Structural evidence is presented for a 'Ca(2+)-bridging' mechanism, proposed for Ca(2+)-binding interfacial membrane proteins such as annexins, protein kinase C, and certain coagulation proteins. Crystal structures of Ca(2+)-annexin V complexes with phospholipid polar heads provide molecular details of 'Ca(2+)-bridges' as key features in the membrane attachment exhibited by these proteins. Distinct binding sites for phospholipid head groups are observed, including a novel, double-Ca2+ recognition site for phosphoserine that may serve as a phosphatidylserine receptor site in vivo.

Threonylcarbamoyladenosine (t6A) is a universal modification found at position 37 of ANN decoding tRNAs, which imparts a unique structure to the anticodon loop enhancing its binding to ribosomes in vitro. Using a combination of bioinformatic, genetic, structural and biochemical approaches, the universal protein family YrdC/Sua5 (COG0009) was shown to be involved in the biosynthesis of this hypermodified base. Contradictory reports on the essentiality of both the yrdC wild-type gene of Escherichia coli and the SUA5 wild-type gene of Saccharomyces cerevisiae led us to reconstruct null alleles for both genes and prove that yrdC is essential in E. coli, whereas SUA5 is dispensable in yeast but results in severe growth phenotypes. Structural and biochemical analyses revealed that the E. coli YrdC protein binds ATP and preferentially binds RNAThr lacking only the t6A modification. This work lays the foundation for elucidating the function of a protein family found in every sequenced genome to date and understanding the role of t6A in vivo.

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The enzyme YkvM from Bacillus subtilis was identified previously along with three other enzymes (YkvJKL) in a bioinformatics search for enzymes involved in the biosynthesis of queuosine, a 7-deazaguanine modified nucleoside found in tRNA GUN of Bacteria and Eukarya. Genetic analysis of ykvJKLM mutants in Acinetobacter confirmed that each was essential for queuosine biosynthesis, and the genes were renamed queCDEF. QueF exhibits significant homology to the type I GTP cyclohydrolases characterized by FolE. Given that GTP is the precursor to queuosine and that a cyclohydrolase-like reaction was postulated as the initial step in queuosine biosynthesis, QueF was proposed to be the putative cyclohydrolase-like enzyme responsible for this reaction. We have cloned the queF genes from B. subtilis and Escherichia coli and characterized the recombinant enzymes. Contrary to the predictions based on sequence analysis, we discovered that the enzymes, in fact, catalyze a mechanistically unrelated reaction, the NADPHdependentreductionof7-cyano-7-deazaguanineto7-aminomethyl-7-deazaguanine, a late step in the biosynthesis of queuosine. We report here in vitro and in vivo studies that demonstrate this catalytic activity, as well as preliminary biochemical and bioinformatics analysis that provide insight into the structure of this family of enzymes.tRNA ͉ modified base T he avalanche of new protein structures that have been reported over the last decade (see summary at www.rcsb. org͞pdb͞holdings.html) has made it clear that the number of scaffolds that are used to produce all of the proteins in a cell is surprisingly limited, with Ϸ80% of the proteins using one of the 400 structural folds identified to date (1, 2). Specific functions evolve by duplication, recombination, and divergence of this core repertoire (3). Analysis of the functions of the different members of a protein structural family reveal that, in general, catalytic mechanisms and chemistries are conserved in a given family whereas substrate specificity changes (4). Much rarer are the cases in which the reactions catalyzed differ among members of the family (3); the best characterized examples being the TIM barrel superfamilies (5) and the enolase superfamily (6). Understanding the molecular paths that lead to the evolution of one function from another in a given superfamily is one of the next challenges of structural biology, impacting not only our understanding of how proteins evolve, but also the task of correctly annotating the genes identified by whole-genome sequencing (7,8).We recently used comparative genomic techniques (9) to discover four previously uncharacterized bacterial genes families (queCDEF) involved in the biosynthesis of the modified nucleoside queuosine (10). Three of these families (queCDE) have homologs in Archaea and are therefore implicated in the biosynthesis of the related modified nucleoside archaeosine (Fig. 1). Both nucleosides share an unusual 7-deazaguanosine core structure but diverge in their phylogenetic distribution, location in the ...