Structure of the receptor-binding carboxy-terminal domain of bacteriophage T7 tail fibers

The six bacteriophage T7 tail fibers, homo-trimers of gene product 17, are thought to be responsible for the first specific, albeit reversible, attachment to Escherichia coli lipopolysaccharide. The protein trimer forms kinked fibers comprised of an amino-terminal tail-attachment domain, a slender shaft, and a carboxyl-terminal domain composed of several nodules. Previously, we expressed, purified, and crystallized a carboxyl-terminal fragment comprising residues 371–553. Here, we report the structure of this protein trimer, solved using anomalous diffraction and refined at 2 Å resolution. Amino acids 371–447 form a tapered pyramid with a triangular cross-section composed of interlocked β-sheets from each of the three chains. The triangular pyramid domain has three α-helices at its narrow end, which are connected to a carboxyl-terminal three-blade β-propeller tip domain by flexible loops. The monomers of this tip domain each contain an eight-stranded β-sandwich. The exact topology of the β-sandwich fold is novel, but similar to that of knob domains of other viral fibers and the phage Sf6 needle. Several host-range change mutants have been mapped to loops located on the top of this tip domain, suggesting that this surface of the tip domain interacts with receptors on the cell surface.     

Structure of BipA in GTP form bound to the ratcheted ribosome

BPI-inducible protein A (BipA) is a member of the family of ribosome-dependent translational GTPase (trGTPase) factors along with elongation factors G and 4 (EF-G and EF4). Despite being highly conserved in bacteria and playing a critical role in coordinating cellular responses to environmental changes, its structures (isolated and ribosome bound) remain elusive. Here, we present the crystal structures of apo form and GTP analog, GDP, and guanosine-3′,5′-bisdiphosphate (ppGpp)-bound BipA. In addition to having a distinctive domain arrangement, the C-terminal domain of BipA has a unique fold. Furthermore, we report the cryo-electron microscopy structure of BipA bound to the ribosome in its active GTP form and elucidate the unique structural attributes of BipA interactions with the ribosome and A-site tRNA in the light of its possible function in regulating translation.Bacterial protein synthesis involves four main translational GTPase (trGTPase) factors: initiation factor 2 (IF2), elongation factors Tu and G (EF-Tu and EF-G), and release factor 3 (RF3). These factors catalyze major steps in translation initiation, elongation (both decoding and mRNA–tRNA complex translocation), and termination, in a GTP-dependent manner. Several additional GTPase factors, including EF4 (formerly known as LepA), BipA, and RelA, have been revealed to be associated with ribosomes under stress conditions (1).Both EF4 and BipA are paralogs of EF-G (1–5). Although EF4 is highly conserved in bacteria (4), deletion of ef4 gene causes no evident phenotype in Escherichia coli under optimal growth conditions (6). However, EF4 was shown to notably improve protein synthesis under stress conditions (7). Qin et al. (2) reported a unique function of EF4 promoting the back translocation of the elongation complex by one codon, hence presumably providing a second chance for EF-G to carry out a correct translocation.BipA (BPI-inducible protein A) gene is highly conserved among bacterial and chloroplast genomes (4) and has been implicated in regulating a variety of cellular processes including bacterial virulence, symbiosis, various stress responses, resistance to host defenses, swarming motility, biofilm, and capsule formation (8–10). As is the case with EF4, BipA is not required under optimal growth conditions but becomes an essential factor for bacterial survival at low temperature, nutrient depletion, and various other stress conditions (1, 9). The diverse nature of these processes underscores the global regulatory properties of BipA. Similarity to classical trGTPases and EF4 led to the speculation that BipA affects translation through directly interacting with the ribosome. For example, wild-type (fully modified) ribosomes seem to depend on BipA for translation of specific mRNAs (11). Furthermore, as with EF4, overexpression of BipA inhibits transfer-messenger mRNA (tmRNA)-dependent peptide tagging activity of nonstop messages on ribosome (6). Thus, BipA likely functions as an elongation factor as well. Consistent with this notion, BipA is able to bind to 70S ribosome in a GTP-dependent manner and its GTPase activity is enhanced in the presence of ribosomes, a characteristic feature of classical trGTPase factors (5, 12). Salmonella enterica BipA has been shown to interact with either 70S ribosomes or 30S subunits depending on the relative abundance of GTP and of the stress alarmone guanosine-3′,5′-bisdiphosphate (ppGpp), respectively (12). In addition, a recent study links BipA to ribosome biogenesis because bipA gene deletion results in perturbed 50S subunit processing and assembly, particularly at low temperatures (13). Although the evidence for BipA involvement in ribosome biosynthesis and/or functioning in translation is mounting, its exact role remains elusive.As a member of the ribosome-dependent trGTPase family, BipA is proposed to share structural similarity with EF4 and EF-G (4, 5). Indeed, all three consist of five domains, of which the N-terminal G domain (nucleotide-binding domain), the β-barrel domain (domain II), and the two α/β-domains (domains III and IV) are topologically equivalent (5) (Fig. 1). EF-G has G′ domain inserted into its G domain and a unique domain IV, whereas unique C-terminal domains (CTDs) are present in BipA and EF4 (Fig. 1). Despite the similarity, the three proteins have distinct functions probably attributed to their varied domain arrangements and ribosome-binding modes.Open in a separate windowFig. 1.Comparison of domain arrangement and overall structure of EF-G, EF4, and BipA. (A) Structures of isolated EF-G and EF4 are obtained from Protein Data Bank (PDB ID codes: 2BM0 and 3CB4, respectively). Structure of BipA apo form is presented. Domain I (green), also known as the G domain, is the nucleotide-binding region. G′ domain insertion (dark blue) is a characteristic feature of the EF-G protein. Domain II (violet) contains the translation factor signature β-barrel motif. Domains III (yellow) and V (sky blue) contain α/β-motifs. EF-G has a unique domain IV (brown), whereas EF4 and BipA have unique C-terminal domains (warm pink and red, respectively). The same color scheme is used throughout this work. (B) Schematic diagram depicting the domain arrangement of EF-G, EF4, and BipA.Extensive structural studies (14–22) of EF-G bound to ribosome have generated a wealth of atomic or near-atomic resolution information on how EF-G, in particular the positioning of its domain IV in ribosome decoding center, facilitates translocation. Mutagenesis study of EF-G revealed that the highly conserved loops I and II of domain IV disrupt the interactions between the decoding center and the codon–anticodon duplex that act as the barrier for mRNA–tRNA complex translocation (23). Structural studies have also shed light on the molecular basis of how EF4 reverses EF-G catalyzed translocation through its CTD reaching into the PTC and interacting with the acceptor stem of the peptidyl-tRNA in the P site (24, 25). In contrast, structures of neither the isolated BipA nor BipA bound to ribosome, which could illuminate the molecular basis of BipA functioning in protein translation, have been characterized yet. Hence, we aimed to structurally characterize the various biologically relevant states of BipA on and off the ribosome, toward a better understanding of the detailed function of BipA. Note that during the revision process, a paper was published reporting the structure of isolated BipA (26).     

Structure of the 70S ribosome bound to release factor 2 and a substrate analog provides insights into catalysis of peptide release

We report the crystal structure of release factor 2 bound to ribosome with an aminoacyl tRNA substrate analog at the ribosomal P site, at 3.1 Å resolution. The structure shows that upon stop-codon recognition, the universally conserved GGQ motif packs tightly into the peptidyl transferase center. Nucleotide A2602 of 23S rRNA, implicated in peptide release, packs with the GGQ motif in release factor 2. The ribose of A76 of the peptidyl-tRNA adopts the C2′-endo conformation, and the 2′ hydroxyl of A76 is within hydrogen-bond distance of the 2′ hydroxyl of A2451. The structure suggests how a catalytic water can be coordinated in the peptidyl transferase center and, together with previous biochemical and computational data, suggests a model for how the ester bond between the peptidyl tRNA and the nascent peptide is hydrolyzed.     

Connecting actin monomers by iso-peptide bond is a toxicity mechanism of the Vibrio cholerae MARTX toxin

The Gram-negative bacterium Vibrio cholerae is the causative agent of a severe diarrheal disease that afflicts three to five million persons annually, causing up to 200,000 deaths. Nearly all V. cholerae strains produce a large multifunctional-autoprocessing RTX toxin (MARTX_Vc), which contributes significantly to the pathogenesis of cholera in model systems. The actin cross-linking domain (ACD) of MARTX_Vc directly catalyzes a covalent cross-linking of monomeric G-actin into oligomeric chains and causes cell rounding, but the nature of the cross-linked bond and the mechanism of the actin cytoskeleton disruption remained elusive. To elucidate the mechanism of ACD action and effect on actin, we identified the covalent cross-link bond between actin protomers using limited proteolysis, X-ray crystallography, and mass spectrometry. We report here that ACD catalyzes the formation of an intermolecular iso-peptide bond between residues E270 and K50 located in the hydrophobic and the DNaseI-binding loops of actin, respectively. Mutagenesis studies confirm that no other residues on actin can be cross-linked by ACD both in vitro and in vivo. This cross-linking locks actin protomers into an orientation different from that of F-actin, resulting in strong inhibition of actin polymerization. This report describes a microbial toxin mechanism acting via iso-peptide bond cross-linking between host proteins and is, to the best of our knowledge, the only known example of a peptide linkage between nonterminal glutamate and lysine side chains.     

Electronic properties of the highly ruffled heme bound to the heme degrading enzyme IsdI

IsdI, a heme-degrading protein from Staphylococcus aureus, binds heme in a manner that distorts the normally planar heme prosthetic group to an extent greater than that observed so far for any other heme-binding protein. To understand better the relationship between this distinct structural characteristic and the functional properties of IsdI, spectroscopic, electrochemical, and crystallographic results are reported that provide evidence that this heme ruffling is essential to the catalytic activity of the protein and eliminates the need for the water cluster in the distal heme pocket that is essential for the activity of classical heme oxygenases. The lack of heme orientational disorder in (1)H-NMR spectra of the protein argues that the catalytic formation of β- and δ-biliverdin in nearly equal yield results from the ability of the protein to attack opposite sides of the heme ring rather than from binding of the heme substrate in two alternative orientations.     

Structural polymorphism in the N-terminal oligomerization domain of NPM1

Nucleophosmin (NPM1) is a multifunctional phospho-protein with critical roles in ribosome biogenesis, tumor suppression, and nucleolar stress response. Here we show that the N-terminal oligomerization domain of NPM1 (Npm-N) exhibits structural polymorphism by populating conformational states ranging from a highly ordered, folded pentamer to a highly disordered monomer. The monomer–pentamer equilibrium is modulated by posttranslational modification and protein binding. Phosphorylation drives the equilibrium in favor of monomeric forms, and this effect can be reversed by Npm-N binding to its interaction partners. We have identified a short, arginine-rich linear motif in NPM1 binding partners that mediates Npm-N oligomerization. We propose that the diverse functional repertoire associated with NPM1 is controlled through a regulated unfolding mechanism signaled through posttranslational modifications and intermolecular interactions.Nucleophosmin (NPM1) is a highly abundant nucleolar phosphoprotein with functions associated with ribosome biogenesis (1, 2), maintenance of genome stability (1), nucleolar stress response (3), modulation of the p53 tumor suppressor pathway (4), and regulation of apoptosis (5). Importantly, genetic alterations that affect the NPM1 protein sequence or expression level are associated with oncogenesis. For example, NPM1 overexpression was observed in a variety of solid tumors, and mutations within the protein and genetic translocations involving NPM1 are associated with hematological malignancies (reviewed in ref. 6).NPM1 primarily resides in the nucleolus which is a membrane-less compartment and the site of rRNA synthesis, processing, and assembly with ribosomal proteins (7). In the nucleolus, NPM1 is involved in processing preribosomal RNA (4), chaperoning the nucleolar entry of ribosomal (1, 8) and viral (9) proteins, and stabilizing the alternate reading frame (ARF) tumor suppressor protein (4, 5, 10, 11), while also playing a role in the shuttling of preribosomal particles assembled in the nucleolus to the cytoplasm (12–14).NPM1 is a member of the nucleoplasmin protein family, which includes the histone chaperones NPM2 and NPM3. These proteins share a conserved N-terminal oligomerization domain that mediates homopentamerization (15). Disruption of NPM1 oligomerization by a small molecule (16) or an RNA aptamer (17) causes exclusive nucleoplasmic localization, loss of colocalization with ARF, and induction of p53-dependent apoptosis (16, 17). These observations suggest that changes in the oligomeric state of NPM1 may influence its biological functions. However, although it is hypothesized (1) that NPM1 function is modulated through control of its oligomeric state, experimental data are currently lacking. Intriguingly, NPM1 exhibits 40 putative phosphorylation sites, the majority of which are evolutionarily conserved (18, 19). Modification of these sites that is influenced by subcellular localization and cell cycle phase (20, 21) modulates the biological function of NPM1 (1, 6). Approximately one third of these phosphorylation sites are located within the N-terminal oligomerization domain, indicating their possible involvement in regulation of the oligomerization state of NPM1.What is the molecular mechanism that underlies the various functions of NPM1? Here we show by using in vitro biophysical and structural methods that the N-terminal oligomerization domain of NPM1 (Npm-N) exhibits structural polymorphism by populating a range of conformations with various degrees of structural disorder that span two extreme structural states: a folded pentameric state and a disordered monomeric state. Several conserved phosphorylation sites in pentameric Npm-N are positioned within the hydrophobic interior of the pentameric structure (18) and therefore are inaccessible to kinases. Interestingly, other conserved sites are solvent accessible, and we show that these posttranslational modification (PTM) sites serve as molecular switches for modulating the oligomerization and the folding state of Npm-N. We propose that, when exposed through initial destabilizing phosphorylation events, the otherwise inaccessible sites act as molecular locks that, when phosphorylated (22–24), destabilize the pentameric form and lock Npm-N in the monomeric state. We demonstrate that the monomer–pentamer equilibrium is modulated by protein binding partners and have identified a short, arginine-rich (R-rich) linear motif that mediates this interaction. Our results suggest that the diverse cellular functions and subcellular localization of NPM1 are influenced through a regulated unfolding mechanism, signaled through PTMs and intermolecular interactions.     

Structure of human Mad1 C-terminal domain reveals its involvement in kinetochore targeting

The spindle checkpoint prevents aneuploidy by delaying anaphase onset until all sister chromatids achieve proper microtubule attachment. The kinetochore-bound checkpoint protein complex Mad1-Mad2 promotes the conformational activation of Mad2 and serves as a catalytic engine of checkpoint signaling. How Mad1 is targeted to kinetochores is not understood. Here, we report the crystal structure of the conserved C-terminal domain (CTD) of human Mad1. Mad1 CTD forms a homodimer and, unexpectedly, has a fold similar to those of the kinetochore-binding domains of Spc25 and Csm1. Nonoverlapping Mad1 fragments retain detectable kinetochore targeting. Deletion of the CTD diminishes, does not abolish, Mad1 kinetochore localization. Mutagenesis studies further map the functional interface of Mad1 CTD in kinetochore targeting and implicate Bub1 as its receptor. Our results indicate that CTD is a part of an extensive kinetochore-binding interface of Mad1, and rationalize graded kinetochore targeting of Mad1 during checkpoint signaling.     

Autoimmunity to RNA polymerase II is focused at the carboxyl terminal domain of the large subunit

Objective. Previous studies have demonstrated antibodies to the large (220 kd) polypeptide subunit of RNA polymerase II (Pol II) in sera from certain patients with scleroderma. In the present study, we sought to identify the autoantigenic region on this polypeptide. Methods. A recombinant fusion protein, corresponding to the 52-heptapeptide repeat found in the carboxyl terminal domain (CTD) of the large Pol II subunit, was used to identify 15 patient sera that contained autoantibodies. Synthetic peptides CTD7 (representing a single heptapeptide) and CTD18 (representing 2½ heptapeptide repeats) were used in a competitive inhibition assay to define the specificity of these sera and the importance of the CTD as an autoantigen. Results. All 15 sera immunoprecipitated the Pol II subunit from radiolabeled cell extracts, and 11 of them bound the CTD fusion protein in immunoblots. Immunoprecipitation of Pol II was completely inhibited by CTD18 in 5 sera and partially inhibited in 4 additional sera. Conclusion. These results indicate that the CTD heptapeptide repeat is a focal point for autoimmune responses in scleroderma. It is likely that the repetitive sequence and high content of charged residues of this structure contribute to its role as an autoantigen.