Escherichia coliMethionyl-tRNA Formyltransferase:  Role of Amino Acids Conserved in the Linker Region and in the C-Terminal Domain on the Specific Recognition of the Initiator tRNA†

The reduced folate carrier (RFC; SLC19A1) is closely related to the thiamine transporter, SLC19A2 (ThTr1). Hydropathy models for these homologous transporters predict up to 12 transmembrane domains (TMDs), with internally oriented N- and C-termini and a large central loop between TMDs 6 and 7. The homologies are localized mostly in the TMDs. However, there is little similarity in their N- and C-terminal domains and the central peptide linkers connecting putative TMDs 1—6 and TMDs 7—12. To explore the functional role of the 61-amino acid central linker in the human RFC (hRFC), we introduced deletions of 49 and 60 amino acids into this region, differing by the presence of a stretch of 11 highly conserved amino acids between the human and rodent RFCs (positions 204—214). An additional hRFC construct was prepared in which only the 11 conserved amino acids were deleted. The resulting hRFCD215—R263Δ, hRFCK204—R263Δ and hRFCK204—R214Δ proteins were transfected into transport-impaired K562 cells. The deletion constructs were all expressed in plasma membranes; however, they were completely inactive for methotrexate and (6S)5-formyl tetrahydrofolate transport. Insertion of non-homologous 73- and 84-amino acid fragments from the structurally analogous ThTr1 linker region into position 204 of hRFCK204—R263Δ restored low levels of transport (16—21% of the wild type). Insertion of the ThTr1 linkers into hRFCD215—R263Δ at position 215 restored 60—80% of wild-type levels of transport. Collectively, our results suggest that the role of the hRFC linker peptide is to provide the proper spatial orientation between the two halves of the hRFC protein for optimal function, and that this is largely independent of amino acid sequence. Our results also demonstrate a critical transport role for the stretch of 11 conserved amino acids starting at position 204 of hRFC.

Download Full-text

Mutation of Hydrophobic Residues in the C-Terminal Domain of the Marburg Virus Matrix Protein VP40 Disrupts Trafficking to the Plasma Membrane

Viruses ◽

10.3390/v12040482 ◽

2020 ◽

Vol 12 (4) ◽

pp. 482 ◽

Cited By ~ 2

Author(s):

Kaveesha J. Wijesinghe ◽

Luke McVeigh ◽

Monica L. Husby ◽

Nisha Bhattarai ◽

Jia Ma ◽

...

Keyword(s):

Amino Acids ◽

Plasma Membrane ◽

Matrix Protein ◽

Marburg Virus ◽

Membrane Localization ◽

Hydrophobic Residues ◽

Plasma Membrane Localization ◽

Terminal Domain ◽

Loop Region

Marburg virus (MARV) is a lipid-enveloped negative sense single stranded RNA virus, which can cause a deadly hemorrhagic fever. MARV encodes seven proteins, including VP40 (mVP40), a matrix protein that interacts with the cytoplasmic leaflet of the host cell plasma membrane. VP40 traffics to the plasma membrane inner leaflet, where it assembles to facilitate the budding of viral particles. VP40 is a multifunctional protein that interacts with several host proteins and lipids to complete the viral replication cycle, but many of these host interactions remain unknown or are poorly characterized. In this study, we investigated the role of a hydrophobic loop region in the carboxy-terminal domain (CTD) of mVP40 that shares sequence similarity with the CTD of Ebola virus VP40 (eVP40). These conserved hydrophobic residues in eVP40 have been previously shown to be critical to plasma membrane localization and membrane insertion. An array of cellular experiments and confirmatory in vitro work strongly suggests proper orientation and hydrophobic residues (Phe281, Leu283, and Phe286) in the mVP40 CTD are critical to plasma membrane localization. In line with the different functions proposed for eVP40 and mVP40 CTD hydrophobic residues, molecular dynamics simulations demonstrate large flexibility of residues in the EBOV CTD whereas conserved mVP40 hydrophobic residues are more restricted in their flexibility. This study sheds further light on important amino acids and structural features in mVP40 required for its plasma membrane localization as well as differences in the functional role of CTD amino acids in eVP40 and mVP40.

Download Full-text

Critical Role of the PA-X C-Terminal Domain of Influenza A Virus in Its Subcellular Localization and Shutoff Activity

Journal of Virology ◽

10.1128/jvi.00954-16 ◽

2016 ◽

Vol 90 (16) ◽

pp. 7131-7141 ◽

Cited By ~ 27

Author(s):

Tsuyoshi Hayashi ◽

Chutikarn Chaimayo ◽

James McGuinness ◽

Toru Takimoto

Keyword(s):

Amino Acids ◽

Subcellular Localization ◽

Nuclear Localization ◽

Influenza A ◽

Critical Role ◽

Terminal Region ◽

Terminal Deletion ◽

Link Type ◽

Terminal Domain

ABSTRACTPA-X is a recently identified influenza virus protein that is composed of the PA N-terminal 191 amino acids and unique C-terminal 41 or 61 residues. We and others showed that PA-X has a strong ability to suppress host protein synthesis via host mRNA decay, which is mediated by endonuclease activity in its N-terminal domain (B. W. Jagger, H. M. Wise, J. C. Kash, K. A. Walters, N. M. Wills, Y. L. Xiao, R. L. Dunfee, L. M. Schwartzman, A. Ozinsky, G. L. Bell, R. M. Dalton, A. Lo, S. Efstathiou, J. F. Atkins, A. E. Firth, J. K. Taubenberger, and P. Digard, 2012, Science337:199–204,http://dx.doi.org/10.1126/science.1222213, and E. A. Desmet, K. A. Bussey, R. Stone, and T. Takimoto, 2013, J Virol87:3108–3118,http://dx.doi.org/10.1128/JVI.02826-12). However, the mechanism of host mRNA degradation, especially where and how PA-X targets mRNAs, has not been analyzed. In this study, we determined the localization of PA-X and the role of the C-terminal unique region in shutoff activity. Quantitative subcellular localization analysis revealed that PA-X was located equally in both cytoplasm and nucleus. By characterizing a series of PA-X C-terminal deletion mutants, we found that the first 9 amino acids were sufficient for nuclear localization, but an additional 6 residues were required to induce the maximum shutoff activity observed with intact PA-X. Importantly, forced nuclear localization of the PA-X C-terminal deletion mutant enhanced shutoff activity, highlighting the ability of nuclear PA-X to degrade host mRNAs more efficiently. However, PA-X also inhibited luciferase expression from transfected mRNAs synthesizedin vitro, suggesting that PA-X also degrades mRNAs in the cytoplasm. Among the basic amino acids in the PA-X C-terminal region, 3 residues, 195K, 198K, and 199R, were identified as key residues for inducing host shutoff and nuclear localization. Overall, our data indicate a critical role for the 15 residues in the PA-X C-terminal domain in degrading mRNAs in both the cytoplasm and nucleus.IMPORTANCEInfluenza A viruses express PA-X proteins to suppress global host gene expression, including host antiviral genes, to allow efficient viral replication in infected cells. However, little is known about how PA-X induces host shutoff. In this study, we showed that PA-X localized equally in both the cytoplasm and nucleus of the cells, but the nuclear localization of PA-X mediated by its C-terminal region has a significant impact on shutoff activity. Three basic residues at the C-terminal region play a critical role in nuclear localization, but additional basic residues were required for maximum shutoff activity. Our findings indicate that PA-X targets and degrades mRNAs in both the nucleus and cytoplasm, and that the first 15 residues of the PA-X unique C-terminal region play a critical role in shutoff activity.

Download Full-text

ONIOM and ab-initio calculations on the mechanism of uncatalyzed peptide bond formation

Biochemistry and Cell Biology ◽

10.1139/o2012-027 ◽

2012 ◽

Vol 90 (6) ◽

pp. 691-700 ◽

Cited By ~ 1

Author(s):

Hadieh Monajemi ◽

Mohammad Noh Daud ◽

Sharifuddin Mohd. Zain ◽

Wan Ahmad Tajuddin Wan Abdullah

Keyword(s):

Amino Acids ◽

Bond Formation ◽

Computational Cost ◽

Peptide Bond ◽

Peptide Bond Formation ◽

Initiator Trna ◽

Reduced Rate ◽

Surface Scan ◽

A Site

Finding a proper transition structure for the peptide bond formation process can lead one to a better understanding of the role of ribosome in catalyzing this reaction. Using computer simulations, we performed the potential energy surface scan on the ester bond dissociation of P-site aminoacyl-tRNA and the peptide bond formation of P-site and A-site amino acids. The full fragments of initiator tRNAimet and elongator tRNAphe are attached to both cognate and non-cognate amino acids as the P-site substrate. The A-site amino acid for all four calculations is methionine. We used ONIOM calculations to reduce the computational cost. Our study illustrates the reduced rate of peptide bond formation for misacylated tRNAimet in the absence of ribosomal bases. The misacylated elongator tRNAphe, however, did not show any difference in its PES compared with that for the phe-tRNAphe. This demonstrates the structural specification of initiator tRNAimet for the amino acids side chain.

Download Full-text

Characterization of Chitinase C from a Marine Bacterium, Alteromonas sp. Strain O-7, and Its Corresponding Gene and Domain Structure

Applied and Environmental Microbiology ◽

10.1128/aem.64.2.472-478.1998 ◽

1998 ◽

Vol 64 (2) ◽

pp. 472-478 ◽

Cited By ~ 51

Author(s):

Hiroshi Tsujibo ◽

Hideyuki Orikoshi ◽

Kayoko Shiotani ◽

Miyuki Hayashi ◽

Junko Umeda ◽

...

Keyword(s):

Amino Acids ◽

Fusion Protein ◽

Catalytic Domain ◽

Transferase Activity ◽

Chitin Binding Domain ◽

Reading Frame ◽

Terminal Domain ◽

Fibronectin Type Iii ◽

Chitinase Genes

ABSTRACT One of the chitinase genes of Alteromonas sp. strain O-7, the chitinase C-encoding gene (chiC), was cloned, and the nucleotide sequence was determined. An open reading frame coded for a protein of 430 amino acids with a predicted molecular mass of 46,680 Da. Alignment of the deduced amino acid sequence demonstrated that ChiC contained three functional domains, the N-terminal domain, a fibronectin type III-like domain, and a catalytic domain. The N-terminal domain (59 amino acids) was similar to that found in the C-terminal extension of ChiA (50 amino acids) of this strain and furthermore showed significant sequence homology to the regions found in several chitinases and cellulases. Thus, to evaluate the role of the domain, we constructed the hybrid gene that directs the synthesis of the fusion protein with glutathione S-transferase activity. Both the fusion protein and the N-terminal domain itself bound to chitin, indicating that the N-terminal domain of ChiC constitutes an independent chitin-binding domain.

Download Full-text

The N-terminal Domain of the Yeast Mitochondrial RNA Polymerase Regulates Multiple Steps of Transcription

Journal of Biological Chemistry ◽

10.1074/jbc.m111.228023 ◽

2011 ◽

Vol 286 (18) ◽

pp. 16109-16120 ◽

Cited By ~ 16

Author(s):

Swaroopa Paratkar ◽

Aishwarya P. Deshpande ◽

Guo-Qing Tang ◽

Smita S. Patel

Keyword(s):

Amino Acids ◽

Rna Polymerase ◽

Transcription Initiation ◽

Rna Synthesis ◽

Full Length ◽

Mitochondrial Rna ◽

Terminal Domain ◽

Mitochondrial Rna Polymerase

Transcription of the yeast (Saccharomyces cerevisiae) mitochondrial (mt) genome is catalyzed by nuclear-encoded proteins that include the core RNA polymerase (RNAP) subunit Rpo41 and the transcription factor Mtf1. Rpo41 is homologous to the single-subunit bacteriophage T7/T3 RNAP. Its ∼80-kDa C-terminal domain is highly conserved among mt RNAPs, but its ∼50-kDa N-terminal domain (NTD) is less conserved and not present in T7/T3 RNAP. To understand the role of the NTD, we have biochemically characterized a series of NTD deletion mutants of Rpo41. Our studies show that NTD regulates multiple steps of transcription initiation. Interestingly, NTD functions in an autoinhibitory manner during initiation, and its partial deletion increases the efficiency of RNA synthesis. Deletion of 1–270 amino acids (DN270) reduces abortive synthesis and increases full-length to abortive RNA ratio relative to full-length (FL) Rpo41. A larger deletion of 1–380 amino acids (DN380), decreases RNA synthesis on duplex but not on premelted promoter. We show that DN380 is defective in promoter opening near the transcription start site. Most strikingly, both DN270 and DN380 catalyze highly processive RNA synthesis on the premelted promoter, and unlike the FL Rpo41, the mutants are not inhibited by Mtf1. Both mutants show weaker interactions with Mtf1, which explains many of our results, and particularly the ability of the mutants to efficiently transition from initiation to elongation. We propose that in vivo the accessory proteins that bind NTD may modulate interactions of Rpo41 with the promoter/Mtf1 to activate and allow timely release from Mtf1 for transition into elongation.

Download Full-text

Mutation of Hydrophobic Residues in the C-Terminal Domain of the Marburg Virus Matrix Protein VP40 Disrupts Trafficking to the Plasma Membrane

10.20944/preprints202002.0467.v1 ◽

2020 ◽

Author(s):

Kaveesha J. Wijesinghe ◽

Luke McVeigh ◽

Monica L. Husby ◽

Nisha Bhattarai ◽

Jia Ma ◽

...

Keyword(s):

Amino Acids ◽

Plasma Membrane ◽

Matrix Protein ◽

Marburg Virus ◽

Membrane Localization ◽

Hydrophobic Residues ◽

Plasma Membrane Localization ◽

Terminal Domain ◽

Loop Region

Marburg virus (MARV) is a lipid-enveloped negative sense single stranded RNA virus, which can cause a deadly hemorrhagic fever. MARV encodes seven proteins, including VP40 (mVP40), a matrix protein that interacts with the cytoplasmic leaflet of the host cell plasma membrane. VP40 traffics to the plasma membrane inner leaflet, where it assembles to facilitate the budding of viral particles. VP40 is a multifunctional protein that interacts with several host proteins and lipids to complete the viral replication cycle, but many of these host-interactions remain unknown or are poorly characterized. In this study, we investigated the role of a hydrophobic loop region in the carboxy-terminal domain (CTD) of mVP40 that shares sequence similarity with the CTD of Ebola virus VP40 (eVP40). These conserved hydrophobic residues in eVP40 have been previously shown to be critical to plasma membrane localization and membrane insertion. An array of cellular experiments and confirmatory in vitro work strongly suggests proper orientation and hydrophobic residues (Phe281, Leu283, and Phe286) in the mVP40 CTD are critical to plasma membrane localization. In line with the different functions proposed for eVP40 and mVP40 CTD hydrophobic residues, molecular dynamics simulations demonstrate large flexibility of residues in the EBOV CTD whereas conserved mVP40 hydrophobic residues are more restricted in their flexibility. This study sheds further light on important amino acids and structural features in mVP40 required for its plasma membrane localization as well as differences in the functional role of CTD amino acids in eVP40 and mVP40.

Download Full-text

Functional domains of chlamydial histone H1-like protein

Biochemical Journal ◽

10.1042/bj3150481 ◽

1996 ◽

Vol 315 (2) ◽

pp. 481-486 ◽

Cited By ~ 6

Author(s):

Miguel REMACHA ◽

Ravi KAUL ◽

Richard SHERBURNE ◽

Wanda M. WENMAN

Keyword(s):

Amino Acids ◽

Protein Interactions ◽

Histone H1 ◽

Protein Protein Interactions ◽

Amino Acid Similarity ◽

Terminal Domain ◽

Double Stranded Dna ◽

Unique Protein ◽

Dna Protein Interaction

Chlamydia trachomatis is one of the few prokaryotic organisms known to contain proteins that bear amino acid similarity to eukaryotic histone H1. It is also appreciated that chlamydial histone-like proteins, designated Hc1 and Hc2, can bind DNA and are presumably involved in the condensation of infectious elementary bodies. However, there is no information on either the orientation of Hc1 and Hc2 or the mechanism of their DNA–protein and protein–protein interactions. Whereas the C-terminal domain of Hc1 between amino acids 63 and 125 shows best alignment with sea-urchin histone H1, the N-terminus between amino acids 1 and 62 is highly conserved among various chlamydial species, suggesting a bifunctional role for this unique protein. In order to delineate the regions responsible for the Hc1 characteristics, we have expressed these two fragments independently in Escherichia coli and studied the binding of double-stranded DNA to either whole Hc1 protein or its two termini. Our results support the role of the carboxyl portion in DNA–protein interaction, a function similar to its eukaryotic counterpart. Although this interaction initiates DNA condensation in the absence of the N-terminal domain, it is not sufficient to produce complete compaction. Intra- or inter-molecular protein–protein interactions may be necessary to achieve such an effect.

Download Full-text