Translation initiation factors eIF4A and eIF4G form, together with the cap-binding factor eIF4E, the eIF4F complex, which is crucial for recruiting the small ribosomal subunit to the mRNA 5 end and for subsequent scanning and searching for the start codon. eIF4A is an ATP-dependent RNA helicase whose activity is stimulated by binding to eIF4G. We report here the structure of the complex formed by yeast eIF4G's middle domain and full-length eIF4A at 2.6-Å resolution. eIF4A shows an extended conformation where eIF4G holds its crucial DEAD-box sequence motifs in a productive conformation, thus explaining the stimulation of eIF4A's activity. A hitherto undescribed interaction involves the amino acid Trp-579 of eIF4G. Mutation to alanine results in decreased binding to eIF4A and a temperature-sensitive phenotype of yeast cells that carry a Trp579Ala mutation as its sole source for eIF4G. Conformational changes between eIF4A's closed and open state provide a model for its RNA-helicase activity.translation initiation ͉ DEAD-box protein ͉ X-ray structure ͉ eIF4F T ranslation initiation in eukarya is usually the rate-limiting and most tightly controlled stage of polypeptide synthesis (reviewed in refs. 1-3). For the majority of eukaryotic mRNAs, the cap-dependent pathway is used for translation initiation (3). It comprises four consecutive steps: (i) formation of the 43S preinitiation complex consisting of the 40S ribosomal subunit, initiation factors (eIF2, eIF3), and Met-tRNA i ; (ii) recruitment of the 43S preinitiation complex to the capped 5Ј end of the mRNA; (iii) scanning of the 5Ј untranslated region of the mRNA and start codon recognition; and (iv) joining of the large 60S ribosomal subunit and assembly of the 80S ribosome.Approximately a dozen eukaryotic translation initiation factors (eIFs) are needed for this process. A central component of the second and third step is eIF4F, a heterotrimeric stable complex consisting of the cap-binding protein eIF4E, the DEAD-box helicase eIF4A, and the central multiscaffold protein eIF4G, which possesses additional binding sites for the poly(A)-binding protein PABP and, in mammalia, for eIF3 (Fig. 1A). Mammalian eIF4G possesses a second eIF4A binding site in its C-terminal region in proximity to a binding site for protein kinase Mnk1 (mitogen-activated protein kinase-interacting kinase), which phosphorylates eIF4E. Crystal structures of the central and the C-terminal region of human eIF4GII reveal the formation of one or two HEAT domains, respectively (4, 5)Saccharomyces cerevisiae possesses two genes encoding for eIF4G, TIF4631 and TIF4632. The gene products, eIF4GI and eIF4GII, are 952 and 914 aa long and share Ϸ50% sequence identity. Deletion of one of these genes is tolerated by yeast cells, but double deletion of both genes causes lethality. Interaction of eIF4G with eIF4A is essential for the cell (6, 7). The 45-kDa initiation factor 4A (eIF4A) is a prototypical DEAD-box helicase (8). Its ATPase activity is RNA-dependent and its activity is substantially enhanced in ...

SummaryBis‐(3′,5′) cyclic di‐guanylate (c‐di‐GMP) is a key bacterial second messenger that is implicated in the regulation of many crucial processes that include biofilm formation, motility and virulence. Cellular levels of c‐di‐GMP are controlled through synthesis by GGDEF domain diguanylate cyclases and degradation by two classes of phosphodiesterase with EAL or HD‐GYP domains. Here, we have determined the structure of an enzymatically active HD‐GYP domain protein from Persephonella marina (PmGH) alone, in complex with substrate (c‐di‐GMP) and final reaction product (GMP). The structures reveal a novel trinuclear iron binding site, which is implicated in catalysis and identify residues involved in recognition of c‐di‐GMP. This structure completes the picture of all domains involved in c‐di‐GMP metabolism and reveals that the HD‐GYP family splits into two distinct subgroups containing bi‐ and trinuclear metal centres.

The ATP-dependent integral membrane protease FtsH is universally conserved in bacteria. Orthologs exist in chloroplasts and mitochondria, where in humans the loss of a close FtsH-homolog causes a form of spastic paraplegia. FtsH plays a crucial role in quality control by degrading unneeded or damaged membrane proteins, but it also targets soluble signaling factors like 32 and -CII. We report here the crystal structure of a soluble FtsH construct that is functional in caseinolytic and ATPase assays. The molecular architecture of this hexameric molecule consists of two rings where the protease domains possess an all-helical fold and form a flat hexagon that is covered by a toroid built by the AAA domains. The active site of the protease classifies FtsH as an Asp-zincin, contrary to a previous report. The different symmetries of protease and AAA rings suggest a possible translocation mechanism of the target polypeptide chain into the interior of the molecule where the proteolytic sites are located.AAA ͉ protease ͉ protein degradation ͉ x-ray A TP-dependent proteases play crucial roles in protein quality control and regulation (for reviews, see refs. 1 and 2). One of these is FtsH, initially described as a temperature-sensitive and cell-division-defective mutant, which is also called HflB, named after a high-frequency of lysogenization locus of bacteriophage . FtsH is an integral membrane protease found in bacteria, chloroplasts, and mitochondria (reviewed in ref.3) (Fig. 6, which is published as supporting information on the PNAS web site). In bacteria, FtsH malfunction causes severe phenotypes like cell division defects and growth arrest (4, 5) (reviewed in ref.3). Deletion of the human mitochondrial homolog paraplegin, which shares 40% sequence identity with FtsH from Escherichia coli, is responsible for an autosomal recessive form of hereditary spastic paraplegia (6). The N terminus of FtsH contains two transmembrane helices followed by an AAA module (ATPases associated with various cellular activities), including the SRH (second region of homology) (3). The C-terminal part of the polypeptide chain bears the HEXXH motif that is characteristic for Zn-dependent metalloproteases, where the two histidines coordinate to the zinc ion and the glutamate serves as a catalytic base. In bacteria, the AAA and protease domains are located on the cytosolic side of the membrane. Of the five ATP-dependent proteases in E. coli, HslVU, Lon, ClpXP, ClpAP, and FtsH, the last is the only one that is essential and universally conserved in bacteria. It degrades membrane proteins like the uncomplexed SecY subunit of translocase (7), the a-subunit of F o F 1 -ATPase (8), and the photosystem in chloroplasts (9), therefore playing an important role in the quality control of membrane proteins. Further targets comprise regulatory soluble proteins such as 32 (10, 11) or -CII transcriptional activator protein (12). All these substrates are degraded in an ATP-dependent manner where the energy is used for pulling the proteins out of the membran...

Commensal lactobacilli frequently produce bile salt hydrolase (Bsh) enzymes whose roles in intestinal survival are unclear. Twenty-six Lactobacillus salivarius strains from different sources all harbored a bsh1 allele on their respective megaplasmids. This allele was related to the plasmid-borne bsh1 gene of the probiotic strain UCC118. A second locus (bsh2) was found in the chromosomes of two strains that had higher bile resistance levels. Four Bsh1-encoding allele groups were identified, defined by truncations or deletions involving a conserved residue. In vitro analyses showed that this allelic variation was correlated with widely varying bile deconjugation phenotypes. Despite very low activity of the UCC118 Bsh1 enzyme, a mutant lacking this protein had significantly lower bile resistance, both in vitro and during intestinal transit in mice. However, the overall bile resistance phenotype of this and other strains was independent of the bsh1 allele type. Analysis of the L. salivarius transcriptome upon exposure to bile and cholate identified a multiplicity of stress response proteins and putative efflux proteins that appear to broadly compensate for, or mask, the effects of allelic variation of bsh genes. Bsh enzymes with different bile-degrading kinetics, though apparently not the primary determinants of bile resistance in L. salivarius, may have additional biological importance because of varying effects upon bile as a signaling molecule in the host.

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Ypr118w is a non-essential, low copy number gene product from Saccharomyces cerevisiae. It belongs to the PFAM family PF01008, which contains the ␣-, ␤-, and ␦-subunits of eukaryotic translation initiation factor eIF2B, as well as proteins of unknown function from all three kingdoms. Recently, one of those latter proteins from Bacillus subtilis has been characterized as a 5-methylthioribose-1-phosphate isomerase, an enzyme of the methionine salvage pathway. We report here the crystal structure of Ypr118w, which reveals a dimeric protein with two domains and a putative active site cleft. The C-terminal domain resembles ribose-5-phosphate isomerase from Escherichia coli with a similar location of the active site. In vivo, Ypr118w protein is required for yeast cells to grow on methylthioadenosine in the absence of methionine, showing that Ypr118w is involved in the methionine salvage pathway. The crystal structure of Ypr118w reveals for the first time the fold of a PF01008 member and allows a deeper discussion of an enzyme of the methionine salvage pathway, which has in the past attracted interest due to tumor suppression and as a target of aniprotozoal drugs.The Saccharomyces cerevisiae gene YPR118W is a non-essential gene on chromosome 16 encoding an acidic protein (pI 4.89) of 411 amino acids, Ypr118w, the function of which is unknown (1) and which is of rather low abundance (922 molecules/cell (2). Ypr118w belongs to the PFAM family PF01008 and the TIGR 00512 and 00524 families, the latter also being called eIF2B-related (eIF2B_rel) (3-5). Members of PF01008 contain the ␣-, ␤-, and ␦-, but not the catalytically active ⑀-, subunits of eukaryotic translation initiation factor 2B (eIF2B) 1 from yeast and mammals (4, 5) (Fig. 1). eIF2B is an important regulator of translation initiation. In eukaryotic translation initiation, a ternary complex consisting of Met-tRNA i , GTP, and the heterotrimeric initiation factor eIF2 (6, 7) associates with the 40 S ribosomal subunit together with other initiation factors to form the 43 S preinitiation complex. This complex binds close to the 5Ј-end of mRNA and scans it in the 5Ј-to 3Ј-direction to localize the AUG initiation codon. AUG recognition is mediated by codon-anticodon interaction and involves GTP hydrolysis stimulated by eIF5. This is a prerequisite for the 80 S initiation complex formation, i.e. the joining of the large ribosomal subunit and the release of initiation factors bound to the 40 S ribosomal subunit. After its release, the eIF2-GDP complex is bound by eIF2B, which catalyzes the exchange of GDP for GTP.

The metzincins constitute a subclan of metalloproteases possessing a HEXXHXXGXXH/D zinc-binding consensus sequence where the three histidines are zinc ligands and the glutamic acid is the catalytic base. A completely conserved methionine is located downstream of this motif. Families of the metzincin clan comprise, besides others, astacins, adamalysins proteases, matrix metalloproteases, and serralysins. The latter are extracellular 50 kDa proteases secreted by Gram-negative bacteria via a type I secretion system. While there is a large body of structural and biochemical information available, the function of the conserved methionine has not been convincingly clarified yet. Here, we present the crystal structures of a number of mutants of the serralysin member protease C with the conserved methionine being replaced by Ile, Ala, and His. Together with our former report on the leucine and cysteine mutants, we demonstrate here that replacement of the methionine side chain results in an increasing distortion of the zinc-binding geometry, especially pronounced in the x 2 angles of the first and third histidine of the consensus sequence. This is correlated with an increasing loss of proteolytic activity and a sharp increase of flexibility of large segments of the polypeptide chain.