Journal of molecular biology [journal]
- Processive DNA Unwinding by RecBCD Helicase in the Absence of Canonical Motor Translocation. [JOURNAL ARTICLE]
- J Mol Biol 2016 Jul 12.
Escherichia coli RecBCD is a DNA helicase/nuclease that functions in double-stranded DNA break repair. RecBCD possesses two motors (RecB, a 3´ to 5´ translocase, and RecD, a 5´ to 3´ translocase). Current DNA unwinding models propose that motor translocation is tightly coupled to base pair (bp) melting. However, some biochemical evidence suggests that DNA melting of multiple bp may occur separately from single stranded DNA translocation. To test this hypothesis, we designed DNA substrates containing reverse backbone polarity (RP) linkages that prevent ssDNA translocation of the canonical RecB and RecD motors. Surprisingly, we find that RecBCD can processively unwind DNA for at least 80bp beyond the RP linkages. This ability requires an ATPase active RecB motor, the RecB "arm" domain and also the RecB nuclease domain, but not its nuclease activity. These results indicate that RecBCD can unwind duplex DNA processively in the absence of ssDNA translocation by the canonical motors and that the nuclease domain regulates the helicase activity of RecBCD.
- Structure-activity studies of β-hairpin peptide inhibitors of the Plasmodium falciparum AMA1-RON2 interaction. [JOURNAL ARTICLE]
- J Mol Biol 2016 Jul 12.
The interaction between apical membrane antigen 1 (AMA1) and rhoptry neck protein 2 (RON2) plays a key role in the invasion of red blood cells by Plasmodium parasites. Disruption of this critical protein-protein interaction represents a promising avenue for antimalarial drug discovery. In this work, we exploited a 13-residue β-hairpin based on the C-terminal loop of RON2 to probe a conserved binding site on P. falciparum AMA1. A series of mutations was synthetically engineered into β-hairpin peptides to establish structure-activity relationships. The best mutations improved the binding affinity of the β-hairpin peptide by ~7-fold for 3D7 AMA1 and ~14-fold for FVO AMA1. We determined the crystal structures of several β-hairpin peptides in complex with AMA1 in order to define the structural features and specific interactions that contribute to improved binding affinity. The same mutations in the longer RON2sp2 peptide (residue 2027-2055 of RON2) increased the binding affinity by >30-fold for 3D7 and FVO AMA1, producing KD values of 2.1nM and 0.4nM, respectively. To our knowledge this is the most potent strain-transcending peptide yet reported, and represents a valuable tool to characterize the AMA1-RON2 interaction.
- Signal transduction at the single-cell level: Approaches to study the dynamic nature of signaling networks. [REVIEW, JOURNAL ARTICLE]
- J Mol Biol 2016 Jul 15.
Signal transduction, or how cells interpret and react to external events, is a fundamental aspect of cellular function. Traditional study of signal transduction pathways involves mapping cellular signaling pathways at the population level. However, population averaged readouts do not adequately illuminate the complex dynamics and heterogeneous responses found at the single-cell level. Recent technological advances to observe cellular response, computationally model signaling pathways, and experimentally manipulate cells now enables studying signal transduction at the single-cell level. These studies will enable deeper insights into the dynamic nature of signaling networks.
- Molecular Mechanisms of Signaling in Myxococcus xanthus Development. [REVIEW, JOURNAL ARTICLE]
- J Mol Biol 2016 Jul 15.
Myxococcus xanthus is an environmental bacterium that displays a complex lifecycle that includes motility, predation, multicellular fruiting body development, and sporulation. Given the elaborate fruiting body development of this bacterial species, M. xanthus has served as a model organism for the study of multicellular development of bacteria and a remarkable number of genes have been identified that contribute to the regulation of this highly dynamic process. Included amongst these developmental factors is a robust repertoire of signaling proteins, which have arisen from extensive gene duplication in M. xanthus and related species. In this review we explore several aspects of the molecular mechanisms of signaling in M. xanthus development. This includes mechanisms of kin selection, single cell sensing of nutrient depletion and the stringent response, the production of and response to extracellular population cues, and the contribution of several two-component signaling systems regulating developmental transcriptional programs. Collectively, these signaling mechanisms function to tightly regulate the sensing of nutrient depletion, the aggregation of populations of cells, and the temporal and spatial formation of complex fruiting bodies and sporulation of M. xanthus.
- Tuning the Catalytic Activity of Subcellular Nanoreactors. [JOURNAL ARTICLE]
- J Mol Biol 2016 Jul 14.
Bacterial microcompartments are naturally occurring subcellular organelles of bacteria, and serve as a promising scaffold for the organization of heterologous biosynthetic pathways. A critical element in the design of custom biosynthetic organelles is quantitative control over the loading of heterologous enzymes to the interior of the organelles. We demonstrate that the loading of heterologous proteins to the 1,2-propanediol utilization microcompartment of Salmonella enterica can be controlled using two strategies: by modulating the transcriptional activation of the microcompartment container, and by coordinating the expression of the microcompartment container and the heterologous cargo. These strategies allow general control over the loading of heterologous proteins localized by two different N-terminal targeting peptides, and represent an important step towards tuning the catalytic activity of bacterial microcompartments for increased biosynthetic productivity.
- Chopping and changing: The evolution of the flavin-dependent monooxygenases. [JOURNAL ARTICLE]
- J Mol Biol 2016 Jul 13.
Flavin-dependent monooxygenases play a variety of key physiological roles and are also very powerful biotechnological tools. These enzymes have been classified into eight different classes (A-H) based on their sequences and biochemical features. By combining structural and sequence analysis, as well as phylogenetic inference, we have explored the evolutionary history of classes A, B, E, F and G, and demonstrate that their multi-domain architectures reflect their phylogenetic relationships, suggesting that the main evolutionary steps in their divergence are likely to have arisen from the recruitment of different domains. Additionally, the functional divergence within in each class appears to have been the result of other mechanisms such as a complex set of single-point mutations. Our results reinforce the idea that a main constraint on the evolution of cofactor-dependent enzymes is the functional binding of the cofactor. Additionally, a remarkable feature of this family is that the sequence of the key FAD-binding domain is split into at least two parts in all classes studied here. We propose a complex set of evolutionary events that gave rise to the origin of the different classes within this family.
- Systems Glycobiology: Integrating glycogenomics, glycoproteomics, glycomics and other 'omics data sets to characterize cellular glycosylation processes. [REVIEW, JOURNAL ARTICLE]
- J Mol Biol 2016 Jul 13.
The number of proteins encoded in the human genome has been estimated at between 20,000 and 25,000 despite estimates that the entire proteome contains more than a million proteins. One reason for this difference is due to the many protein post-translational modifications that contribute to proteome complexity. Among them, glycosylation is of particular relevance because it serves to modify a large number of cellular proteins. Glycogenomics, glycoproteomics, glycomics and glycoinformatics are helping to accelerate our understanding of the cellular events involved in generating the glycoproteome, the variety of glycan structures possible, and the importance glycans play in therapeutics and disease. Indeed, interest in glycosylation has expanded rapidly over the past decade as large amounts of experimental 'omics data relevant to glycosylation processing has accumulated. Furthermore, new and more sophisticated glycoinformatics tools and databases are now available for glycan and glycosylation pathway analysis. Here, we summarize some of the recent advances in both experimental profiling and analytical methods involving N- and O-linked glycosylation processing for biotechnological and medically-relevant cells together with the unique opportunities and challenges associated with interrogating and assimilating multiple disparate high-throughput glycosylation data sets. This emerging era of advanced glycomics will lead to the discovery of key glycan biomarkers linked to diseases and help establish a better understanding of physiology and improved control of glycosylation processing in diverse cells and tissues important to disease and production of recombinant therapeutics. Furthermore, methodologies that facilitate the integration of glycomics measurements together with other 'omics data sets will lead to a deeper understanding and greater insights into the nature of glycosylation as a complex cellular process.
- Order-Disorder Transitions in the Cardiac Troponin Complex. [REVIEW, JOURNAL ARTICLE]
- J Mol Biol 2016 Jul 6.
The troponin complex is a molecular switch that ties shifting intracellular calcium concentration to association and dissociation of actin and myosin, effectively allowing excitation-contraction coupling in striated muscle. Although there is a long history of muscle biophysics and structural biology, many of the mechanistic details that enable troponin's function remain incompletely understood. This review summarizes the current structural understanding of the troponin complex on the muscle thin filament, focusing on conformational changes in flexible regions of the troponin I subunit. In particular, we focus on order-disorder transitions in the C-terminal domain of troponin I, which have important implications in cardiac disease and could also have potential as a model system for the study of coupled binding and folding.
- Pironetin Binds Covalently to αCys316 and Perturbs a Major Loop and Helix of α-Tubulin to Inhibit Microtubule Formation. [JOURNAL ARTICLE]
- J Mol Biol 2016 Jul 6.
Microtubule-targeting agents are among the most powerful drugs used in chemotherapy to treat cancer patients. Pironetin is a natural product that displays promising anticancer properties by binding to and potently inhibiting tubulin assembly into microtubules; however, its molecular mechanism of action remained obscure. Here, we solved the crystal structure of the tubulin-pironetin complex and found that the compound covalently binds to Cys316 of α-tubulin. The structure further revealed that pironetin perturbs the T7 loop and helix H8 of α-tubulin. Since both these elements are essential for establishing longitudinal tubulin contacts in microtubules, this result explains how pironetin inhibits the formation of microtubules. Together, our data define the molecular details of the pironetin binding site on α-tubulin and thus offer a promising basis for the rational design of pironetin variants with improved activity profiles. They further extend our knowledge on strategies evolved by natural products to target and perturb the microtubule cytoskeleton.
- Polysaccharide degradation by the intestinal microbiota and its influence on human health and disease. [REVIEW, JOURNAL ARTICLE]
- J Mol Biol 2016 Jul 5.
Carbohydrates comprise a large fraction of the typical diet, yet humans are only able to directly process some types of starch and simple sugars. The remainder transits the large intestine where it becomes food for the commensal bacterial community. This is an environment of both intense competition, but also impressive cooperation for available glycans as these bacteria work to maximize their energy harvest from these carbohydrates during their limited transit time through the gut. The species within the gut microbiota use a variety of strategies to process and scavenge both dietary and host produced glycans such as mucins. Some act as generalists, able to degrade a wide range of polysaccharides, while others are specialists only able to target a few select glycans. All are members of a metabolic network where substantial cross-feeding takes place as by-products of one organism serve as important resources for another. Much of this metabolic activity influences host physiology, as secondary metabolites and fermentation end products are absorbed either by the epithelial layer or transit via the portal vein to the liver where they can have additional effects. These microbially derived compounds influence cell proliferation and apoptosis, modulate the immune response and can alter host metabolism. This review summarizes the molecular underpinnings of these polysaccharide degradation processes, their impact on human health and how we can manipulate them through the use of prebiotics.