutant. We found that the zea1 mutant has a point mutation of the 1337th nucleotide of the ZEP sequence (a change from guanine to adenine), resulting in a change of glycine to aspartate in a highly conserved region in the catalytic domain. Similar expression levels of ZEP mRNA and protein in both wild-type and zea1 were confirmed by using qRT-PCR and western blot analysis, respectively. Additionally, the enzyme activity analysis of ZEPs in the presence of cofactors showed that the inactivation of ZEP in zea1 was not caused by deficiency in the levels of cofactors. From the predicted three-dimensional ZEP structure of zea1, we observed a conformational change on the substrate-binding site in the ZEP. A comparative analysis of the ZEP structures suggested that the conformational change induced by a single amino acid mutation might impact the interaction between the substrate and substrate-binding site, resulting in loss of zeaxanthin epoxidase function.

Download Full-text

Single amino acid substitutions in one Ca2+ binding site of uvomorulin abolish the adhesive function

Cell ◽

10.1016/0092-8674(90)90506-a ◽

1990 ◽

Vol 63 (5) ◽

pp. 1033-1038 ◽

Cited By ~ 201

Author(s):

Masayuki Ozawa ◽

Jürgen Engel ◽

Rolf Kemler

Keyword(s):

Amino Acid ◽

Binding Site ◽

Single Amino Acid ◽

Amino Acid Substitutions

Download Full-text

Catalytic Strategy of Citrate Synthase: Subunit Interactions Revealed as a Consequence of a Single Amino Acid Change in the Oxaloacetate Binding Site

Biochemistry ◽

10.1021/bi00041a003 ◽

1995 ◽

Vol 34 (41) ◽

pp. 13278-13288 ◽

Cited By ~ 13

Author(s):

Linda C. Kurz ◽

Saurabh Shah ◽

Carl Frieden ◽

Tanuj Nakra ◽

Richard E. Stein ◽

...

Keyword(s):

Amino Acid ◽

Binding Site ◽

Amino Acid Change ◽

Citrate Synthase ◽

Single Amino Acid ◽

Subunit Interactions ◽

Single Amino Acid Change

Download Full-text

A single-amino-acid in-frame deletion in CYP17A1 results in combined 17-hydroxylase and 17,20-lyase deficiency in an Iranian family despite the protein mutation site

Human Genome Variation ◽

10.1038/s41439-021-00160-y ◽

2021 ◽

Vol 8 (1) ◽

Author(s):

Ashkan Habib ◽

Alireza Shojazadeh ◽

Mohadeseh Molayemat ◽

Hossein Jafari Khamirani ◽

Sina Zoghi ◽

...

Keyword(s):

Amino Acid ◽

Binding Site ◽

Single Amino Acid ◽

Previous Description ◽

Mutation Site ◽

Protein Mutation ◽

Lyase Deficiency ◽

Redox Partner ◽

Iranian Family ◽

Homozygous Mutations

AbstractIn this study, we detected homozygous mutations in the CYP17A1 gene (NM_000102.4:c.1053_1055delCCT; p.Leu353del; SCV001479329) in a 28-year-old female patient (46,XX) and her phenotypically female 30-year-old sister (46,XY) who had phenotypes consistent with combined 17-hydroxylase and 17,20-lyase deficiency. The phenotypes were not expected based on the location of the mutation in the CYP17A1 redox partner-binding site and a previous description of the same mutation linked with isolated 17,20-lyase deficiency.

Download Full-text

Attenuation of Getah virus by a single amino acid substitution at residue 253 of the E2 protein that might be part of a new heparan sulfate binding site on alphaviruses

Journal of Virology ◽

10.1128/jvi.01751-21 ◽

2022 ◽

Author(s):

Ningning Wang ◽

Xiaofeng Zhai ◽

Xiaoling Li ◽

Yu Wang ◽

Wan-ting He ◽

...

Keyword(s):

Amino Acid ◽

Mouse Model ◽

Heparan Sulfate ◽

Binding Site ◽

Amino Acid Substitution ◽

Single Amino Acid ◽

Virus Clearance ◽

E2 Glycoprotein ◽

Attachment Factor

The emergence of new epidemic variants of alphaviruses poses a public health risk. It is associated with adaptive mutations that often cause increased pathogenicity. Getah virus (GETV), a neglected and re-emerging mosquito-borne alphavirus, poses threat to many domestic animals and probably even humans. At present, the underlying mechanisms of GETV pathogenesis are not well defined. We identified a residue in the E2 glycoprotein that is critical for viral adsorption to cultured cells and pathogenesis in vivo . Viruses containing an arginine instead of a lysine at residue 253 displayed enhanced infectivity in mammalian cells and diminished virulence in a mouse model of GETV disease. Experiments in cell culture show that heparan sulfate (HS) is a new attachment factor for GETV, and the exchange Lys253Arg improves virus attachment by enhancing binding to HS. The mutation also results in more effective binding to glycosaminoglycan (GAG), linked to low virulence due to rapid virus clearance from the circulation. Localization of residue 253 in the 3D structure of the spike revealed several other basic residues in E2 and E1 in close vicinity that might constitute an HS-binding site different from sites previously identified in other alphaviruses. Overall, our study reveals that HS acts as the attachment factor of GETV and provides convincing evidence for an HS-binding determinant at residue 253 in the E2 glycoprotein of GETV, which contributes to infectivity and virulence. IMPORTANCE Due to decades of inadequate monitoring and lack of vaccines and specific treatment, a large number of people have been infected with alphaviruses. GETV is a re-emerging alphavirus that has the potential to infect humans. This specificity of the GETV disease, particularly its propensity for chronic musculoskeletal manifestations, underscores the need to identify the genetic determinants that govern GETV virulence in the host. Using a mouse model, we show that a single amino acid substitution at residue 253 in the E2 glycoprotein causes attenuation of the virus. Residue 253 might be part of a binding site for HS, a ubiquitous attachment factor on the cell surface. The substitution of Lys by Arg improves cell attachment of the virus in vitro and virus clearance from the blood in vivo by enhancing binding to HS. In summary, we have identified HS as a new attachment factor for GETV and the corresponding binding site in the E2 protein for the first time. Our research potentially improved understanding of the pathogenic mechanism of GETV and provided a potential target for the development of new attenuated vaccines and antiviral drugs.

Download Full-text

Comparative Activity of Adenosine Deaminase Acting on RNA (ADARs) Isoforms for Correction of Genetic Code in Gene Therapy

Current Gene Therapy ◽

10.2174/1566523218666181114122116 ◽

2019 ◽

Vol 19 (1) ◽

pp. 31-39 ◽

Cited By ~ 3

Author(s):

Md. Thoufic A. Azad ◽

Umme Qulsum ◽

Toshifumi Tsukahara

Keyword(s):

Amino Acid ◽

Adenosine Deaminase ◽

Rna Binding ◽

Stop Codon ◽

Single Amino Acid ◽

Fluorescence Signal ◽

Artificial Enzyme ◽

Western Blots ◽

Stem Loop ◽

Hek 293

Introduction: Members of the adenosine deaminase acting on RNA (ADAR) family of enzymes consist of double-stranded RNA-binding domains (dsRBDs) and a deaminase domain (DD) that converts adenosine (A) into inosine (I), which acts as guanosine (G) during translation. Using the MS2 system, we engineered the DD of ADAR1 to direct it to a specific target. The aim of this work was to compare the deaminase activities of ADAR1-DD and various isoforms of ADAR2-DD. Materials and Methods: We measured the binding affinity of the artificial enzyme system on a Biacore ™ X100. ADARs usually target dsRNA, so we designed a guide RNA complementary to the target RNA, and then fused the guide sequence to the MS2 stem-loop. A mutated amber (TAG) stop codon at 58 amino acid (TGG) of EGFP was targeted. After transfection of these three factors into HEK 293 cells, we observed fluorescence signals of various intensities. Results: ADAR2-long without the Alu-cassette yielded a much higher fluorescence signal than ADAR2-long with the Alu-cassette. With another isoform, ADAR2-short, which is 81 bp shorter at the C-terminus, the fluorescence signal was undetectable. A single amino acid substitution of ADAR2-long-DD (E488Q) rendered the enzyme more active than the wild type. The results of fluorescence microscopy suggested that ADAR1-DD is more active than ADAR2-long-DD. Western blots and sequencing confirmed that ADAR1-DD was more active than any other DD. Conclusion: This study provides information that should facilitate the rational use of ADAR variants for genetic restoration and treatment of genetic diseases.

Download Full-text