Project description:Assembly of viruses that have hundreds of subunits or folding of proteins that have hundreds of amino acids-complex biological reactions-are often spontaneous and rapid. Here, we examine the complete set of intermediates available for the assembly of a hypothetical viruslike particle and the connectivity between these intermediates in a graph-theory-inspired study. Using a build-up procedure, assuming ideal geometry, we enumerated the complete set of 2,423,313 species for formation of an icosahedron from 30 dimeric subunits. Stability of each n-subunit intermediate was defined by the number of contacts between subunits. The probability of forming an intermediate was based on the number of paths to it from its precedecessors. When defining population subsets predicted to have the greatest impact on assembly, both stability- and probability-based criteria select a small group of compact and degenerate species; ergo, only a few hundred intermediates make a measurable contribution to assembly. Though the number of possible intermediates grows combinatorially with the number of subunits in the capsid, the number of intermediates that make a significant contribution to the reaction grows by a much smaller function, a result that may contribute to our understanding of assembly and folding reactions.
Project description:The Escherichia coli type I-E CRISPR-Cas system Cascade effector is a multisubunit complex that binds CRISPR RNA (crRNA). Through its 32-nucleotide spacer sequence, Cascade-bound crRNA recognizes protospacers in foreign DNA, causing its destruction during CRISPR interference or acquisition of additional spacers in CRISPR array during primed CRISPR adaptation. Within Cascade, the crRNA spacer interacts with a hexamer of Cas7 subunits. We show that crRNAs with a spacer length reduced to 14 nucleotides cause primed adaptation, while crRNAs with spacer lengths of more than 20 nucleotides cause both primed adaptation and target interference in vivo Shortened crRNAs assemble into altered-stoichiometry Cascade effector complexes containing less than the normal amount of Cas7 subunits. The results show that Cascade assembly is driven by crRNA and suggest that multisubunit type I CRISPR effectors may have evolved from much simpler ancestral complexes.
Project description:Proteins interact with one another across a broad spectrum of affinities. Our understanding of the low end of this spectrum, as characterized by millimolar dissociation constants, relies on a handful of cases in which weak encounters have experimentally been identified. These weak interactions away from the specific target binding site can lead toward a higher-affinity complex. Recently, we detected weak encounters between two paralogous phosphotransferase pathways of Escherichia coli, which regulate various metabolic processes and stress responses. In addition to encounters that are known to occur between cognate proteins, i.e., those that can exchange phosphate groups with each other, surprisingly, encounters involving noncognates were also observed. It is not clear whether these "futile" encounters have a cooperative or competitive role. Using agent-based simulations, we find that the encounter complexes can be cooperative or competitive so as to increase or lower the effective binding affinity of the specific complex under different circumstances. This finding invites further questions into how organisms might exploit such low affinities to connect their signaling components.
Project description:There are two paralogous Escherichia coli phosphotransferase systems, one for sugar import (PTSsugar) and one for nitrogen regulation (PTSNtr), that utilize proteins enzyme Isugar (EIsugar) and HPr, and enzyme INtr (EINtr) and NPr, respectively. The enzyme I proteins have similar folds, as do their substrates HPr and NPr, yet they show strict specificity for their cognate partner both in stereospecific protein-protein complex formation and in reversible phosphotransfer. Here, we investigate the mechanism of specific EINtr:NPr complex formation by the study of transient encounter complexes. NMR paramagnetic relaxation enhancement experiments demonstrated transient encounter complexes of EINtr not only with the expected partner, NPr, but also with the unexpected partner, HPr. HPr occupies transient sites on EINtr but is unable to complete stereospecific complex formation. By occupying the non-productive transient sites, HPr promotes NPr transient interaction to productive sites closer to the stereospecific binding site and actually enhances specific complex formation between NPr and EINtr. The cellular level of HPr is approximately 150 times higher than that of NPr. Thus, our finding suggests a potential mechanism for cross-regulation of enzyme activity through formation of competitive encounter complexes.
Project description:As a signature of reproductive interference (RI), we reviewed hybrid production in eusocial bumblebees in Japan, by comparing introduced Bombus terrestris with native B. ignitus in Honshu (main island of Japan) and with native B. hypocrita sapporoensis in Hokkaido (northern island of Japan). In this review, we present additional new data showing hybrid production between introduced B. terrestris and native B. ignitus in Honshu. Interspecific mating with introduced B. terrestris disrupts the reproduction of native B. h. sapporoensis and B. ignitus, which belong to the same subgenus of Bombus, through inviable egg production. This interference appears to facilitate species replacement on Hokkaido. Simultaneously, the mating frequencies for queens of B. terrestris have increased, suggesting that polyandry might evolve in response to the extent of RI between B. terrestris and B. h. sapporoensis. To suppress the population size of B. terrestris in Hokkaido, two methods have been proposed: the mass release of B. h. sapporoensis males to induce RI between the two species and the spraying of insecticides against foraging workers so that the workers will carry the insecticides back to their colonies, killing the immature bees within the colonies. A candidate insecticide type is insect growth regulator, which may disrupt larval development without any apparent effect on foraging workers.
Project description:The intricacy of nuclear pore complex (NPC) biogenesis imposes risks of failure that can cause defects in nuclear transport and nuclear envelope (NE) morphology; however, cellular mechanisms used to alleviate NPC assembly stress are not well defined. In the budding yeast Saccharomyces cerevisiae, we demonstrate that NVJ1- and MDM1-enriched NE-vacuole contacts increase when NPC assembly is compromised in several nup mutants, including nup116ΔGLFG cells. These interorganelle nucleus-vacuole junctions (NVJs) cooperate with lipid droplets to maintain viability and enhance NPC formation in assembly mutants. Additionally, NVJs function with ATG1 to remodel the NE and promote vacuole-dependent degradation of specific nucleoporins in nup116ΔGLFG cells. Importantly, NVJs significantly improve the physiology of NPC assembly mutants, despite having only negligible effects when NPC biogenesis is unperturbed. These results therefore define how NE-vacuole interorganelle contacts coordinate responses to mitigate deleterious cellular effects caused by disrupted NPC assembly.
Project description:The Human Silencing Hub (HuSH) complex silences retrotransposable elements in vertebrates. Here, we identify a second HuSH complex, designated HuSH2, which is centered around TASOR2, a paralog of the core TASOR protein in HuSH. Our findings reveal that HuSH and HuSH2 localize to distinct and non-overlapping genomic loci. Specifically, HuSH localizes to and represses LINE-1 retrotransposons, whereas HuSH2 targets and represses KRAB-ZNFs and interferon signaling and response genes. We use in silico protein structure predictions to simulate MPP8 interactions with TASOR paralogs, guiding amino acid substitutions that disrupted binding to HuSH complexes. These MPP8 transgenes and other constructs reveal the importance of HuSH complex quantities in regulating LINE-1 activity. Furthermore, our results suggest that dynamic changes in TASOR and TASOR2 expression enable cells to finely tune HuSH-mediated silencing. This study offers insights into the interplay of HuSH complexes, highlighting their vital role in retrotransposon regulation.
Project description:The Human Silencing Hub (HuSH) complex is composed of TASOR, MPP8, and PPHLN1 subunits and serves as a conserved protein complex responsible for silencing transposable elements in vertebrate animals. Despite its importance, the regulatory mechanisms and recruitment dynamics governing this complex remain poorly understood. In this study, we have identified a second HuSH complex, termed HuSH2, centered around TASOR2, a paralog of the core TASOR protein in HuSH. Our findings indicate that every subunit in both HuSH and HuSH2 has an important role in achieving precise genomic localization to distinct, non-overlapping genomic loci. We utilized in silico protein structure prediction to simulate the interactions between MPP8 and both TASOR paralogs. Drawing on the insights gained from these predictions, we implemented amino acid substitutions that interfered with the binding of MPP8 to each HuSH complex. Leveraging these MPP8 transgenes and other constructs, we identified an important role played by the relative quantities of HuSH complexes in controlling the activity of LINE-1 elements. Furthermore, our results suggest that dynamic changes in TASOR and TASOR2 expression enable cells to finely tune the extent of HuSH-mediated silencing. Our study provides insights into the intricate interplay between HuSH complexes, illuminating their important role in the regulation of retrotransposon silencing.
Project description:The well-established presence of histidine donors in binding sites of Ni-containing biomolecules prompts the study of orientational preference and stereodynamic nature of flat monodentate ligands (L = imidazoles, pyridine and an N-heterocyclic carbene) bound to planar N(2)SNi moieties. Square planar [N(2)SNiL](n+) complexes are accessed through bridge-splitting reactions of dimeric, thiolate-S bridged [N(2)SNi](2) complexes. The solid state molecular structures of three mononuclear products, and three monothiolate bridged dinickel complexes, reveal that the plane of the added monodentate ligand orients largely orthogonal to the N(2)SNiL square plane. Variable temperature (1)H NMR characterization of dynamic processes and ground state isomer ratios of imidazole complexes in their stopped exchange limiting spectra, readily correlate with density functional theory (DFT)-guided interpretation of Ni-L rotational activation barriers. Full DFT characterization finds Ni-L bond lengthening as well as a tetrahedral twist distortion in the transition state, reaching a maximum in the NHC complex, and relating mainly to the steric hindrance derived both from the ligand and the binding pocket. In the case of the imidazole ligands a minor electronic contribution derives from intramolecular electrostatic interactions (imidazole C-2 C-H(delta+)- - S(delta-) interaction). Computational studies find this donor-acceptor interaction is magnified in O-analogues, predicting coplanar arrangements in the ground state of N(2)ON(imid)Ni complexes.
Project description:The CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) adaptive immune systems use small guide RNAs, the CRISPR RNAs (crRNAs), to mark foreign genetic material, e.g. viral nucleic acids, for degradation. Archaea and bacteria encode a large variety of Cas proteins that bind crRNA molecules and build active ribonucleoprotein surveillance complexes. The evolution of CRISPR-Cas systems has resulted in a diversification of cas genes and a classification of the systems into three types and additional subtypes characterized by distinct surveillance and interfering complexes. Recent crystallographic and biochemical advances have revealed detailed insights into the assembly and DNA/RNA targeting mechanisms of the various complexes. Here, we review our knowledge on the molecular mechanism involved in the DNA and RNA interference stages of type I (Cascade: CRISPR-associated complex for antiviral defense), type II (Cas9) and type III (Csm, Cmr) CRISPR-Cas systems. We further highlight recently reported structural and mechanistic themes shared among these systems.