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Journal of molecular biology [journal]
- Dynactin 3D structure: Implications for assembly and dynein binding. [JOURNAL ARTICLE]
- J Mol Biol 2014 Jul 18.
The multisubunit protein complex, dynactin, is an essential component of the cytoplasmic dynein motor. High resolution structural work on dynactin and the dynein/dynactin supercomplex has been limited to small subunits and recombinant fragments that do not report fully on either≈1 MDa assembly. In the present study, we used negative stain electron microscopy and image analysis based on random conical tilt reconstruction to obtain a three-dimensional structure of native vertebrate dynactin. The 35nm long dynactin molecule has a V-shaped shoulder at one end and a flattened tip at the other, both offset relative to the long axis of the actin-related protein (Arp) backbone. The shoulder projects dramatically away from the Arp filament core in a way that cannot be appreciated in 2D images, which has implications for the mechanism of dynein binding. The 3D structure allows the helical parameters of the entire Arp filament core, which includes the actin capping protein, CP, to be determined for the first time. This structure exhibits near identity to F-actin and can be well fitted into the dynactin envelope. Molecular fitting of modeled CP-Arp polymers into the envelope shows that the filament contains between 7 and 9 Arp protomers and is capped at both ends. In the 7-Arp model, which agrees best with measured Arp stoichiometry and other structural information, actin capping protein (CP) is not present at the distal tip of the structure, unlike what is seen in the other models. The 3D structure suggests a mechanism for dynactin assembly and length specification.
- Adaptive remodelling by FliN in the bacterial rotary motor. [JOURNAL ARTICLE]
- J Mol Biol 2014 Jul 18.
Sensory adaptation in the E. coli chemosensory pathway has been the subject of interest for decades, with investigation focusing on the receptors that process extracellular inputs. Recent studies demonstrate that the flagellar motors responsible for cell locomotion also play a role, adding or subtracting FliM subunits to maximise sensitivity to pathway signals. It is difficult to reconcile this FliM remodelling with the observation that partner FliN subunits are relatively static fixtures in the motor. By fusing a fluorescent protein internally to FliN, we show that there is in fact significant FliN remodelling. The kinetics and stoichiometry of FliN in steady-state and in adapting motors are investigated and found to match the behaviour of FliM in all respects except for timescale, where FliN rates are about four times slower. We notice that motor adaptation is slower in the presence of the fluorescent protein, indicating a possible source for the difference. The behaviour of FliM and FliN is consistent with a kinetic and stoichiometric model that contradicts the traditional view of a packed, rigid motor architecture.
- Structural and biochemical characterization of the natural product kendomycin and the 20S proteasome. [JOURNAL ARTICLE]
- J Mol Biol 2014 Jul 16.
Natural products are a valuable source for novel lead structures in drug discovery, but for the majority of isolated bioactive compounds the cellular targets are unknown. The structurally unique ansa-polyketide kendomycin was reported to exert its potent cytotoxic effects via impairment of the ubiquitin proteasome system, but the exact mode of action remained unclear. Here, we present a systematic biochemical characterization of kendomycin-proteasome interactions in vitro as well as in vivo, including complex structures of wildtype and mutant yeast 20S proteasome with kendomycin. Our results provide evidence for a polypharmacological mode of action for kendomycin's cytotoxic effect on cancer cells.
- Structural characterization of anti-inflammatory Immunoglobulin G Fc proteins. [JOURNAL ARTICLE]
- J Mol Biol 2014 Jul 15.
Immunoglobulin G (IgG) is a central mediator of host defense due to its ability to recognize and eliminate pathogens. The recognition and effector responses are encoded on distinct regions of IgGs. The diversity of the antigen recognition Fab domains accounts for IgG's ability to bind with high specificity to essentially any antigen. Recent studies have indicated that the Fc effector domain also displays considerable heterogeneity, accounting for its complex effector functions of inflammation, modulation and immune suppression. Therapeutic anti-tumor antibodies, for example, require the pro-inflammatory properties of the IgG Fc to eliminate tumor cells, while the anti-inflammatory activity of Intravenous Immunoglobulin G (IVIG) requires specific Fc glycans for activity. In particular, the anti-inflammatory activity of IVIG is ascribed to a small population of IgGs in which the Asn297-linked complex N-glycans attached to each Fc CH2 domain include terminal α2,6-linked sialic acids. We used chemoenzymatic glycoengineering to prepare fully di-sialylated IgG Fc and solved its crystal structure. Comparison of the structures of asialylated Fc, sialylated Fc, and F241A Fc, a mutant that displays increased glycan sialylation, suggests that increased conformational flexibility of the CH2 domain is associated with the switch from pro- to anti-inflammatory activity of the Fc.
- Pathways for virus assembly around nucleic acids. [JOURNAL ARTICLE]
- J Mol Biol 2014 Jul 15.
Understanding the pathways by which viral capsid proteins assemble around their genomes could identify key intermediates as potential drug targets. In this work we use computer simulations to characterize assembly over a wide range of capsid protein-protein interaction strengths and solution ionic strengths. We find that assembly pathways can be categorized into two classes, in which intermediates are either predominantly ordered or disordered. Our results suggest that estimating the protein-proteinand the protein-genome binding affinities may be sufficient to predict which pathway occurs. Furthermore, the calculated phase diagrams suggest that knowledge of the dominant assembly pathway and its relationship to control parameters could identify optimal strategies to thwart or redirect assembly to block infection. Finally, analysis of simulation trajectories suggests that the two classes of assembly pathways can be distinguished in single molecule fluorescence correlation spectroscopy or bulk time resolved small angle x-ray scattering experiments.
- Combining single-molecule imaging and single-channel electrophysiology. [REVIEW]
- J Mol Biol 2014 Jul 12.
Combining simultaneous single-molecule fluorescence measurements of ion channel conformational change with single-channel electrophysiology would enable a direct link between structure and function. Such methods would help us to create a truly molecular 'movie' of how these important biomolecules work. Here we review past and recent progress towards this goal.
- The devil lies in the details: How variations in polysaccharide fine-structure impact the physiology and evolution of gut microbes. [REVIEW]
- J Mol Biol 2014 Jul 12.
The critical importance of gastrointestinal microbes to digestion of dietary fiber in humans and other mammals has been appreciated for decades. Symbiotic microorganisms expand mammalian digestive physiology by providing an armament of diverse polysaccharide degrading enzymes, which are largely absent in mammalian genomes. By out-sourcing this aspect of digestive physiology to our gut microbes, we maximize our ability to adapt to different carbohydrate nutrients on time scales as short as several hours, due to the ability of the gut microbial community to rapidly alter its physiology from meal-to-meal. Because of their ability to pick up new traits by lateral gene transfer, our gut microbes also enable adaption over time periods as long as centuries and millennia by adjusting their gene content to reflect cultural dietary trends. Despite a vast amount of sequence-based insight into the metabolic potential of gut microbes, the specific mechanisms by which symbiotic gut microorganisms recognize and attack complex carbohydrates remain largely undefined. Here, we review the recent literature on this topic and posit that numerous, subtle variations in polysaccharides diversify the spectrum of available nutrient niches, each of which may be best filled by a subset of microorganisms that possess the corresponding proteins to recognize and degrade different carbohydrates. Understanding these relationships at precise mechanistic levels will be essential to obtain a complete understanding of the forces shaping gut microbial ecology and genomic evolution, as well as devising strategies to intentionally manipulate the composition and physiology of the gut microbial community to improve health.
- Structural Insights into the Substrate Specificity of (S)-Ureidoglycolate Amidohydrolase and Its Comparison with Allantoate Amidohydrolase. [JOURNAL ARTICLE]
- J Mol Biol 2014 Jul 11.
In plants, the ureide pathway is a metabolic route that converts the ring nitrogen atoms of purine to ammonia via sequential enzymatic reactions, playing an important role in nitrogen recovery. In the final step of the pathway, (S)-ureidoglycolate amidohydrolase (UAH) catalyzes the conversion of (S)-ureidoglycolate into glyoxylate and releases two molecules of ammonia as by-products. UAH is homologous in structure and sequence with allantoate amidohydrolase (AAH), an upstream enzyme in the pathway with a similar function as that of an amidase but with a different substrate. Both enzymes exhibit strict substrate specificity and catalyze reactions in a concerted manner, resulting in purine degradation. Here, we report three crystal structures of Arabidopsis thaliana UAH: bound with substrate, reaction intermediate, and product, and a structure of Escherichia coli AAH complexed with allantoate. Structural analyses of UAH revealed a distinct binding mode for each ligand in a bimetal reaction center with the active site in a closed conformation. The ligand directly participates in the coordination shell of two metal ions and is stabilized by the surrounding residues. In contrast, AAH, which exhibits a substrate-binding site similar to that of UAH, requires a larger active site due to the additional ureido group in allantoate. Structural analyses and mutagenesis revealed that both enzymes undergo an open-to-closed conformational transition in response to ligand binding, and that the active site size as well as the interaction environment in UAH and AAH are determinants of the substrate specificities of these two structurally homologous enzymes.
- Structural basis for the specific recognition of the major antigenic peptide from the Japanese cedar pollen allergen Cry j 1 by HLA-DP5. [JOURNAL ARTICLE]
- J Mol Biol 2014 Jul 11.
The major allergen, Cry j 1, was isolated from Japanese cedar Cryptomeria japonica (Cry j) pollen, and shown to react with immunoglobulin E (IgE) antibodies in the sera from pollinosis patients. We previously reported that the frequency of HLA-DP5 was significantly higher in pollinosis patients, and the immunodominant peptides from Cry j 1 bound to HLA-DP5 to activate Th2 cells. In the present study, we determined the crystal structure of the HLA-DP5 heterodimer in complex with a Cry j 1-derived nine-residue peptide, at 2.4Å resolution. The peptide-binding groove recognizes the minimal peptide with ten hydrogen bonds, including those between the negatively charged P1 pocket and the Lys side chain at the first position in the peptide sequence. We confirmed that HLA-DP5 exhibits the same Cry j 1-binding mode in solution, through pull-down experiments using structure-based mutations of Cry j 1. We also identified the characteristic residues of HLA-DP5 that are responsible for the distinct properties of the groove, by comparing the structure of HLA-DP5 and the previously reported structures of HLA-DP2 in complexes with pDRA of the self-antigen. The comparison revealed that the HLA-DP5•pCry j 1 complex forms several hydrogen-bond/salt bridge networks between the receptor and the antigen that were not observed in the HLA-DP2•pDRA complex. Evolutionary considerations have led us to conclude that HLA-DP5 and HLA-DP2 represent two major groups of the HLA-DP family, in which the properties of the P1 and P4 pockets have evolved and acquired the present ranges of epitope peptide binding specificities.
- Direct interaction of 14-3-3ζ with ezrin promotes cell migration by regulating the formation of membrane ruffle. [JOURNAL ARTICLE]
- J Mol Biol 2014 Jul 11.
14-3-3 proteins have been shown to regulate the actin cytoskeleton remodeling, cell adhesion and migration. In this study,we identified ezirn, a cross-linker between plasma membrane and actin cytoskeleton as a novel 14-3-3ζ interacting partner. The direct interaction between 14-3-3ζ and ezrin was validated in the cells and by in vitro assays. We showed that the 14-3-3ζ binding region in ezrin was located within the N-terminal and central α-helical domains, and that the αG to αI helices of 14-3-3ζ are responsible for the binding to ezrin. Functional analyses revealed that the regulation of cell migration and membrane ruffling by 14-3-3ζ is ezrin-dependent, for which the integrity of ezrin protein was required. Conversely, the knockdown of 14-3-3ζ abrogates also the stimulatory effect of ezrin on cell migration and membrane ruffling. Moreover, we found that the phosphorylation of Thr567 in ezrin facilitates the 14-3-3ζ-ezrin interaction and the formation of membrane ruffles. Taken together, these results suggest strongly that the functions of these two proteins in cell migration are linked, and might be mediated by their direct physical interaction which is important for the formation of membrane ruffles.