Protein fate in higher eukaryotes is controlled by three complexes that share conserved architectural elements: the proteasome, COP9 signalosome, and eukaryotic translation initiation factor 3 (eIF3). Here we reconstitute the 13-subunit human eIF3 in Escherichia coli , revealing its structural core to be the eight subunits with conserved orthologues in the proteasome lid complex and COP9 signalosome. This structural core in eIF3 binds to the small (40S) ribosomal subunit, to translation initiation factors involved in mRNA cap-dependent initiation, and to the hepatitis C viral (HCV) internal ribosome entry site (IRES) RNA. Addition of the remaining eIF3 subunits enables reconstituted eIF3 to assemble intact initiation complexes with the HCV IRES. Negative-stain EM reconstructions of reconstituted eIF3 further reveal how the approximately 400 kDa molecular mass structural core organizes the highly flexible 800 kDa molecular mass eIF3 complex, and mediates translation initiation.

C2 domains are widely-spread protein signaling motifs that in classical PKCs act as Ca 2؉ -binding modules. However, the molecular mechanisms of their targeting process at the plasma membrane remain poorly understood. Here, the crystal structure of PKC␣-C2 domain in complex with Ca 2؉ , 1,2-dihexanoyl-sn-glycero-3-[phospho-L-serine] (PtdSer), and 1,2-diayl-sn-glycero-3-[phosphoinositol-4,5-bisphosphate] [PtdIns(4,5)P2] shows that PtdSer binds specifically to the calcium-binding region, whereas PtdIns(4,5)P2 occupies the concave surface of strands ␤3 and ␤4. Strikingly, the structure reveals a PtdIns(4,5)P2-C2 domain-binding mode in which the aromatic residues Tyr-195 and Trp-245 establish direct interactions with the phosphate moieties of the inositol ring. Mutations that abrogate Tyr-195 and Trp-245 recognition of PtdIns(4,5)P2 severely impaired the ability of PKC␣ to localize to the plasma membrane. Notably, these residues are highly conserved among C2 domains of topology I, and a general mechanism of C2 domain-membrane docking mediated by PtdIns(4,5)P2 is presented.calcium phosphoinositides ͉ peripheral membrane proteins T he C2 domains are considered peripheral proteins that are water-soluble and associate reversibly with lipid bilayers. Recently, evidence has demonstrated that some of these domains are able to interact with the inositol phospholipid 1,2-diacyl-sn-glycero-3-[phosphoinositol-4,5-bisphosphate] [PtdIns(4,5)P 2 ] (1-4), which is able to directly participate in a myriad of functions, including cell signaling at the plasma membrane, regulation of membrane traffic and transport, cytoskeleton dynamics, and nuclear events (5, 6). Despite the number of C2 domain 3D structures currently available, questions about how they interact with the different target phospholipids, their precise spatial position in the lipid bilayer, and their role in transmitting signals downstream have yet to be explored.The main role of the C2 domain in classical PKCs (cPKCs) is to act as the Ca 2ϩ -activated membrane-targeting motif (7, 8). The 3D structure of these C2 domains comprises 8 antiparallel ␤-strands assembled in a ␤-sandwich architecture, with flexible loops on top and at the bottom (9-12). This C2 domain displays 2 functional regions: the Ca 2ϩ -binding region and the polybasic cluster. The former is located in the flexible top loops, binds 2 or 3 Ca 2ϩ ions, depending on the isoenzyme (10,11,13,14), and interacts with 1,2-diacyl-sn-glycero-3-[phospho-L-serine] (PtdSer) (11,15,16). The second region is a polybasic cluster that is located at the concave surface of the C2 domain formed by strands ␤3 and ␤4. Recent studies indicate that this region might bind specifically to PtdIns(4,5)P 2 in a Ca 2ϩ -dependent manner (1,(17)(18)(19)(20)(21).To gain insight into the structural and functional basis for the PtdIns(4,5)P 2 -dependent membrane targeting of the PKC␣-C2 domain, we determined the 3D structures of the ternary and quaternary complexes of the C2 domain of PKC␣, crystallized in presence of Ca 2ϩ and PtdIns(4,5...

The initiation of protein synthesis plays an essential regulatory role in human biology. At the center of the initiation pathway, the 13-subunit eukaryotic translation initiation factor 3 (eIF3) controls access of other initiation factors and mRNA to the ribosome by unknown mechanisms. Using electron microscopy (EM), bioinformatics and biochemical experiments, we identify two highly conserved RNA-binding motifs in eIF3 that direct translation initiation from the hepatitis C virus internal ribosome entry site (HCV IRES) RNA. Mutations in the RNA-binding motif of subunit eIF3a weaken eIF3 binding to the HCV IRES and the 40S ribosomal subunit, thereby suppressing eIF2-dependent recognition of the start codon. Mutations in the eIF3c RNA-binding motif also reduce 40S ribosomal subunit binding to eIF3, and inhibit eIF5B-dependent steps downstream of start codon recognition. These results provide the first connection between the structure of the central translation initiation factor eIF3 and recognition of the HCV genomic RNA start codon, molecular interactions that likely extend to the human transcriptome.

During infection, viruses undergo conformational changes that lead to delivery of their genome into host cytosol. In human rhinovirus A2, this conversion is triggered by exposure to acid pH in the endosome. The first subviral intermediate, the A-particle, is expanded and has lost the internal viral protein 4 (VP4), but retains its RNA genome. The nucleic acid is subsequently released, presumably through one of the large pores that open at the icosahedral twofold axes, and is transferred along a conduit in the endosomal membrane; the remaining empty capsids, termed B-particles, are shuttled to lysosomes for degradation. Previous structural analyses revealed important differences between the native protein shell and the empty capsid. Nonetheless, little is known of A-particle architecture or conformation of the RNA core. Using 3D cryo-electron microscopy and X-ray crystallography, we found notable changes in RNAprotein contacts during conversion of native virus into the A-particle uncoating intermediate. In the native virion, we confirmed interaction of nucleotide(s) with Trp 38 of VP2 and identified additional contacts with the VP1 N terminus. Study of A-particle structure showed that the VP2 contact is maintained, that VP1 interactions are lost after exit of the VP1 N-terminal extension, and that the RNA also interacts with residues of the VP3 N terminus at the fivefold axis. These associations lead to formation of a well-ordered RNA layer beneath the protein shell, suggesting that these interactions guide ordered RNA egress.genome uncoating | X-ray analysis | 3D cryo-EM | picornavirus H uman rhinoviruses (HRVs) cause the common cold. Although seldom severe, this disease is widespread and frequent in man; HRVs thus have considerable economic impact due to expenditure on medication and lost working days. More than 150 serotypes belong to the genus Enteroviruses (EVs) of the Picornaviridae family, which includes serious human and animal pathogens. In addition to phylogenetic classification into species A, -B, and -C, HRVs are divided into a minor receptor group (12 HRV-A) that bind low-density lipoprotein receptors (LDLRs), and a major receptor group (more than 89 HRV-A and -B serotypes) that use intercellular adhesion molecule 1 (ICAM-1) for cell entry (1). HRV-C binds an unknown receptor (2).The EV icosahedral shell is built from four viral proteins (VP1-4) that encase a single-stranded (+)-sense RNA genome. Sixty copies each of these four polypeptides assemble on a T = 1 (pseudo T = 3) lattice, ∼30 nm in diameter. VP1, VP2, and VP3 are surface-exposed; the small myristoylated VP4 is internal. In the mature virion, the N-terminal extensions of VP1, VP2, and VP3, together with the entire VP4, interact in an intricate network beneath the shell (Fig. S1) (3, 4).In the cytosol, the viral RNA is translated into a ∼250 kDa precursor polyprotein that is processed by viral proteinases. Assembly of the viral shell involves immature pentamers built from VP0, VP1, and VP3. VP2 and VP4 arise late in infection through VP0 cl...

birnavirus replication ͉ infectious bursal disease virus ͉ noncanonical palm ͉ virus evolution ͉ double-stranded RNA viruses

SUMMARY Eukaryotic translation initiation factor 3 (eIF3) plays a central role in protein synthesis by organizing the formation of the 43S preinitiation complex. Using genetic tag visualization by electron microscopy, we reveal the molecular organization of ten human eIF3 subunits, including an octameric core. The structure of eIF3 bears a close resemblance to that of the proteasome lid, with a conserved spatial organization of eight core subunits containing PCI and MPN domains that coordinate functional interactions in both complexes. We further show that eIF3 subunits a and c interact with initiation factors eIF1 and eIF1A, which control the stringency of start codon selection. Finally, we find that subunit j, which modulates messenger RNA interactions with the small ribosomal subunit, makes multiple independent interactions with the eIF3 octameric core. These results highlight the conserved architecture of eIF3 and how it scaffolds key factors that control translation initiation in higher eukaryotes, including humans.

TREX1 is the most abundant mammalian 3 3 5 DNA exonuclease. It has been described to form part of the SET complex and is responsible for the Aicardi-Goutières syndrome in humans. Here we show that the exonuclease activity is correlated to the binding preferences toward certain DNA sequences. In particular, we have found three motifs that are selected, GAG, ACA, and CTGC. To elucidate how the discrimination occurs, we determined the crystal structures of two murine TREX1 complexes, with a nucleotide product of the exonuclease reaction, and with a single-stranded DNA substrate. Using confocal microscopy, we observed TREX1 both in nuclear and cytoplasmic subcellular compartments. Remarkably, the presence of TREX1 in the nucleus requires the loss of a C-terminal segment, which we named leucine-rich repeat 3. Furthermore, we detected the presence of a conserved proline-rich region on the surface of TREX1. This observation points to interactions with proline-binding domains. The potential interacting motif "PPPVPRPP" does not contain aromatic residues and thus resembles other sequences that select SH3 and/or Group 2 WW domains. By means of nuclear magnetic resonance titration experiments, we show that, indeed, a polyproline peptide derived from the murine TREX1 sequence interacted with the WW2 domain of the elongation transcription factor CA150. Coimmunoprecipitation studies confirmed this interaction with the full-length TREX1 protein, thereby suggesting that TREX1 participates in more functional complexes than previously thought.

Eukaryotic translation initiation factor 3 (eIF3) is a key regulator of translation initiation, but its in vivo assembly and molecular functions remain unclear. Here we show that eIF3 from Neurospora crassa is structurally and compositionally similar to human eIF3. N. crassa eIF3 forms a stable 12-subunit complex linked genetically and biochemically to the 13th subunit, eIF3j, which in humans modulates mRNA start codon selection. Based on N. crassa genetic analysis, most subunits in eIF3 are essential. Subunits that can be deleted (e, h, k and l) map to the right side of the eIF3 complex, suggesting that they may coordinately regulate eIF3 function. Consistent with this model, subunits eIF3k and eIF3l are incorporated into the eIF3 complex as a pair, and their insertion depends on the presence of subunit eIF3h, a key regulator of vertebrate development. Comparisons to other eIF3 complexes suggest that eIF3 assembles around an eIF3a and eIF3c dimer, which may explain the coordinated regulation of human eIF3 levels. Taken together, these results show that Neurospora crassa eIF3 provides a tractable system for probing the structure and function of human-like eIF3 in the context of living cells.