Binding of human Cdc123 to eIF2γ.

Eukaryotic initiation factor 2 (eIF2) plays a key role in protein synthesis and in its regulation. The assembly of this heterotrimeric factor is facilitated by Cdc123, a member of the ATP grasp family that binds the γ subunit of eIF2. Notably, some mutations related to MEHMO syndrome, an X-linked intellectual disability, affect Cdc123-mediated eIF2 assembly. The mechanism of action of Cdc123 is unclear and structural information for the human protein is awaited. Here, the crystallographic structure of human Cdc123 (Hs-Cdc123) bound to domain 3 of human eIF2γ (Hs-eIF2γD3) was determined. The structure shows that the domain 3 of eIF2γ is bound to domain 1 of Cdc123. In addition, the long C-terminal region of Hs-Cdc123 provides a link between the ATP and Hs-eIF2γD3 binding sites. A thermal shift assay shows that ATP is tightly bound to Cdc123 whereas the affinity of ADP is much smaller. Yeast cell viability experiments, western blot analysis and two-hybrid assays show that ATP is important for the function of Hs-Cdc123 in eIF2 assembly. These data and recent findings allow us to propose a refined model to explain the mechanism of action of Cdc123 in eIF2 assembly.

SpiCee: A Genetic Tool for Subcellular and Cell-Specific Calcium Manipulation.

Calcium is a second messenger crucial to a myriad of cellular processes ranging from regulation of metabolism and cell survival to vesicle release and motility. Current strategies to directly manipulate endogenous calcium signals lack cellular and subcellular specificity. We introduce SpiCee, a versatile and genetically encoded chelator combining low- and high-affinity sites for calcium. This scavenger enables altering endogenous calcium signaling and functions in single cells in vitro and in vivo with biochemically controlled subcellular resolution. SpiCee paves the way to investigate local calcium signaling in vivo and directly manipulate this second messenger for therapeutic use.

Structural basis of the interaction between cyclodipeptide synthases and aminoacylated tRNA substrates.

Cyclodipeptide synthases (CDPSs) catalyze the synthesis of various cyclodipeptides by using two aminoacyl-tRNA (aa-tRNA) substrates in a sequential mechanism. Here, we studied binding of phenylalanyl-tRNAPhe to the CDPS from Candidatus Glomeribacter gigasporarum (Cglo-CDPS) by gel filtration and electrophoretic mobility shift assay. We determined the crystal structure of the Cglo-CDPS:Phe-tRNAPhe complex to 5 Å resolution and further studied it in solution using small-angle X-ray scattering (SAXS). The data show that the major groove of the acceptor stem of the aa-tRNA interacts with the enzyme through the basic ß2 and ß7 strands of CDPSs belonging to the XYP subfamily. A bending of the CCA extremity enables the amino acid moiety to be positioned in the P1 pocket while the terminal A76 adenosine occupies the P2 pocket. Such a positioning indicates that the present structure illustrates the binding of the first aa-tRNA. In cells, CDPSs and the elongation factor EF-Tu share aminoacylated tRNAs as substrates. The present study shows that CDPSs and EF-Tu interact with opposite sides of tRNA. This may explain how CDPSs hijack aa-tRNAs from canonical ribosomal protein synthesis.

Adaptive landscape flattening allows the design of both enzyme: Substrate binding and catalytic power.

Designed enzymes are of fundamental and technological interest. Experimental directed evolution still has significant limitations, and computational approaches are a complementary route. A designed enzyme should satisfy multiple criteria: stability, substrate binding, transition state binding. Such multi-objective design is computationally challenging. Two recent studies used adaptive importance sampling Monte Carlo to redesign proteins for ligand binding. By first flattening the energy landscape of the apo protein, they obtained positive design for the bound state and negative design for the unbound. We have now extended the method to design an enzyme for specific transition state binding, i.e., for its catalytic power. We considered methionyl-tRNA synthetase (MetRS), which attaches methionine (Met) to its cognate tRNA, establishing codon identity. Previously, MetRS and other synthetases have been redesigned by experimental directed evolution to accept noncanonical amino acids as substrates, leading to genetic code expansion. Here, we have redesigned MetRS computationally to bind several ligands: the Met analog azidonorleucine, methionyl-adenylate (MetAMP), and the activated ligands that form the transition state for MetAMP production. Enzyme mutants known to have azidonorleucine activity were recovered by the design calculations, and 17 mutants predicted to bind MetAMP were characterized experimentally and all found to be active. Mutants predicted to have low activation free energies for MetAMP production were found to be active and the predicted reaction rates agreed well with the experimental values. We suggest the present method should become the paradigm for computational enzyme design.

Use of ß3-methionine as an amino acid substrate of Escherichia coli methionyl-tRNA synthetase.

Polypeptides containing ß-amino acids are attractive tools for the design of novel proteins having unique properties of medical or industrial interest. Incorporation of ß-amino acids in vivo requires the development of efficient aminoacyl-tRNA synthetases specific of these non-canonical amino acids. Here, we have performed a detailed structural and biochemical study of the recognition and use of ß3-Met by Escherichia coli methionyl-tRNA synthetase (MetRS). We show that MetRS binds ß3-Met with a 24-fold lower affinity but catalyzes the esterification of the non-canonical amino acid onto tRNA with a rate lowered by three orders of magnitude. Accurate measurements of the catalytic parameters required careful consideration of the presence of contaminating α-Met in ß3-Met commercial samples. The 1.45 Å crystal structure of the MetRS: ß3-Met complex shows that ß3-Met binds the enzyme essentially like α-Met, but the carboxylate moiety is mobile and not adequately positioned to react with ATP for aminoacyl adenylate formation. This study provides structural and biochemical bases for engineering MetRS with improved ß3-Met aminoacylation capabilities.

Recognition of different base tetrads by RHAU (DHX36): X-ray crystal structure of the G4 recognition motif bound to the 3'-end tetrad of a DNA G-quadruplex.

G-quadruplexes (G4) are secondary structures of nucleic acids that can form in cells and have diverse biological functions. Several biologically important proteins interact with G-quadruplexes, of which RHAU (or DHX36) - a helicase from the DEAH-box superfamily, was shown to bind and unwind G-quadruplexes efficiently. We report a X-ray co-crystal structure at 1.5 Šresolution of an N-terminal fragment of RHAU bound to an exposed tetrad of a parallel-stranded G-quadruplex. The RHAU peptide folds into an L-shaped α-helix, and binds to a G-quadruplex through π-stacking and electrostatic interactions. X-ray crystal structure of our complex identified key amino acid residues important for G-quadruplex-peptide binding interaction at the 3'-end G•G•G•G tetrad. Together with previous solution and crystal structures of RHAU bound to the 5'-end G•G•G•G and G•G•A•T tetrads, our crystal structure highlights the occurrence of a robust G-quadruplex recognition motif within RHAU that can adapt to different accessible tetrads.

SponGee: A Genetic Tool for Subcellular and Cell-Specific cGMP Manipulation.

cGMP is critical to a variety of cellular processes, but the available tools to interfere with endogenous cGMP lack cellular and subcellular specificity. We introduce SponGee, a genetically encoded chelator of this cyclic nucleotide that enables in vitro and in vivo manipulations in single cells and in biochemically defined subcellular compartments. SponGee buffers physiological changes in cGMP concentration in various model systems while not affecting cAMP signals. We provide proof-of-concept strategies by using this tool to highlight the role of cGMP signaling in vivo and in discrete subcellular domains. SponGee enables the investigation of local cGMP signals in vivo and paves the way for therapeutic strategies that prevent downstream signaling activation.

The trimeric coiled-coil HSBP1 protein promotes WASH complex assembly at centrosomes.

The Arp2/3 complex generates branched actin networks that exert pushing forces onto different cellular membranes. WASH complexes activate Arp2/3 complexes at the surface of endosomes and thereby fission transport intermediates containing endocytosed receptors, such as α5ß1 integrins. How WASH complexes are assembled in the cell is unknown. Here, we identify the small coiled-coil protein HSBP1 as a factor that specifically promotes the assembly of a ternary complex composed of CCDC53, WASH, and FAM21 by dissociating the CCDC53 homotrimeric precursor. HSBP1 operates at the centrosome, which concentrates the building blocks. HSBP1 depletion in human cancer cell lines and in Dictyostelium amoebae phenocopies WASH depletion, suggesting a critical role of the ternary WASH complex for WASH functions. HSBP1 is required for the development of focal adhesions and of cell polarity. These defects impair the migration and invasion of tumor cells. Overexpression of HSBP1 in breast tumors is associated with increased levels of WASH complexes and with poor prognosis for patients.

Cdc123, a Cell Cycle Regulator Needed for eIF2 Assembly, Is an ATP-Grasp Protein with Unique Features.

Eukaryotic initiation factor 2 (eIF2), a heterotrimeric guanosine triphosphatase, has a central role in protein biosynthesis by supplying methionylated initiator tRNA to the ribosomal translation initiation complex and by serving as a target for translational control in response to stress. Recent work identified a novel step indispensable for eIF2 function: assembly of eIF2 from its three subunits by the cell proliferation protein Cdc123. We report the first crystal structure of a Cdc123 representative, that from Schizosaccharomyces pombe, both isolated and bound to domain III of Saccharomyces cerevisiae eIF2γ. The structures show that Cdc123 resembles enzymes of the ATP-grasp family. Indeed, Cdc123 binds ATP-Mg(2+), and conserved residues contacting ATP-Mg(2+) are essential for Cdc123 to support eIF2 assembly and cell viability. A docking of eIF2αγ onto Cdc123, combined with genetic and biochemical experiments, allows us to propose a model explaining how Cdc123 participates in the biogenesis of eIF2 through facilitating assembly of eIF2γ to eIF2α.

Intrinsic resistance to aminoglycosides in Enterococcus faecium is conferred by the 16S rRNA m5C1404-specific methyltransferase EfmM.

Aminoglycosides are ribosome-targeting antibiotics and a major drug group of choice in the treatment of serious enterococcal infections. Here we show that aminoglycoside resistance in Enterococcus faecium strain CIP 54-32 is conferred by the chromosomal gene efmM, encoding the E. faecium methyltransferase, as well as by the previously characterized aac(6')-Ii that encodes a 6'-N-aminoglycoside acetyltransferase. Inactivation of efmM in E. faecium increases susceptibility to the aminoglycosides kanamycin and tobramycin, and, conversely, expression of a recombinant version of efmM in Escherichia coli confers resistance to these drugs. The EfmM protein shows significant sequence similarity to E. coli RsmF (previously called YebU), which is a 5-methylcytidine (m5C) methyltransferase modifying 16S rRNA nucleotide C1407. The target for EfmM is shown by mass spectrometry to be a neighboring 16S rRNA nucleotide at C1404. EfmM uses the methyl group donor S-adenosyl-L-methionine to catalyze formation of m5C1404 on the 30S ribosomal subunit, whereas naked 16S rRNA and the 70S ribosome are not substrates. Addition of the 5-methyl to C1404 sterically hinders aminoglycoside binding. Crystallographic structure determination of EfmM at 2.28 Å resolution reveals an N-terminal domain connected to a central methyltransferase domain that is linked by a flexible lysine-rich region to two C-terminal subdomains. Mutagenesis of the methyltransferase domain established that two cysteines at specific tertiary locations are required for catalysis. The tertiary structure of EfmM is highly similar to that of RsmF, consistent with m5C formation at adjacent sites on the 30S subunit, while distinctive structural features account for the enzymes' respective specificities for nucleotides C1404 and C1407.

A unique conformation of the anticodon stem-loop is associated with the capacity of tRNAfMet to initiate protein synthesis.

In all organisms, translational initiation takes place on the small ribosomal subunit and two classes of methionine tRNA are present. The initiator is used exclusively for initiation of protein synthesis while the elongator is used for inserting methionine internally in the nascent polypeptide chain. The crystal structure of Escherichia coli initiator tRNA(f)(Met) has been solved at 3.1 A resolution. The anticodon region is well-defined and reveals a unique structure, which has not been described in any other tRNA. It encompasses a Cm32*A38 base pair with a peculiar geometry extending the anticodon helix, a base triple between A37 and the G29-C41 pair in the major groove of the anticodon stem and a modified stacking organization of the anticodon loop. This conformation is associated with the three GC basepairs in the anticodon stem, characteristic of initiator tRNAs and suggests a mechanism by which the translation initiation machinery could discriminate the initiator tRNA from all other tRNAs.

Structural basis of RNA-dependent recruitment of glutamine to the genetic code.

Glutaminyl-transfer RNA (Gln-tRNA(Gln)) in archaea is synthesized in a pretranslational amidation of misacylated Glu-tRNA(Gln) by the heterodimeric Glu-tRNA(Gln) amidotransferase GatDE. Here we report the crystal structure of the Methanothermobacter thermautotrophicus GatDE complexed to tRNA(Gln) at 3.15 angstroms resolution. Biochemical analysis of GatDE and of tRNA(Gln) mutants characterized the catalytic centers for the enzyme's three reactions (glutaminase, kinase, and amidotransferase activity). A 40 angstrom-long channel for ammonia transport connects the active sites in GatD and GatE. tRNA(Gln) recognition by indirect readout based on shape complementarity of the D loop suggests an early anticodon-independent RNA-based mechanism for adding glutamine to the genetic code.

Structure-function relationships of the intact aIF2alpha subunit from the archaeon Pyrococcus abyssi.

Eukaryotic and archaeal initiation factor 2 (e- and aIF2, respectively) are heterotrimeric proteins (alphabetagamma) supplying the small subunit of the ribosome with methionylated initiator tRNA. The gamma subunit forms the core of the heterotrimer. It resembles elongation factor EF1-A and ensures interaction with Met-tRNA(i)(Met). In the presence of the alpha subunit, which is composed of three domains, the gamma subunit expresses full tRNA binding capacity. This study reports the crystallographic structure of the intact aIF2alpha subunit from the archaeon Pyrococcus abyssi and that of a derived C-terminal fragment containing domains 2 and 3. The obtained structures are compared with those of N-terminal domains 1 and 2 of yeast and human eIF2alpha and with the recently determined NMR structure of human eIF2alpha. We show that the three-domain organization in the alpha subunit is conserved in archaea and eukarya. Domains 1 and 2 form a rigid body linked to a mobile third domain. Sequence comparisons establish that the most conserved regions in the aIF2alpha polypeptide lie at opposite sides of the protein, within domain 1 and domain 3, respectively. These two domains are known to exhibit RNA binding capacities. We propose that domain 3, which is known to glue the alpha subunit onto the gamma subunit, participates in Met-tRNA(i)(Met) binding while domain 1 recognizes either rRNA or mRNA on the ribosome. Thereby, the observed structural mobility within the e- and aIF2alpha molecules would be an integral part of the biological function of this subunit in the heterotrimeric e- and aIF2alphabetagamma factors.

Three-dimensional structure of methionyl-tRNA synthetase from Pyrococcus abyssi.

In class 1 aminoacyl-tRNA synthetases, methionyl-tRNA synthetases (MetRS) are homodimers or monomers depending on the presence or absence of a domain appended at the C-side of the polypeptide chain. Beyond this C-domain, all MetRS display a highly conserved catalytic core with a Rossmann fold, the two halves of which are linked by a connective peptide (CP). Three-dimensional folding of CP and its putative zinc content have served as a basis to propose a division of the MetRS family into four subgroups. All subgroups but one, which is predicted to display two zincs per MetRS polypeptide, have been characterized. In the present study, the 3D structure of MetRS from Pyrococcus abyssi could be solved at 2.9 A resolution. The data obtained and atomic absorption spectroscopic measurements establish the presence of two metal ions per polypeptide chain. This finding brings strong support to the above classification. In the crystal, the C-terminal dimerization domain is disordered. This observation is thought to reflect marked flexibility of the two core moieties with respect to the C-domains in the dimer. Gel shift experiments were performed with the isolated C-terminal dimerization domain and a core monomeric MetRS, both derived from the P. abyssi enzyme. Complex formation between the C-domain and the core enzyme could not be evidenced. Moreover, association of tRNA(Met) to the core enzyme is enhanced in the presence of the C-domain. Together, these experiments suggest positive control in trans by the C-domain on recognition of tRNA by the core moiety of MetRS.

Use of analogues of methionine and methionyl adenylate to sample conformational changes during catalysis in Escherichia coli methionyl-tRNA synthetase.

Binding of methionine to methionyl-tRNA synthetase (MetRS) is known to promote conformational changes within the active site. However, the contribution of these rearrangements to enzyme catalysis is not fully understood. In this study, several methionine and methionyl adenylate analogues were diffused into crystals of the monomeric form of Escherichia coli methionyl-tRNA synthetase. The structures of the corresponding complexes were solved at resolutions below 1.9A and compared to those of the enzyme free or complexed with methionine. Residues Y15 and W253 play key roles in the strength of the binding of the amino acid and of its analogues. Indeed, full motions of these residues are required to recover the maximum in free energy of binding. Residue Y15 also controls the size of the hydrophobic pocket where the amino acid side-chain interacts. H301 appears to participate to the specific recognition of the sulphur atom of methionine. Complexes with methionyl adenylate analogues illustrate the shielding by MetRS of the region joining the methionine and adenosine moieties. Finally, the structure of MetRS complexed to a methionine analogue mimicking the tetrahedral carbon of the transition state in the aminoacylation reaction was solved. On the basis of this model, we propose that, in response to the binding of the 3'-end of tRNA, Y15 moves again in order to deshield the anhydride bond in the natural adenylate.

Structure and function of the C-terminal domain of methionyl-tRNA synthetase.

The minimal polypeptide supporting full methionyl-tRNA synthetase (MetRS) activity is composed of four domains: a catalytic Rossmann fold, a connective peptide, a KMSKS domain, and a C-terminal alpha helix bundle domain. The minimal MetRS behaves as a monomer. In several species, MetRS is a homodimer because of a C-terminal domain appended to the core polypeptide. Upon truncation of this C-terminal domain, subunits dissociate irreversibly. Here, the C-terminal domain of dimeric MetRS from Pyrococcus abyssi was isolated and studied. It displays nonspecific tRNA-binding properties and has a crystalline structure closely resembling that of Trbp111, a dimeric tRNA-binding protein found in many bacteria and archaea. The obtained 3D model was used to direct mutations against dimerization of Escherichia coli MetRS. Comparison of the resulting mutants to native and C-truncated MetRS shows that the presence of the appended C-domain improves tRNA(Met) binding affinity. However, dimer formation is required to evidence the gain in affinity.