Membrane Binding and Homodimerization of Atg16 Via Two Distinct Protein Regions is Essential for Autophagy in Yeast

Macroautophagy is a bulk degradation mechanism in eukaryotic cells. Efficiency of an essential step of this process in yeast, Atg8 lipidation, relies on the presence of Atg16, a subunit of the Atg12–Atg5-Atg16 complex acting as the E3-like enzyme in the ubiquitination-like reaction. A current view on the functional structure of Atg16 in the yeast S. cerevisiae comes from the two crystal structures that reveal the Atg5-interacting α-helix linked via a flexible linker to another α-helix of Atg16, which then assembles into a homodimer. This view does not explain the results of previous in vitro studies revealing Atg16-dependent deformations of membranes and liposome-binding of the Atg12–Atg5 conjugate upon addition of Atg16. Here we show that Atg16 acts as both a homodimerizing and peripheral membrane-binding polypeptide. These two characteristics are imposed by the two distinct regions that are disordered in the nascent protein. Atg16 binds to membranes in vivo via the amphipathic α-helix (amino acid residues 113–131) that has a coiled-coil-like propensity and a strong hydrophobic face for insertion into the membrane. The other protein region (residues 64–99) possesses a coiled-coil propensity, but not amphipathicity, and is dispensable for membrane anchoring of Atg16. This region acts as a Leu-zipper essential for formation of the Atg16 homodimer. Mutagenic disruption in either of these two distinct domains renders Atg16 proteins that, in contrast to wild type, completely fail to rescue the autophagy-defective phenotype of atg16Δ cells. Together, the results of this study yield a model for the molecular mechanism of Atg16 function in macroautophagy.     

Statistical evidence for remnants of the primordial code in the acceptor stem of prokaryotic transfer RNA

Summary The specificity of interaction of amino acids with triplets in the acceptor helix stem of tRNA was investigated by means of a statistical analysis of 1400 tRNA sequences. The imprint of a prototypic genetic code at position 3–5 of the acceptor helix was detected, but only for those major amino acids, glycine, alanine, aspartic acid, and valine, that are formed by spark discharges of simple gases in the laboratory. Although remnants of the code at position 3–5 are typical for tRNAs of archaebacteria, eubacteria, and chloroplasts, eukaryotes do not seem to contain this code, and mitochondria take up an intermediary position. A duplication mechanism for the transposition of the original 3–5 code toward its present position in the anticodon stern of tRNA is proposed. From this viewpoint, the mode of evolution of mRNA and functional ribosomes becomes more understandable.Offprint requests to: W. Moller     

Export of proteins across membranes: The helix reversion hypothesis

A model is presented which explains the biological role of the leader peptide in protein export. Along the lines of this model, the conformational changes of a protein with environment serves as a general mechanism for translocation. The leader peptide in the cytoplasm takes a hairpin like conformation which reverts to an extended helix upon integration into the membrane. The essential features of this model are in accord with recent results of protein export.     

Functional roles of amino acid residues involved in forming the alpha-helix-turn-alpha-helix operator DNA binding motif of Tet repressor from Tn10.

The Tn10 derived Tet repressor contains an amino acid segment with high homology to the alpha-helix-turn-alpha-helix motif (HTH) of other DNA binding proteins. The five most conserved amino acids in HTH are probably involved in structural formation of the motif. Their functional role was probed by saturation mutagenesis yielding 95 single amino acid replacement mutants of Tet repressor. Their binding efficiencies to tet operator were quantitatively determined in vivo. All functional mutants contain amino acid substitutions consistent with their proposed role in a HTH. In particular, only the two smallest amino acids (serine, glycine) can substitute a conserved alanine in the proposed first alpha-helix without loss of activity. The last position of the first alpha-helix, the second position in the turn, and the fourth position in the second alpha-helix require mostly hydrophobic residues. The proposed C-terminus of the first alpha-helix is supported by a more active asparagine compared to glutamine replacement mutant of the wt leucine residue. The turn is located close to the protein surface as indicated by functional lysine and arginine replacements for valine. A glycine residue at the first position in the turn can be replaced by any amino acid yielding mutants with at least residual tet operator affinity. A structural model of the HTH of Tet repressor is presented.     

Helix packing of leucine-rich peptides: a parallel leucine ladder in the structure of Boc-Aib-Leu-Aib-Aib-Leu-Leu-Leu-Aib-Leu-Aib-OMe.

The packing of peptide helices in crystals of the leucine-rich decapeptide Boc-Aib-Leu-Aib-Aib-Leu-Leu-Leu-Aib-Leu-Aib-OMe provides an example of ladder-like leucylleucyl interactions between neighboring molecules. The peptide molecule forms a helix with five 5----1 hydrogen bonds and two 4----1 hydrogen bonds near the C terminus. Three head-to-tail NH ... O = C hydrogen bonds between helices form continuous columns of helices in the crystal. The helicial columns associate in an antiparallel fashion, except for the association of Leu ... Leu side chains, which occurs along the diagonal of the cell where the peptide helices are parallel. The peptide, with formula C56H102N10O13, crystallizes in space group P2(1)2(1)2(1) with Z = 4 and cell parameters a = 16.774(3) A, b = 20.032(3) A and c = 20.117(3) A; overall agreement factor R = 10.7% for 2014 data with magnitude of F(obs) greater than 3 sigma (F); resolution 1.0 A.     

Determination of the structure of the DNA binding domain of gamma delta resolvase in solution.

The DNA binding domain (DBD) of gamma delta resolvase (residues 141-183) is responsible for the interaction of this site-specific DNA recombinase with consensus site DNA within the gamma delta transposable element in Escherichia coli. Based on chemical-shift comparisons, the proteolytically isolated DBD displays side-chain interactions within a hydrophobic core that are highly similar to those of this domain when part of the intact enzyme (Liu T, Liu DJ, DeRose EF, Mullen GP, 1993, J Biol Chem 268:16309-16315). The structure of the DBD in solution has been determined using restraints obtained from 2-dimensional proton NMR data and is represented by 17 conformers. Experimental restraints included 458 distances based on analysis of nuclear Overhauser effect connectivities, 17 phi and chi 1 torsion angles based on analysis of couplings, and 17 backbone hydrogen bonds determined from NH exchange data. With respect to the computed average structure, these conformers display an RMS deviation of 0.67 A for the heavy backbone atoms and 1.49 A for all heavy atoms within residues 149-180. The DBD consists of 3 alpha-helices comprising residues D149-Q157, S162-T167, and R172-N183. Helix-2 and helix-3 form a backbone fold, which is similar to the canonical helix-turn-helix motif. The conformation of the NH2-terminal residues, G141-R148, appears flexible in solution. A hydrophobic core is formed by side chains donated by essentially all hydrophobic residues within the helices and turns. Helix-1 and helix-3 cross with a right-handed folding topology. The structure is consistent with a mechanism of DNA binding in which contacts are made by the hydrophilic face of helix-3 in the major groove and the amino-terminal arm in the minor groove. This structure represents an important step toward analysis of the mechanism of DNA interaction by gamma delta resolvase and provides initial structure-function comparisons among the divergent DBDs of related resolvases and invertases.     

Selectively 13C-enriched DNA: 13C and 1H assignments of a triple helix by two-dimensional relayed HMQC experiments

Summary We present NMR studies of an intramolecular triple helix, the three strands of which have been linked by a hexaethylene glycol chain. To overcome the generally encountered difficulties of assignment in the homopyrimidine strands, the carbon C1 of the pyrimidines were selectively ¹³C-enriched. Assignments of the aromatic and sugar protons were obtained from NOESY-HMQC and TOCSY-HMQC spectra. We show that the recognition of a DNA duplex by a third strand via triplex formation is easily carried out in solution by observing the changes of the ¹H1–¹³C1 connectivities as a function of pH. Furthermore, the conformation of the sugars has been found to be C2-endo, on the basis of the coupling constant values directly measured in an HSQC spectrum.     

Analysis of the role of the COL1 domain and its adjacent cysteine-containing sequence in the chain assembly of type IX collagen

The mechanisms of chain selection and assembly of type IX collagen, a heterotrimer 1(IX)2(IX)3(IX), must differ from that of fibrillar collagens since it lacks the characteristic C-propeptide of these latter molecules. We have tested the hypothesis that the information required for this process is contained within the C-terminal triple helical disulfide-bonded region (LMW). The reassociations of the purified LMW fragments of pepsinized bovine type IX collagen were followed by the formation of disulfide-bonded multimers. Our data demonstrate that only three triple helical assemblies form readily, (1)₃, (2)₃, and 123. The information required for chain selection and assembly is thus, at least in part, contained in the studied fragments. Molecular stoichiometries different from the classical heterotrimer may thus also form under certain conditions.     

Dominant role of local dipoles in stabilizing uncompensated charges on a sulfate sequestered in a periplasmic active transport protein.

Electrostatic interactions are among the key factors determining the structure and function of proteins. Here we report experimental results that illuminate the functional importance of local dipoles to these interactions. The refined 1.7-A X-ray structure of the liganded form of the sulfate-binding protein, a primary sulfate active transport receptor of Salmonella typhimurium, shows that the sulfate dianion is completely buried and bound by hydrogen bonds (mostly main-chain peptide NH groups) and van der Waals forces. The sulfate is also closely linked, via one of these peptide units, to a His residue. It is also adjacent to the N-termini of three alpha-helices, of which the two shortest have their C-termini "capped" by Arg residues. Site-directed mutagenesis of the recombinant Escherichia coli sulfate receptor had no effect on sulfate-binding activity when an Asn residue was substituted for the positively charged His and the two Arg (changed singly and together) residues. These results, combined with other observations, further solidify the idea that stabilization of uncompensated charges in a protein is a highly localized process that involves a collection of local dipoles, including those of peptide units confined to the first turns of helices. The contribution of helix macrodipoles appears insignificant.     

Behavioural responses of the vine weevil, Otiorhynchus sulcatus, to semiochemicals from conspecifics, Otiorhynchus salicicola, and host plants

The vine weevil Otiorhynchus sulcatus is a parthenogenetic reproducing species which forages for suitable host plants at night, but is found congregated in dark places during the day. Frass of this weevil species is suspected to contain attractive compounds that are host‐plant related. Using a still‐air olfactometer, we tested adult vine weevils at night for their behavioural response to odours from conspecifics, feeding on a mixture of spindle tree (Euonymus fortunei) and yew (Taxus baccata), and to a sexually reproducing related species (Otiorhynchus salicicola), feeding on a mixture of ivy (Hedera helix) and cherry laurel (Prunus laurocerasus). Their attraction to conspecifics and O. salicicola appeared to be related to frass production. Freshly collected frass from O. sulcatus and from O. salicicola males and females was attractive. Prunus laurocerasus and H. helix have not been observed to be hosts of the vine weevil in the field. However, our tests showed that the vine weevil was attracted to mechanically damaged leaves of both plant species, whereas undamaged leaves were not attractive. Only undamaged young unfolding leaves of H. helix were also attractive. The attraction to odours from mechanically damaged host and non‐host plants suggested the involvement of compounds that are commonly found in many plant species. The involvement of plant compounds and/or aggregation pheromones in attraction to frass of the vine weevil and frass of the related weevil species O. salicicola is discussed.