Crystal structure of the YML079w protein from Saccharomyces cerevisiae reveals a new sequence family of the jelly-roll fold

We determined the three-dimensional crystal structure of the protein YML079wp, encoded by a hypothetical open reading frame from Saccharomyces cerevisiae to a resolution of 1.75 A. The protein has no close homologs and its molecular and cellular functions are unknown. The structure of the protein is a jelly-roll fold consisting of ten beta-strands organized in two parallel packed beta-sheets. The protein has strong structural resemblance to the plant storage and ligand binding proteins (canavalin, glycinin, auxin binding protein) but also to some plant and bacterial enzymes (epimerase, germin). The protein forms homodimers in the crystal, confirming measurements of its molecular mass in solution. Two monomers have their beta-sheet packed together to form the dimer. The presence of a hydrophobic ligand in a well conserved pocket inside the barrel and local sequence similarity with bacterial epimerases may suggest a biochemical function for this protein.     

YbiV from Escherichia coli K12 is a HAD phosphatase

The protein YbiV from Escherichia coli K12 MG1655 is a hypothetical protein with sequence homology to the haloacid dehalogenase (HAD) superfamily of proteins. Although numerous members of this family have been identified, the functions of few are known. Using the crystal structure, sequence analysis, and biochemical assays, we have characterized YbiV as a HAD phosphatase. The crystal structure of YbiV reveals a two-domain protein, one with the characteristic HAD hydrolase fold, the other an inserted alpha/beta fold. In an effort to understand the mechanism, we also solved and report the structures of YbiV in complex with beryllofluoride (BeF3-) and aluminum trifluoride (AlF3), which have been shown to mimic the phosphorylated intermediate and transition state for hydrolysis, respectively, in analogy to other HAD phosphatases. Analysis of the structures reveals the substrate-binding cavity, which is hydrophilic in nature. Both structure and sequence homology indicate YbiV may be a sugar phosphatase, which is supported by biochemical assays that measured the release of free phosphate on a number of sugar-like substrates. We also investigated available genomic and functional data in an effort to determine the physiological substrate.     

Loop relaxation, a mechanism that explains the reduced specificity of rabbit 20alpha-hydroxysteroid dehydrogenase, a member of the aldo-keto reductase superfamily

The aldo-keto reductase rabbit 20alpha-hydroxysteroid dehydrogenase (rb20alpha-HSD; AKR1C5) is less selective than other HSDs, since it exerts its activity both on androgens (C19 steroids) and progestins (C21 steroids). In order to identify the molecular determinants responsible for this reduced selectivity, binary (NADPH) and ternary (NADP(+)/testosterone) complex structures were solved to 1.32A and 2.08A resolution, respectively. Inspection of the cofactor-binding cavity led to the identification of a new interaction between side-chains of residues His222 and Lys270, which cover the central phosphate chain of the cofactor, reminiscent of the "safety-belt" found in other aldo-keto reductases. Testosterone is stabilized by a phenol/benzene tunnel composed of side-chains of numerous residues, among which Phe54, which forces the steroid to take up an orientation markedly contrasting with that found in HSD ternary complexes reported. Combining structural, site-directed mutagenesis, kinetic and fluorescence titration studies, we found that the selectivity of rb20alpha-HSD is mediated by (i) the relaxation of loop B (residues 223-230), partly controlled by the nature of residue 230, (ii) the nature of the residue found at position 54, and (iii) the residues found in the C-terminal tail of the protein especially the side-chain of the amino acid 306.     

Isolation,Characterization, and Evolutionary Divergence of Mouse RNase 6: Evidence for Unusual Evolution in Rodents

The evolution of the ribonuclease A (RNase A) vertebrate-specific enzyme family is interesting in that specific gene lineages appear to be responding to unique selective pressures in wildly diverse manners to generate proteins that are capable of reducing the infectivity of viruses, killing systemic pathogens, and inducing the growth of blood vessels all while maintaining the signature motifs of a ribonuclease. In this paper, we present the DNA sequence and gene structure of Mus musculus RNase 6 and examine the expression pattern and enzymatic activity of the recombinant protein. M. musculus RNase 6 has a limited expression pattern compared to human RNase 6 and is an efficient ribonuclease, with a catalytic efficiency 17-fold higher than that of human protein. Evo- lutionary analysis reveals that RNase 6 was subject to unusual evolutionary forces (d_N/d_S=1.2) in an ancestral rodent lineage before the separation of Mus and Rattus. However, more recent evolution of rodent RNase 6 has been relatively conserved, with an average d_N/d_S of 0.66. These data suggest that the ancestral rodent RNase 6 was subject to accelerated evolution, resulting in the conserved modern gene, which most likely plays an important role in mouse physiology.Reviewing Editor: Dr. Lauren Ancel MeyersThe GenBank accession numbers for the new genes presented here are as follows: Mus musculus, AY545655; Rattus norvegicus, AY545654; Mus spicilegus, AY545653; Mus caroli, AY545651; and Mus pahari, AY545652.     

The crystal structure of Arabidopsis thaliana RAC7/ROP9: the first RAS superfamily GTPase from the plant kingdom

Arabidopsis thaliana RAC/ROP GTPases constitute a plant specific Rho GTPase family in the RAS superfamily, which has been implicated in numerous pivotal signalling cascades in plants. Research has shown that plants in some cases have evolved different modes of regulating Rho GTPase activity as compared to the equivalent systems in animals and yeast. In order to gain structural insight into plant signaling at the molecular level, we have determined the first crystal structure of a RAC-like GTPase belonging to the RAS superfamily from the plant kingdom. The structure of AtRAC7/ROP9 bound to GDP was solved at a resolution of 1.78 A. We have found that the structure of plant Rho GTPases is based upon a conserved G-domain architecture, but structural differences were found concerning the insert region and switch II region of the protein.     

The C. elegans gene dig-1 encodes a giant member of the immunoglobulin superfamily that promotes fasciculation of neuronal processes

The adhesion of growing neurites into appropriate bundles or fascicles is important for the development of correct synaptic connectivity in the nervous system. We describe fasciculation defects of animals with mutations in the C. elegans gene dig-1 and show that dig-1 encodes a giant molecule (13,100 amino acids) of the immunoglobulin superfamily. Five new alleles of dig-1 were isolated in a screen for mutations affecting the morphology or function of several classes of head sensory neurons. Mutants showed process defasciculation of several classes of neurons. Analysis of a temperature-sensitive allele revealed that dig-1 is required during embryogenesis for normal process fasciculation of one class of head sensory neuron. Partial sequencing of two alleles, RNA interference (RNAi) and rescuing experiments showed that dig-1 encodes a giant molecule of the immunoglobulin superfamily. DIG-1 protein contains many domains associated with adhesion, is likely secreted, and has some features of proteoglycans. dig-1 mutants were originally isolated due to their displaced gonads [Thomas, J.H., Stern, M.J., Horvitz, H.R., 1990. Cell interactions coordinate the development of the C. elegans egg-laying system. Cell 62, 1041-52]; thus, dig-1 alleles were also characterized for their effects on gonad placement. Mutant phenotypes suggest that DIG-1 may mediate cell movement as well as process fasciculation and that different regions of the protein may mediate these functions.     

Biophysical characterization of the proton-coupled oligopeptide transporter YjdL

Proton-coupled oligopeptide transporters (POTs) utilize the electrochemical proton gradient to facilitate uptake of di- or tripeptide molecules. YjdL is one of four POTs found in Escherichia coli. It has shown an extraordinary preference for di- rather than tripeptides, and is therefore significantly different from prototypical POTs such as the human hPepT1. Nonetheless YjdL contains several highly conserved POT residues, which include Glu388 that is located in the putative substrate binding cavity. Here we present biophysical characterization of WT-YjdL and Glu388Gln. Isothermal titration calorimetrical studies exhibit a K_d of 14 μM for binding of Ala-Lys to WT-YjdL. Expectedly, no binding could be detected for the tripeptide Ala-Ala-Lys. Surprisingly however, binding could not be detected for Ala-Gln, although earlier studies indicated inhibitory potencies of Ala-Gln to be comparable to Ala-Lys (IC₅₀ values of 0.6 compared to 0.3 mM). Finally, Ala-Lys binding to Glu388Gln was also undetectable which may support a previously suggested role in interaction with the ligand peptide N-terminus.     

Solution structure of the strawberry allergen Fra a 1

The PR10 family protein Fra a 1E from strawberry (Fragaria x ananassa) is down-regulated in white strawberry mutants, and transient RNAi (RNA interference)-mediated silencing experiments confirmed that Fra a 1 is involved in fruit pigment synthesis. In the present study, we determined the solution structure of Fra a 1E. The protein fold is identical with that of other members of the PR10 protein family and consists of a seven-stranded antiparallel β-sheet, two short V-shaped α-helices and a long C-terminal α-helix that encompass a hydrophobic pocket. Whereas Fra a 1E contains the glycine-rich loop that is highly conserved throughout the protein family, the volume of the hydrophobic pocket and the size of its entrance are much larger than expected. The three-dimensional structure may shed some light on its physiological function and may help to further understand the role of PR10 proteins in plants.