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The assembly of a specific polymeric ubiquitin chain on a target protein is a key event in the regulation of numerous cellular processes. Yet, the mechanisms that govern the selective synthesis of particular polyubiquitin signals remain enigmatic. The homologous ubiquitin‐conjugating (E2) enzymes Ubc1 (budding yeast) and Ube2K (mammals) exclusively generate polyubiquitin linked through lysine 48 (K48). Uniquely among E2 enzymes, Ubc1 and Ube2K harbor a ubiquitin‐binding UBA domain with unknown function. We found that this UBA domain preferentially interacts with ubiquitin chains linked through lysine 63 (K63). Based on structural modeling, in vitro ubiquitination experiments, and NMR studies, we propose that the UBA domain aligns Ubc1 with K63‐linked polyubiquitin and facilitates the selective assembly of K48/K63‐branched ubiquitin conjugates. Genetic and proteomics experiments link the activity of the UBA domain, and hence the formation of this unusual ubiquitin chain topology, to the maintenance of cellular proteostasis. SYNOPSIS: Although ubiquitin‐conjugating enzymes Ubc1 and Ube2K have been studied in diverse biological contexts, the function of their UBA domain has remained poorly understood. Here, the UBA domain is found to facilitate the selective targeting of K63‐linked polyubiquitin chains, resulting in formation of branched K48/K63‐linked ubiquitin conjugates. UBA domain binding to an individual ubiquitin moiety in a K63‐linked chain enables reaction of the catalytic UBC domain with the adjacent proximal ubiquitin molecule.This process involves close association of the UBA and UBC domains, which are connected through a flexible linker region.The formation of K48/K63‐branched polyubiquitin is conserved from yeast to humans.Proteomics and genetic data indicate that branched chain formation by Ubc1 regulates resistance to heat stress and DNA replication stress. [ABSTRACT FROM AUTHOR]

To achieve efficient proton pumping in the light‐driven proton pump bacteriorhodopsin (bR), the protein must be tightly coupled to the retinal to rapidly convert retinal isomerization into protein structural rearrangements. Methyl group dynamics of bR embedded in lipid nanodiscs were determined in the dark‐adapted state, and were found to be mostly well ordered at the cytosolic side. Methyl groups in the M145A mutant of bR, which displays only 10 % residual proton pumping activity, are less well ordered, suggesting a link between side‐chain dynamics on the cytosolic side of the bR cavity and proton pumping activity. In addition, slow conformational exchange, attributed to low frequency motions of aromatic rings, was indirectly observed for residues on the extracellular side of the bR cavity. This may be related to reorganization of the water network. These observations provide a detailed picture of previously undescribed equilibrium dynamics on different time scales for ground‐state bR. [ABSTRACT FROM AUTHOR]

Through the canonical LC3 interaction motif (LIR), [W/F/Y]-X1-X2-[I/L/V], protein complexes are recruited to autophagosomes to perform their functions as either autophagy adaptors or receptors. How these adaptors/receptors selectively interact with either LC3 or GABARAP families remains unclear. Herein, we determine the range of selectivity of 30 known core LIR motifs towards individual LC3s and GABARAPs. From these, we define a GABARAP Interaction Motif (GIM) sequence ([W/F]-[V/I]-X2-V) that the adaptor protein PLEKHM1 tightly conforms to. Using biophysical and structural approaches, we show that the PLEKHM1-LIR is indeed 11-fold more specific for GABARAP than LC3B. Selective mutation of the X1 and X2 positions either completely abolished the interaction with all LC3 and GABARAPs or increased PLEKHM1-GIM selectivity 20-fold towards LC3B. Finally, we show that conversion of p62/SQSTM1, FUNDC1 and FIP200 LIRs into our newly defined GIM, by introducing two valine residues, enhances their interaction with endogenous GABARAP over LC3B. The identification of a GABARAP-specific interaction motif will aid the identification and characterization of the expanding array of autophagy receptor and adaptor proteins and their in vivo functions. [ABSTRACT FROM AUTHOR]

Selective labeling with stable isotopes has long been recognized as a valuable tool in protein NMR to alleviate signal overlap and sensitivity limitations. In this study, combinatorial 15N-, 13Cα-, and 13C'-selective labeling has been used during the backbone assignment of human cyclophilin D to explore binding of an inhibitor molecule. Using a cell-free expression system, a scheme that involves 15N, 1-13C, 2-13C, fully 15N/13C, and unlabeled amino acids was optimized to gain a maximum of assignment information from three samples. This scheme was combined with time-shared triple-resonance NMR experiments, which allows a fast and efficient backbone assignment by giving the unambiguous assignment of unique amino acid pairs in the protein, the identity of ambiguous pairs and information about all 19 non-proline amino acid types. It is therefore well suited for binding studies where de novo assignments of amide 1H and 15N resonances need to be obtained, even in cases where sensitivity is the limiting factor. [ABSTRACT FROM AUTHOR]

Most members of the p53 family of transcription factors form tetramers. Responsible for determining the oligomeric state is a short oligomerization domain consisting of one β-strand and one α-helix. With the exception of human p53 all other family members investigated so far contain a second α-helix as part of their tetramerization domain. Here we have used nuclear magnetic resonance spectroscopy to characterize the oligomerization domains of the two p53-like proteins from the tunicate Ciona intestinalis, representing the closest living relative of vertebrates. Structure determination reveals for one of the two proteins a new type of packing of this second α-helix on the core domain that was not predicted based on the sequence, while the other protein does not form a second helix despite the presence of crucial residues that are conserved in all other family members that form a second helix. By mutational analysis, we identify a proline as well as large hydrophobic residues in the hinge region between both helices as the crucial determinant for the formation of a second helix. [ABSTRACT FROM AUTHOR]

Highlights: [•] The CcP–Cc encounter complex was studied with paramagnetic NMR. [•] MTSL spin labels were attached to Cc and PRE was observed in the CcP spectrum. [•] Our data matched the previous encounter complex model well, with a few exceptions. [•] Novel interactions were observed that were not seen in previous studies. [•] This highlights the importance of obtaining a comprehensive data set. [ABSTRACT FROM AUTHOR]

NMR coupling constants, both direct one-bond (1 J) and geminal two-bond (2 J), are employed to analyze the protein secondary structure of human oxidized ERp18. Coupling constants collected and evaluated for the 18 kDa protein comprise 1268 values of 1 JCαHα, 1 JCαCβ, 1 JCαC′, 1 JC′N′, 1 JN′Cα, 1 JN′HN, 2 JCαN′, 2 JHNCα, 2 JC′HN, and 2 JHαC′. Comparison with 1 J and 2 J data from reference proteins and pattern analysis on a per-residue basis permitted main-chain φ,ψ torsion-angle combinations of many of the 149 amino-acid residues in ERp18 to be narrowed to particular secondary-structure motifs. J-coupling indexing is here being developed on statistical criteria and used to devise a ternary grid for interpreting patterns of relative values of J. To account for the influence of the varying substituent pattern in different amino-acid sidechains, a table of residue-type specific threshold values was compiled for discriminating small, medium, and large categories of J. For the 15-residue insertion that distinguishes the ERp18 fold from that of thioredoxin, the J-coupling data hint at a succession of five isolated Type-I β turns at progressively shorter sequence intervals, in agreement with the crystal structure. Proteins 2011. © 2010 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

Geminal two-bond couplings (2 J) in proteins were analyzed in terms of correlation with protein secondary structure. NMR coupling constants measured and evaluated for a total six proteins comprise 3999 values of 2 JCαN′, 2 JC′HN, 2 JHNCα, 2 JC′Cα, 2 JHαC′, 2 JHαCα, 2 JCβC′, 2 JN′Hα, 2 JN′Cβ, and 2 JN′C′, encompassing an aggregate 969 amino-acid residues. A seamless chain of pattern comparisons across the spectrum datasets recorded allowed the absolute signs of all 2 J coupling constants studied to be retrieved. Grouped by their mediating nucleus, C′, N′ or Cα, 2 J couplings related to C′ and N′ depend significantly on ϕ,ψ torsion-angle combinations. β turn types I, I′, II and II′, especially, can be distinguished on the basis of relative-value patterns of 2 JCαN′, 2 JHNCα, 2 JC′HN, and 2 JHαC′. These coupling types also depend on planar or tetrahedral bond angles, whereas such dependences seem insignificant for other types. 2 JHαCβ appears to depend on amino-acid type only, showing negligible correlation with torsion-angle geometry. Owing to its unusual properties, 2 JCαN′ can be considered a 'one-bond' rather than two-bond interaction, the allylic analog of 1 JN′Cα, as it were. Of all protein J coupling types, 2 JCαN′ exhibits the strongest dependence on molecular conformation, and among the 2 J types, 2 JHNCα comes second in terms of significance, yet was hitherto barely attended to in protein structure work. Proteins 2010. © 2009 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

Autophagy is the cellular homeostatic pathway that delivers large cytosolic materials for degradation in the lysosome. Recent evidence indicates that autophagy mediates selective removal of protein aggregates, organelles and microbes in cells. Yet, the specificity in targeting a particular substrate to the autophagy pathway remains poorly understood. Here, we show that the mitochondrial protein Nix is a selective autophagy receptor by binding to LC3/GABARAP proteins, ubiquitin-like modifiers that are required for the growth of autophagosomal membranes. In cultured cells, Nix recruits GABARAP-L1 to damaged mitochondria through its amino-terminal LC3-interacting region. Furthermore, ablation of the Nix:LC3/GABARAP interaction retards mitochondrial clearance in maturing murine reticulocytes. Thus, Nix functions as an autophagy receptor, which mediates mitochondrial clearance after mitochondrial damage and during erythrocyte differentiation. [ABSTRACT FROM AUTHOR]

The tetrameric state of p53, p63, and p73 has been considered one of the hallmarks of this protein family. While the DNA binding domain (DBD) is highly conserved among vertebrates and invertebrates, sequences C-terminal to the DBD are highly divergent. In particular, the oligomerization domain (OD) of the p53 forms of the model organisms Caenorhabditis elegans and Drosophila cannot be identified by sequence analysis. Here, we present the solution structures of their ODs and show that they both differ significantly from each other as well as from human p53. CEP-1 contains a composite domain of an OD and a sterile alpha motif (SAM) domain, and forms dimers instead of tetramers. The Dmp53 structure is characterized by an additional N-terminal β-strand and a C-terminal helix. Truncation analysis in both domains reveals that the additional structural elements are necessary to stabilize the structure of the OD, suggesting a new function for the SAM domain. Furthermore, these structures show a potential path of evolution from an ancestral dimeric form over a tetrameric form, with additional stabilization elements, to the tetramerization domain of mammalian p53. [ABSTRACT FROM AUTHOR]

Membrane proteins are highly underrepresented in structural data banks due to tremendous difficulties that occur upon approaching their structural analysis. Inefficient sample preparation from conventional cellular expression systems is in many cases the first major bottleneck. Preparative scale cell-free expression has now become an emerging alternative tool for the high level production of integral membrane proteins. Many toxic effects attributed to the overproduction of recombinant proteins are eliminated by cell-free expression as viable host cells are no longer required. A unique characteristic is the open nature of cell-free systems that offers a variety of options to manipulate the reaction conditions in order to protect or to stabilize the synthesized recombinant proteins. Detergents or lipids can easily be supplemented and membrane proteins can therefore be synthesized directly into a defined hydrophobic environment of choice that permits solubility and allows the functional folding of the proteins. Alternatively, cell-free produced precipitates of membrane proteins can efficiently be solubilized in mild detergents after expression. Highly valuable for structural approaches is the fast and efficient cell-free production of uniformly or specifically labeled proteins. A considerable number of membrane proteins from diverse families like prokaryotic small multidrug transporters or eukaryotic G-protein coupled receptors have been produced in cell-free systems in high amounts and in functionally active forms. We will give an overview about the current state of the art of this new approach with special emphasis on technical aspects as well as on the functional and structural characterization of cell-free produced membrane proteins. [ABSTRACT FROM AUTHOR]

We demonstrate the high level expression of integral membrane proteins (IMPs) in a cell-free coupled transcription/translation system using a modified Escherichia coli S30 extract preparation and an optimized protocol. The expression of the E. coli small multidrug transporters EmrE and SugE containing four transmembrane segments (TMS), the multidrug transporter TehA with 10 putative TMS, and the cysteine transporter YfiK with six putative TMS, were analysed. All IMPs were produced at high levels yielding up to 2.7 mg of protein per mL of reaction volume. Whilst the vast majority of the synthesized IMPs were precipitated in the reaction mixture, the expression of a fluorescent EmrE-sgGFP fusion construct showed evidence that a small part of the synthesized protein ‘remained soluble and this amount could be significantly increased by the addition of E. coli lipids into the cell-free reaction. Alternatively, the majority of the precipitated IMPs could be solubilized in detergent micelles, and modifications to the solubilization procedures yielded proteins that were almost pure. The folding induced by formation of the proposed α-helical secondary structures of the IMPs after solubilization in various micelles was monitored by CD spectroscopy. Furthermore, the reconstitution of EmrE, SugE and TehA into proteoliposomes was demonstrated by freeze-fracture electron microscopy, and the function of EmrE was additionally analysed by the specific transport of ethidium. The cell-free expression technique allowed efficient amino acid specific labeling of the IMPs with 15N isotopes, and the recording of solution NMR spectra of the solubilized EmrE, SugE and YfiK proteins further indicated a correctly folded conformation of the proteins. [ABSTRACT FROM AUTHOR]

Heteronuclear high-resolution NMR spectroscopy was employed to determine the solution structure of the excisionase protein (Xis) from the λ-like bacteriophage HK022 and to study its sequence-specific DNA interaction. As wild-type Xis was previously characterized as a generally unstable protein, a biologically active HK022 Xis mutant with a single amino acid substitution Cys28→Ser was used in this work. This substitution has been shown to diminish the irreversibility of Xis denaturation and subsequent degradation, but does not affect the structural or thermodynamic properties of the protein, as evidenced by NMR and differential scanning calorimetry. The solution structure of HK022 Xis forms a compact, highly ordered protein core with two well-defined α-helices (residues 5–11 and 18–27) and five β-strands (residues 2–4, 30–31, 35–36, 41–44 and 48–49). These data correlate well with 1H2O-2H2O exchange experiments and imply a different organization of the HK022 Xis secondary structure elements in comparison with the previously determined structure of the bacteriophage λ excisionase. Superposition of both Xis structures indicates a better correspondence of the full-length HK022 Xis to the typical ‘winged-helix’ DNA-binding motif, as found, for example, in the DNA-binding domain of the Mu-phage repressor. Residues 51–72, which were not resolved in the λ Xis, do not show any regular structure in HK022 Xis and thus appear to be completely disordered in solution. The resonance assignments have shown, however, that an unusual connectivity exists between residues Asn66 and Gly67 owing to asparagine-isoaspartyl isomerization. Such an isomerization has been previously observed and characterized only in eukaryotic proteins. [ABSTRACT FROM AUTHOR]

Examines the solution structure of recombinant mouse major urinary protein. Capacity to bind hydrophobic molecules; Role pheromones in the reproductive cycle of rodents; Description on the geometric calculations to the experimental procedures.

Examines the solution conformation of oxidized flavodoxin from the gram-negative bacteria desulfovibrio vulgaris using nuclear magnetic resonance. Hydrogen exchange and J-coupling analysis; Determination of tertiary structure; Detection of protein-bound water molecules.

Recombinant Desulfovibrio vulgaris flavodoxin (molecular mass 16.3 kDa) was produced in Escherichia coli. The oxidized protein has been investigated with a combination of homonuclear and heteronuclear two-dimensional and heteronuclear three-dimensional NMR spectroscopy. Sequence-specific assignment of all backbone and most of the side chain ¹H and 15N resonances has been obtained. The secondary structure has been inferred from the pattern of sequential, medium-, and long-range NOEs, together with information about slowly exchanging amide hydrogens and HN-H α spin-spin coupling constants. In solution, flavodoxin consists of a five-stranded parallel β-sheet and four alpha-helices. Residues 3-9, 32-36, 52-58, 87-96, and 123-128 are involved in the β-sheet whereas the a-helical regions comprise residues 13-28, 69-76, 104-114, and 134-148. Several proton resonances of the bound flavin mononucleotide cofactor have been assigned. NOE contacts between the prosthetic group and the apoprotein have been detected. [ABSTRACT FROM AUTHOR]