Biomedical subjects

Assen Marintchev

Publications and source records attributed to Assen Marintchev.

10 recordsLinked to original sources

PubMed2006

Mapping of the auto-inhibitory interactions of protein kinase R by nuclear magnetic resonance.

The dsRNA-dependent protein kinase (PKR) is a key mediator of the anti-viral and anti-proliferative effects of interferon. Unphosphorylated PKR is characterized by inhibitory interactions between the kinase and RNA binding domains (RBDs), but the structural details of the latent state and its unraveling during activation are not well understood. To study PKR regulation by NMR we assigned a large portion of the backbone resonances of the catalytically inactive K296R kinase domain, and performed (15)N-heteronuclear single quantum coherence (HSQC) titrations of this kinase domain with the RBDs. Chemical shift perturbations in the kinase indicate that RBD2 binds to the substrate eIF2alpha docking site in the kinase C-lobe. Consistent with these results, a mutation in the eIF2alpha docking site, F495A, displays weaker interactions with the RBD. The full-length RBD1+2 binds more strongly to the kinase domain than RBD2 alone. The observed chemical shift changes extend from the eIF2alpha binding site into the kinase N-lobe and inside the active site, consistent with weak interactions between the N-terminal part of the RBD and the kinase.

Amino Acid Sequence↗

PubMed2005

The eukaryotic initiation factor (eIF) 5 HEAT domain mediates multifactor assembly and scanning with distinct interfaces to eIF1, eIF2, eIF3, and eIF4G.

Eukaryotic translation initiation factor (eIF) 5 is crucial for the assembly of the eukaryotic preinitiation complex. This activity is mediated by the ability of its C-terminal HEAT domain to interact with eIF1, eIF2, and eIF3 in the multifactor complex and with eIF4G in the 48S complex. However, the binding sites for these factors on eIF5-C-terminal domain (CTD) have not been known. Here we present a homology model for eIF5-CTD based on the HEAT domain of eIF2Bepsilon. We show that the binding site for eIF2beta is located in a surface area containing aromatic and acidic residues (aromatic/acidic boxes), that the binding sites for eIF1 and eIF3c are located in a conserved surface region of basic residues, and that eIF4G binds eIF5-CTD at an interface overlapping with the acidic area. Mutations in these distinct eIF5 surface areas impair GCN4 translational control by disrupting preinitiation complex interactions. These results indicate that the eIF5 HEAT domain is a critical nucleation core for preinitiation complex assembly and function.

Binding Sites↗

PubMed2005

eIF4G and CBP80 share a common origin and similar domain organization: implications for the structure and function of eIF4G.

Eukaryotic translation initiation factor 4G (eIF4G) plays a critical role in protein expression, and is at the center of a complex regulatory network. Together with the cap-binding protein eIF4E, it recruits the small ribosomal subunit to the 5'-end of mRNA and promotes the assembly of a functional translation initiation complex, which scans along the mRNA to the translation start codon. Human eIF4G contains three consecutive HEAT domains, as well as long unstructured regions involved in multiple protein-protein interactions. Despite the accumulating data about the structure and function of eIF4G, the mechanisms of coordination and regulation of its interactions with other factors have remained largely unknown. Here, we present evidence that eIF4G and the large subunit of the nuclear cap-binding complex, CBP80, share a common origin and domain structure. We propose that the organization of the individual domains in eIF4G and CBP80 could also be conserved. The structure of CBP80, in complex with the nuclear cap-binding protein CBP20, is used to build a model for the mutual orientation of the domains in eIF4G and their interactions with other factors. The organization of the CBP80-CBP20 complex suggests how the activity of eIF4G in translation initiation could be regulated through a dynamic network of overlapping intra- and intermolecular interactions centered around the eIF4G HEAT domains.

Animals↗

PubMed2005

Structural basis for the enhancement of eIF4A helicase activity by eIF4G.

The eukaryotic translation initiation factors 4A (eIF4A) and 4G (eIF4G) are crucial for the assembly of the translationally active ribosome. Together with eIF4E, they form the eIF4F complex, which recruits the 40S subunit to the 5' cap of mRNA. The two-domain RNA helicase eIF4A is a very weak helicase by itself, but the activity is enhanced upon interaction with the scaffolding protein eIF4G. Here we show that, albeit both eIF4A domains play a role in binding the middle domain of eIF4G (eIF4G-m, amino acids 745-1003), the main interaction surface is located on the C-terminal domain. We use NMR spectroscopy to define the binding site and find that the contact surface is adjacent to the RNA-, ATP-, and eIF4A-NTD-interacting regions. Mutations of interface residues abrogated binding, confirmed the interface, and showed that the N-terminal end of eIF4G-m interacts with the C-terminal domain of eIF4A. The data suggest that eIF4G-m forms a soft clamp to stabilize the closed interdomain orientation of eIF4A. This model can explain the cooperativity between all binding partners of eIF4A (eIF4G, RNA, ATP) and stimulation of eIF4A activity in the eIF4F complex.

Amino Acid Motifs↗

PubMed2004

Solution structure of human initiation factor eIF2alpha reveals homology to the elongation factor eEF1B.

The GTP-bound form of the trimeric eukaryotic translation initiation factor 2 (eIF2) transfers aminoacylated initiator methionyl tRNA onto the 40S ribosome. We have solved with solution NMR the structure of the alpha subunit of human eIF2 (heIF2alpha). The protein consists of two domains that are mobile relative to each other. The N-terminal domain has an S1-type oligonucleotide/oligosaccharide binding-fold subdomain and an alpha-helical subdomain. The C-terminal domain adopts an alphabeta-fold very similar to the C-terminal domain of elongation factor (eEF) 1Balpha, the guanine-nucleotide exchange factor for eEF1A. The structural and functional similarities found between eIF2alpha/eIF2gamma and eEF1Balpha/eEF1A suggest a model for the interaction of eIF2alpha with eIF2gamma, and eIF2 with Met-tRNAiMet. It further indicates a previously unrecognized evolutionary lineage of eIF2alpha/gamma from the functionally related elongation factor eEF1Balpha/eEF1A complex.

Amino Acid Sequence↗

PubMed2003

Position of eukaryotic initiation factor eIF1 on the 40S ribosomal subunit determined by directed hydroxyl radical probing.

Eukaryotic initiation factor (eIF) eIF1 maintains the fidelity of initiation codon selection by enabling 43S complexes to reject codon-anticodon mismatches, to recognize the initiation codon context, and to discriminate against establishing a codon-anticodon interaction with AUGs located <8 nt from the 5'-end of mRNA. To understand how eIF1 plays its discriminatory role, we determined its position on the 40S ribosomal subunit using directed hydroxyl radical cleavage. The cleavage of 18S rRNA in helices 23b, 24a, and 44 by hydroxyl radicals generated from Fe(II) tethered to seven positions on the surface of eIF1 places eIF1 on the interface surface of the platform of the 40S subunit in the proximity of the ribosomal P-site. The position of eIF1 on the 40S subunit suggests that although eIF1 is unable to inspect the region of initiation codon directly, its position close to the P-site is very favorable for an indirect mechanism of eIF1's action by influencing the conformation of the platform of the 40S subunit and the positions of mRNA and initiator tRNA in initiation complexes. Unexpectedly, the position of eIF1 on the 40S subunit was strikingly similar to the position on the 30S ribosomal subunit of the sequence and structurally unrelated C-terminal domain of prokaryotic initiation factor IF3, which also participates in initiation codon selection in prokaryotes.

Animals↗

PubMed2003

Mapping the binding interface between human eukaryotic initiation factors 1A and 5B: a new interaction between old partners.

The translation initiation factors (IFs) IF1/eIF1A and IF2e/IF5B have been conserved throughout all kingdoms. Although the central roles of the bacterial factors IF1 and IF2 were established long ago, the importance of their eukaryotic homologs, eukaryotic IFs (eIFs) eIF1A and eIF5B, has only recently become evident. The translation machinery in eukaryotes is more complex and accordingly, eIF1A and eIF5B seem to have acquired a number of new functions while also retaining many of the roles of bacterial IF1 and IF2. IF1 and IF2 have been shown to interact on the ribosome but no binding has been detected for the free factors. In contrast, yeast eIF1A and eIF5B have been reported to interact in the absence of ribosomes. Here, we have identified the binding interface between human eIF1A and the C-terminal domain of eIF5B by using solution NMR. That interaction interface involves the C termini of the two proteins, which are not present in bacterial IF1 and IF2. The interaction is, therefore, unique to eukaryotes. A structural model for the interaction of eIF1A and eIF5B in the context of the ribosome is presented. We propose that eIF1A and eIF5B simultaneously interact at two sites that are >50 A apart: through their C termini as reported here, and through an interface previously identified in bacterial IF1 and IF2. The binding between the C termini of eIF1A and eIF5B has implications for eukaryote-specific mechanisms of recruitment and release of translation IFs from the ribosome.

Binding Sites↗

PubMed2003

Site-directed mutagenesis analysis of the structural interaction of the single-strand-break repair protein, X-ray cross-complementing group 1, with DNA polymerase beta.

Human X-ray cross-complementing group 1 (XRCC1) is a single-strand DNA break repair protein which forms a base excision repair (BER) complex with DNA polymerase beta (beta-Pol). Here we report a site- directed mutational analysis in which 16 mutated versions of the XRCC1 N-terminal domain (XRCC1-NTD) were constructed on the basis of previous NMR results that had implicated the proximity of various surface residues to beta-Pol. Mutant proteins defective in XRCC1-NTD interaction with beta-Pol and with a beta-Pol-gapped DNA complex were determined by gel filtration chromatography and a gel mobility shift assay. The interaction surface determined from the mutated residues was found to encompass beta-strand D and E of the five-stranded beta-sheet (betaABGDE) and the protruding alpha2 helix of the XRCC1-NTD. Mutations that included F67A (betaD), E69K (betaD), V86R (betaE) on the five-stranded beta-sheet and deletion of the alpha2 helix, but not mutations within alpha2, abolished binding of the XRCC1-NTD to beta-Pol. A Y136A mutant abolished beta-Pol binding, and a R109S mutant reduced beta-Pol binding. E98K, E98A, N104A, Y136A, R109S, K129E, F142A, R31A/K32A/R34A and delta-helix-2 mutants displayed temperature dependent solubility. These findings confirm the importance of the alpha2 helix and the betaD and betaE strands of XRCC1-NTD to the energetics of beta-Pol binding. Establishing the direct contacts in the beta-Pol XRCC1 complex is a critical step in understanding how XRCC1 fulfills its numerous functions in DNA BER.

Amino Acid Substitution↗

PubMed2002

Mapping of the interaction interface of DNA polymerase beta with XRCC1.

Residues of DNA polymerase beta (beta-Pol) that interact with the DNA repair protein XRCC1 have been determined by NMR chemical shift mapping (CSM) and mutagenesis. 15N/(13)C/(2)H/(1)H,(13)C-methyl(Leu,Ile,Val)-labeled beta-Pol palm-thumb domain was used for assignments of the 1H, 15N, and 13C resonances used for CSM of the palm-thumb on forming the 40 kDa complex with the XRCC1 N-terminal domain (NTD). Large chemical shift changes were observed in the thumb on complexation. 15N relaxation data indicate reduction in high-frequency motion for a thumb loop and three palm turn/loops, which showed concomitant chemical shift changes on complexation. A deltaV303-V306 deletion and an L301R/V303R/V306R triple mutation abolished complex formation due to loss in hydrophobicity. In an updated model, the thumb-loop of beta-Pol contacts an edge/face region of the beta sheet of the XRCC1 NTD, while the beta-Pol palm weakly contacts the alpha2 helix.

Amino Acid Sequence↗

PubMedDate not supplied

Translation initiation: structures, mechanisms and evolution.

Translation, the process of mRNA-encoded protein synthesis, requires a complex apparatus, composed of the ribosome, tRNAs and additional protein factors, including aminoacyl tRNA synthetases. The ribosome provides the platform for proper assembly of mRNA, tRNAs and protein factors and carries the peptidyl-transferase activity. It consists of small and large subunits. The ribosomes are ribonucleoprotein particles with a ribosomal RNA core, to which multiple ribosomal proteins are bound. The sequence and structure of ribosomal RNAs, tRNAs, some of the ribosomal proteins and some of the additional protein factors are conserved in all kingdoms, underlying the common origin of the translation apparatus. Translation can be subdivided into several steps: initiation, elongation, termination and recycling. Of these, initiation is the most complex and the most divergent among the different kingdoms of life. A great amount of new structural, biochemical and genetic information on translation initiation has been accumulated in recent years, which led to the realization that initiation also shows a great degree of conservation throughout evolution. In this review, we summarize the available structural and functional data on translation initiation in the context of evolution, drawing parallels between eubacteria, archaea, and eukaryotes. We will start with an overview of the ribosome structure and of translation in general, placing emphasis on factors and processes with relevance to initiation. The major steps in initiation and the factors involved will be described, followed by discussion of the structure and function of the individual initiation factors throughout evolution. We will conclude with a summary of the available information on the kinetic and thermodynamic aspects of translation initiation.

Biophysical Phenomena↗