Amino acid residues Leu135 and Tyr236 are required for RNA binding activity of CFIm25 in Entamoeba histolytica

AbstractA mouse monoclonal antibody reacting in ELISA with a synthetic peptide representing a linear amino acid stretch of the protein antigen was tested on all overlap ping 5-mer to 9-mer fragments of the peptide, as prepared by multi-pin synthesis. Analysis of the binding data suggests that several residues in the peptide might be relatively unrelevant for recognition, while few others seem to play a critical role as key residues. On the basis of such observations, we attempted to reconstruct an alternative essential epitope by introducing multiple amino acid substitutions in the 9-mer peptide exhibiting the best binding activity, and then tested its ability to be recognized by the monoclonal antibody.

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Characterization of amino acid residues essential for tetramer formation and DNA-binding activity of ssDNA-binding protein of Mycobacterium tuberculosis

Biochemistry (Moscow) ◽

10.1134/s000629791106006x ◽

2011 ◽

Vol 76 (6) ◽

pp. 658-665 ◽

Cited By ~ 2

Author(s):

Hua Zhang ◽

Yuxi Tian ◽

Ziduo Liu ◽

Feng Huang ◽

Lihua Hu ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Amino Acid ◽

Dna Binding ◽

Binding Activity ◽

Amino Acid Residues ◽

Ssdna Binding ◽

Dna Binding Activity ◽

Tetramer Formation ◽

Ssdna Binding Protein

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Amino acid residues involved in the heparin-binding activity of murine IL-12 in the context of an antibody-cytokine fusion protein

Cytokine ◽

10.1016/j.cyto.2019.04.004 ◽

2019 ◽

Vol 120 ◽

pp. 220-226 ◽

Cited By ~ 1

Author(s):

Rosendo Luria-Pérez ◽

Pierre V. Candelaria ◽

Tracy R. Daniels-Wells ◽

José A. Rodríguez ◽

Gustavo Helguera ◽

...

Keyword(s):

Amino Acid ◽

Fusion Protein ◽

Binding Activity ◽

Amino Acid Residues ◽

Heparin Binding

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A double-stranded RNA platform is required for the interaction between a host restriction factor and the NS1 protein of influenza A virus

Nucleic Acids Research ◽

10.1093/nar/gkz1094 ◽

2019 ◽

Vol 48 (1) ◽

pp. 304-315 ◽

Cited By ~ 3

Author(s):

Guifang Chen ◽

Li-Chung Ma ◽

Shanshan Wang ◽

Ryan L Woltz ◽

Emily M Grasso ◽

...

Keyword(s):

Amino Acid ◽

Influenza A ◽

Binding Activity ◽

Restriction Factor ◽

Amino Acid Residues ◽

Ns1 Protein ◽

Double Stranded Rna ◽

Host Restriction ◽

Host Restriction Factor ◽

Dsrna Binding

Abstract Influenza A viruses cause widespread human respiratory disease. The viral multifunctional NS1 protein inhibits host antiviral responses. This inhibition results from the binding of specific cellular antiviral proteins at various positions on the NS1 protein. Remarkably, binding of several proteins also requires the two amino-acid residues in the NS1 N-terminal RNA-binding domain (RBD) that are required for binding double-stranded RNA (dsRNA). Here we focus on the host restriction factor DHX30 helicase that is countered by the NS1 protein, and establish why the dsRNA-binding activity of NS1 is required for its binding to DHX30. We show that the N-terminal 152 amino-acid residue segment of DHX30, denoted DHX30N, possesses all the antiviral activity of DHX30 and contains a dsRNA-binding domain, and that the NS1-DHX30 interaction in vivo requires the dsRNA-binding activity of both DHX30N and the NS1 RBD. We demonstrate why this is the case using bacteria-expressed proteins: the DHX30N-NS1 RBD interaction in vitro requires the presence of a dsRNA platform that binds both NS1 RBD and DHX30N. We propose that a similar dsRNA platform functions in interactions of the NS1 protein with other proteins that requires these same two amino-acid residues required for NS1 RBD dsRNA-binding activity.

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A Novel Function of the MA-3 Domains in Transformation and Translation Suppressor Pdcd4 Is Essential for Its Binding to Eukaryotic Translation Initiation Factor 4A

Molecular and Cellular Biology ◽

10.1128/mcb.24.9.3894-3906.2004 ◽

2004 ◽

Vol 24 (9) ◽

pp. 3894-3906 ◽

Cited By ~ 141

Author(s):

Hsin-Sheng Yang ◽

Myung-Haing Cho ◽

Halina Zakowicz ◽

Glenn Hegamyer ◽

Nahum Sonenberg ◽

...

Keyword(s):

Amino Acid ◽

Translation Initiation ◽

Binding Activity ◽

Translation Initiation Factor ◽

Initiation Factor ◽

Amino Acid Residues ◽

Eukaryotic Translation Initiation Factor ◽

Stem Loop ◽

Eukaryotic Translation ◽

Eukaryotic Translation Initiation

ABSTRACT Αn α-helical MA-3 domain appears in several translation initiation factors, including human eukaryotic translation initiation factor 4G (eIF4G) and DAP-5/NAT1/p97, as well as in the tumor suppressor Pdcd4. The function of the MA-3 domain is, however, unknown. C-terminal eIF4G (eIG4Gc) contains an MA-3 domain that is located within the eIF4A-binding region, suggesting a role for eIF4A binding. Interestingly, C-terminal DAP-5/NAT1/p97 contains an MA-3 domain, but it does not bind to eIF4A. Mutation of amino acid residues conserved between Pdcd4 and eIF4Gc but not in DAP-5/NAT1/p97 to the amino acid residues found in the DAP-5/NAT1/p97 indicates that some of these amino acid residues within the MA-3 domain are critical for eIF4A-binding activity. Six Pdcd4 mutants (Pdcd4E249K, Pdcd4D253A, Pdcd4D414K, Pdcd4D418A, Pdcd4E249K,D414K, and Pdcd4D253A,D418A) lost >90% eIF4A-binding activity. Mutation of the corresponding amino acid residues in the eIF4Gc also produced similar results, as seen for Pdcd4. These results demonstrate that the MA-3 domain is important for eIF4A binding and explain the ability of Pdcd4 or eIF4Gc but not DAP-5/NAT1/p97 to bind to eIF4A. Competition experiments indicate that Pdcd4 prevents ca. 60 to 70% of eIF4A binding to eIF4Gc at a Pdcd4/eIF4A ratio of 1:1, but mutants Pdcd4D253A and Pdcd4D253A,D418A do not. Translation of stem-loop structured mRNA is susceptible to inhibition by wild-type Pdcd4 but not by Pdcd4D253A, Pdcd4D418A, or Pdcd4D235A,D418A. Together, these results indicate that not only binding to eIF4A but also prevention of eIF4A binding to the MA-3 domain of eIF4Gc contributes to the mechanism by which Pdcd4 inhibits translation.

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Identification of the Binding Domain of Streptococcus oralis Glyceraldehyde-3-Phosphate Dehydrogenase for Porphyromonas gingivalis Major Fimbriae

Infection and Immunity ◽

10.1128/iai.00439-09 ◽

2009 ◽

Vol 77 (11) ◽

pp. 5130-5138 ◽

Cited By ~ 24

Author(s):

Hideki Nagata ◽

Mio Iwasaki ◽

Kazuhiko Maeda ◽

Masae Kuboniwa ◽

Ei Hashino ◽

...

Keyword(s):

Amino Acid ◽

Porphyromonas Gingivalis ◽

Biofilm Formation ◽

Binding Activity ◽

Binding Domain ◽

Laser Scanning Microscopy ◽

Dependent Manner ◽

Oral Streptococci ◽

Amino Acid Residues ◽

Streptococcus Oralis

ABSTRACT Porphyromonas gingivalis forms communities with antecedent oral biofilm constituent streptococci. P. gingivalis major fimbriae bind to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) present on the streptococcal surface, and this interaction plays an important role in P. gingivalis colonization. This study identified the binding domain of Streptococcus oralis GAPDH for P. gingivalis fimbriae. S. oralis recombinant GAPDH (rGAPDH) was digested with lysyl endopeptidase. Cleaved fragments of rGAPDH were applied to a reverse-phase high-pressure liquid chromatograph equipped with a C18 column. Each peak was collected; the binding activity toward P. gingivalis recombinant fimbrillin (rFimA) was analyzed with a biomolecular interaction analysis system. The fragment displaying the strongest binding activity was further digested with various proteinases, after which the binding activity of each fragment was measured. The amino acid sequence of each fragment was determined by direct sequencing, mass spectrometric analysis, and amino acid analysis. Amino acid residues 166 to 183 of S. oralis GAPDH exhibited the strongest binding activity toward rFimA; confocal laser scanning microscopy revealed that the synthetic peptide corresponding to amino acid residues 166 to 183 of S. oralis GAPDH (pep166-183, DNFGVVEGLMTTIHAYTG) inhibits S. oralis-P. gingivalis biofilm formation in a dose-dependent manner. Moreover, pep166-183 inhibited interbacterial biofilm formation by several oral streptococci and P. gingivalis strains with different types of FimA. These results indicate that the binding domain of S. oralis GAPDH for P. gingivalis fimbriae exists within the region encompassing amino acid residues 166 to 183 of GAPDH and that pep166-183 may be a potent inhibitor of P. gingivalis colonization in the oral cavity.

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The Respiratory Syncytial Virus M2-1 Protein Forms Tetramers and Interacts with RNA and P in a Competitive Manner

Journal of Virology ◽

10.1128/jvi.00335-09 ◽

2009 ◽

Vol 83 (13) ◽

pp. 6363-6374 ◽

Cited By ~ 52

Author(s):

Thi-Lan Tran ◽

Nathalie Castagné ◽

Virginie Dubosclard ◽

Sylvie Noinville ◽

Emmanuelle Koch ◽

...

Keyword(s):

Respiratory Syncytial Virus ◽

Amino Acid ◽

Rna Binding ◽

Amino Acid Residues ◽

Polymerase Complex ◽

Bacterial Rna ◽

Infected Cells ◽

Syncytial Virus ◽

Α Helix ◽

Zinc Binding Domain

ABSTRACT The respiratory syncytial virus (RSV) M2-1 protein is an essential cofactor of the viral RNA polymerase complex and functions as a transcriptional processivity and antitermination factor. M2-1, which exists in a phosphorylated or unphosphorylated form in infected cells, is an RNA-binding protein that also interacts with some of the other components of the viral polymerase complex. It contains a CCCH motif, a putative zinc-binding domain that is essential for M2-1 function, at the N terminus. To gain insight into its structural organization, M2-1 was produced as a recombinant protein in Escherichia coli and purified to >95% homogeneity by using a glutathione S-transferase (GST) tag. The GST-M2-1 fusion proteins were copurified with bacterial RNA, which could be eliminated by a high-salt wash. Circular dichroism analysis showed that M2-1 is largely α-helical. Chemical cross-linking, dynamic light scattering, sedimentation velocity, and electron microscopy analyses led to the conclusion that M2-1 forms a 5.4S tetramer of 89 kDa and ∼7.6 nm in diameter at micromolar concentrations. By using a series of deletion mutants, the oligomerization domain of M2-1 was mapped to a putative α-helix consisting of amino acid residues 32 to 63. When tested in an RSV minigenome replicon system using a luciferase gene as a reporter, an M2-1 deletion mutant lacking this region showed a significant reduction in RNA transcription compared to wild-type M2-1, indicating that M2-1 oligomerization is essential for the activity of the protein. We also show that the region encompassing amino acid residues 59 to 178 binds to P and RNA in a competitive manner that is independent of the phosphorylation status of M2-1.

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The p23 Protein of Citrus Tristeza Virus Controls Asymmetrical RNA Accumulation

Journal of Virology ◽

10.1128/jvi.76.2.473-483.2002 ◽

2002 ◽

Vol 76 (2) ◽

pp. 473-483 ◽

Cited By ~ 62

Author(s):

Tatineni Satyanarayana ◽

Siddarame Gowda ◽

María A. Ayllón ◽

María R. Albiach-Martí ◽

Shailaja Rabindran ◽

...

Keyword(s):

Amino Acid ◽

Zinc Finger ◽

Citrus Tristeza Virus ◽

Rna Binding ◽

Fold Increase ◽

Amino Acid Residues ◽

Rna Accumulation ◽

P23 Gene ◽

Tristeza Virus ◽

Citrus Tristeza

ABSTRACT Citrus tristeza virus (CTV), a member of the Closteroviridae, has a 19.3-kb positive-stranded RNA genome that is organized into 12 open reading frames (ORFs) with the 10 3′ genes expressed via a nested set of nine or ten 3′-coterminal subgenomic mRNAs (sgRNAs). Relatively large amounts of negative-stranded RNAs complementary to both genomic and sgRNAs accumulate in infected cells. As is characteristic of RNA viruses, wild-type CTV produced more positive than negative strands, with the plus-to-minus ratios of genomic and sgRNAs estimated at 10 to 20:1 and 40 to 50:1, respectively. However, a mutant with all of the 3′ genes deleted replicated efficiently, but produced plus to minus strands at a markedly decreased ratio of 1 to 2:1. Deletion analysis of 3′-end genes revealed that the p23 ORF was involved in asymmetric RNA accumulation. A mutation which caused a frameshift after the fifth codon resulted in nearly symmetrical RNA accumulation, suggesting that the p23 protein, not a cis-acting element within the p23 ORF, controls asymmetric accumulation of CTV RNAs. Further in-frame deletion mutations in the p23 ORF suggested that amino acid residues 46 to 180, which contained RNA-binding and zinc finger domains, were indispensable for asymmetrical RNA accumulation, while the N-terminal 5 to 45 and C-terminal 181 to 209 amino acid residues were not absolutely required. Mutation of conserved cysteine residues to alanines in the zinc finger domain resulted in loss of activity of the p23 protein, suggesting involvement of the zinc finger in asymmetric RNA accumulation. The absence of p23 gene function was manifested by substantial increases in accumulation of negative-stranded RNAs and only modest decreases in positive-stranded RNAs. Moreover, the substantial decrease in the accumulation of negative-stranded coat protein (CP) sgRNA in the presence of the functional p23 gene resulted in a 12- to 15-fold increase in the expression of the CP gene. Apparently the excess negative-stranded sgRNA reduces the availability of the corresponding positive-stranded sgRNA as a messenger. Thus, the p23 protein controls asymmetric accumulation of CTV RNAs by downregulating negative-stranded RNA accumulation and indirectly increases expression of 3′ genes.

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Fibronectin Binding to the Salmonella enterica Serotype Typhimurium ShdA Autotransporter Protein Is Inhibited by a Monoclonal Antibody Recognizing the A3 Repeat

Journal of Bacteriology ◽

10.1128/jb.186.15.4931-4939.2004 ◽

2004 ◽

Vol 186 (15) ◽

pp. 4931-4939 ◽

Cited By ~ 27

Author(s):

Robert A. Kingsley ◽

Daad Abi Ghanem ◽

Nahum Puebla-Osorio ◽

A. Marijke Keestra ◽

Luc Berghman ◽

...

Keyword(s):

Monoclonal Antibody ◽

Amino Acid ◽

Binding Activity ◽

Immune Serum ◽

Amino Acid Residues ◽

Repeat Region ◽

Passenger Domain ◽

Amino Acid Repeats ◽

Primary Amino ◽

Fibronectin Binding

ABSTRACT ShdA is a large outer membrane protein of the autotransporter family whose passenger domain binds the extracellular matrix proteins fibronectin and collagen I, possibly by mimicking the host ligand heparin. The ShdA passenger domain consists of ∼1,500 amino acid residues that can be divided into two regions based on features of the primary amino acid sequence: an N-terminal nonrepeat region followed by a repeat region composed of two types of imperfect direct amino acid repeats, called type A and type B. The repeat region bound bovine fibronectin with an affinity similar to that for the complete ShdA passenger domain, while the nonrepeat region exhibited comparatively low fibronectin-binding activity. A number of fusion proteins containing truncated fragments of the repeat region did not bind bovine fibronectin. However, binding of the passenger domain to fibronectin was inhibited in the presence of immune serum raised to one truncated fragment of the repeat region that contained repeats A2, B8, A3, and B9. Furthermore, a monoclonal antibody that specifically recognized an epitope in a recombinant protein containing the A3 repeat inhibited binding of ShdA to fibronectin.

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