The variable domain from dynamin-related protein 1 promotes liquid-liquid phase separation that enhances its interaction with cardiolipin-containing membranes.

Dynamins are an essential superfamily of mechanoenzymes that remodel membranes and often contain a "variable domain" important for regulation. For the mitochondrial fission dynamin, dynamin-related protein 1, a regulatory role for the variable domain (VD) is demonstrated by gain- and loss-of-function mutations, yet the basis for this is unclear. Here, the isolated VD is shown to be intrinsically disordered and undergo a cooperative transition in the stabilizing osmolyte trimethylamine N-oxide. However, the osmolyte-induced state is not folded and surprisingly appears as a condensed state. Other co-solutes including known molecular crowder Ficoll PM 70, also induce a condensed state. Fluorescence recovery after photobleaching experiments reveal this state to be liquid-like indicating the VD undergoes a liquid-liquid phase separation under crowding conditions. These crowding conditions also enhance binding to cardiolipin, a mitochondrial lipid, which appears to promote phase separation. Since dynamin-related protein 1 is found assembled into discrete punctate structures on the mitochondrial surface, the inference from the present work is that these structures might arise from a condensed state involving the VD that may enable rapid tuning of mechanoenzyme assembly necessary for fission.

Kinetic Growth of Multicomponent Microcompartment Shells.

An important goal of systems and synthetic biology is to produce high value chemical species in large quantities. Microcompartments, which are protein nanoshells encapsulating catalytic enzyme cargo, could potentially function as tunable nanobioreactors inside and outside cells to generate these high value species. Modifying the morphology of microcompartments through genetic engineering of shell proteins is one viable strategy to tune cofactor and metabolite access to encapsulated enzymes. However, this is a difficult task without understanding how changing interactions between the many different types of shell proteins and enzymes affect microcompartment assembly and shape. Here, we use multiscale molecular dynamics and experimental data to describe assembly pathways available to microcompartments composed of multiple types of shell proteins with varied interactions. As the average interaction between the enzyme cargo and the multiple types of shell proteins is weakened, the shell assembly pathway transitions from (i) nucleating on the enzyme cargo to (ii) nucleating in the bulk and then binding the cargo as it grows to (iii) an empty shell. Atomistic simulations and experiments using the 1,2-propanediol utilization microcompartment system demonstrate that shell protein interactions are highly varied and consistent with our multicomponent, coarse-grained model. Furthermore, our results suggest that intrinsic bending angles control the size of these microcompartments. Overall, our simulations and experiments provide guidance to control microcomparmtent size and assembly by modulating the interactions between shell proteins.

The variable domain from the mitochondrial fission mechanoenzyme Drp1 promotes liquid-liquid phase separation.

Dynamins are an essential superfamily of mechanoenzymes that remodel membranes and often contain a "variable domain" (VD) important for regulation. For the mitochondrial fission dynamin, Drp1, a regulatory role for the VD is demonstrated by mutations that can elongate, or fragment, mitochondria. How the VD encodes inhibitory and stimulatory activity is unclear. Here, isolated VD is shown to be intrinsically disordered (ID) yet undergoes a cooperative transition in the stabilizing osmolyte TMAO. However, the TMAO stabilized state is not folded and surprisingly appears as a condensed state. Other co-solutes including known molecular crowder Ficoll PM 70, also induce a condensed state. Fluorescence recovery after photobleaching experiments reveal this state to be liquid-like indicating the VD undergoes a liquid-liquid phase separation under crowding conditions. These crowding conditions also enhance binding to cardiolipin, a mitochondrial lipid, raising the possibility that phase separation may enable rapid tuning of Drp1 assembly necessary for fission.

Theoretical and Practical Aspects of Multienzyme Organization and Encapsulation.

The advent of biotechnology has enabled metabolic engineers to assemble heterologous pathways in cells to produce a variety of products of industrial relevance, often in a sustainable way. However, many pathways face challenges of low product yield. These pathways often suffer from issues that are difficult to optimize, such as low pathway flux and off-target pathway consumption of intermediates. These issues are exacerbated by the need to balance pathway flux with the health of the cell, particularly when a toxic intermediate builds up. Nature faces similar challenges and has evolved spatial organization strategies to increase metabolic pathway flux and efficiency. Inspired by these strategies, bioengineers have developed clever strategies to mimic spatial organization in nature. This review explores the use of spatial organization strategies, including protein scaffolding and protein encapsulation inside of proteinaceous shells, toward overcoming bottlenecks in metabolic engineering efforts.

Vertex protein PduN tunes encapsulated pathway performance by dictating bacterial metabolosome morphology.

Engineering subcellular organization in microbes shows great promise in addressing bottlenecks in metabolic engineering efforts; however, rules guiding selection of an organization strategy or platform are lacking. Here, we study compartment morphology as a factor in mediating encapsulated pathway performance. Using the 1,2-propanediol utilization microcompartment (Pdu MCP) system from Salmonella enterica serovar Typhimurium LT2, we find that we can shift the morphology of this protein nanoreactor from polyhedral to tubular by removing vertex protein PduN. Analysis of the metabolic function between these Pdu microtubes (MTs) shows that they provide a diffusional barrier capable of shielding the cytosol from a toxic pathway intermediate, similar to native MCPs. However, kinetic modeling suggests that the different surface area to volume ratios of MCP and MT structures alters encapsulated pathway performance. Finally, we report a microscopy-based assay that permits rapid assessment of Pdu MT formation to enable future engineering efforts on these structures.

Linking the Salmonella enterica 1,2-Propanediol Utilization Bacterial Microcompartment Shell to the Enzymatic Core via the Shell Protein PduB.

Bacterial microcompartments (MCPs) are protein-based organelles that house the enzymatic machinery for metabolism of niche carbon sources, allowing enteric pathogens to outcompete native microbiota during host colonization. While much progress has been made toward understanding MCP biogenesis, questions still remain regarding the mechanism by which core MCP enzymes are enveloped within the MCP protein shell. Here, we explore the hypothesis that the shell protein PduB is responsible for linking the shell of the 1,2-propanediol utilization (Pdu) MCP from Salmonella enterica serovar Typhimurium LT2 to its enzymatic core. Using fluorescent reporters, we demonstrate that all members of the Pdu enzymatic core are encapsulated in Pdu MCPs. We also demonstrate that PduB is critical for linking the entire Pdu enzyme core to the MCP shell. Using MCP purifications, transmission electron microscopy, and fluorescence microscopy, we find that shell assembly can be decoupled from the enzymatic core, as apparently empty MCPs are formed in Salmonella strains lacking PduB. Mutagenesis studies reveal that PduB is incorporated into the Pdu MCP shell via a conserved, lysine-mediated hydrogen bonding mechanism. Finally, growth assays and system-level pathway modeling reveal that unencapsulated pathway performance is strongly impacted by enzyme concentration, highlighting the importance of minimizing polar effects when conducting these functional assays. Together, these results provide insight into the mechanism of enzyme encapsulation within Pdu MCPs and demonstrate that the process of enzyme encapsulation and shell assembly are separate processes in this system, a finding that will aid future efforts to understand MCP biogenesis. IMPORTANCE MCPs are unique, genetically encoded organelles used by many bacteria to survive in resource-limited environments. There is significant interest in understanding the biogenesis and function of these organelles, both as potential antibiotic targets in enteric pathogens and also as useful tools for overcoming metabolic engineering bottlenecks. However, the mechanism by which these organelles are formed natively is still not completely understood. Here, we provide evidence of a potential mechanism in S. enterica by which a single protein, PduB, links the MCP shell and metabolic core. This finding is critical for those seeking to disrupt MCPs during pathogenic infections or for those seeking to harness MCPs as nanobioreactors in industrial settings.

Density-based binning of gene clusters to infer function or evolutionary history using GeneGrouper.

MOTIVATION: Identifying variant forms of gene clusters of interest in phylogenetically proximate and distant taxa can help to infer their evolutionary histories and functions. Conserved gene clusters may differ by only a few genes, but these small differences can in turn induce substantial phenotypes, such as by the formation of pseudogenes or insertions interrupting regulation. Particularly as microbial genomes and metagenomic assemblies become increasingly abundant, unsupervised grouping of similar, but not necessarily identical, gene clusters into consistent bins can provide a population-level understanding of their gene content variation and functional homology. RESULTS: We developed GeneGrouper, a command-line tool that uses a density-based clustering method to group gene clusters into bins. GeneGrouper demonstrated high recall and precision in benchmarks for the detection of the 23-gene Salmonella enterica LT2 Pdu gene cluster and four-gene Pseudomonas aeruginosa PAO1 Mex gene cluster among 435 genomes spanning mixed taxa. In a subsequent application investigating the diversity and impact of gene-complete and -incomplete LT2 Pdu gene clusters in 1130 S.enterica genomes, GeneGrouper identified a novel, frequently occurring pduN pseudogene. When investigated in vivo, introduction of the pduN pseudogene negatively impacted microcompartment formation. We next demonstrated the versatility of GeneGrouper by clustering distant homologous gene clusters and variable gene clusters found in integrative and conjugative elements. AVAILABILITY AND IMPLEMENTATION: GeneGrouper software and code are publicly available at https://pypi.org/project/GeneGrouper/. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Bacterial microcompartments: tiny organelles with big potential.

Organization of metabolic processes within the space of a cell is critical for the survival of many organisms. In bacteria, spatial organization is achieved via proteinaceous organelles called bacterial microcompartments, which encapsulate pathway enzymes, substrates, and co-factors to drive the safe and efficient metabolism of niche carbon sources. Microcompartments are self-assembled from shell proteins that encapsulate a core comprising various enzymes. This review discusses how recent advances in understanding microcompartment structure and assembly have informed engineering efforts to repurpose compartments and compartment-based structures for non-native functions. These advances, both in understanding of the native structure and function of compartments, as well as in the engineering of new functions, will pave the way for the use of these structures in bacterial cell factories.

Computational and Experimental Approaches to Controlling Bacterial Microcompartment Assembly.

Bacterial microcompartments compartmentalize the enzymes that aid chemical and energy production in many bacterial species. They are postulated to help bacteria survive in hostile environments. Metabolic engineers are interested in repurposing these organelles for non-native functions. Here, we use computational, theoretical, and experimental approaches to determine mechanisms that effectively control microcompartment self-assembly. We find, via multiscale modeling and mutagenesis studies, the interactions responsible for the binding of hexamer-forming proteins in a model system, the propanediol utilization bacterial microcompartments from Salmonella enterica serovar Typhimurium LT2. We determine how the changes in the microcompartment hexamer protein preferred angles and interaction strengths can modify the assembled morphologies. We demonstrate that such altered strengths and angles are achieved via amino acid mutations. A thermodynamic model provides guidelines to design microcompartments of various morphologies. These findings yield insight in controlled protein assembly and provide principles for assembling microcompartments for biochemical or energy applications as nanoreactors.

Self-assembling Shell Proteins PduA and PduJ have Essential and Redundant Roles in Bacterial Microcompartment Assembly.

Protein self-assembly is a common and essential biological phenomenon, and bacterial microcompartments present a promising model system to study this process. Bacterial microcompartments are large, protein-based organelles which natively carry out processes important for carbon fixation in cyanobacteria and the survival of enteric bacteria. These structures are increasingly popular with biological engineers due to their potential utility as nanobioreactors or drug delivery vehicles. However, the limited understanding of the assembly mechanism of these bacterial microcompartments hinders efforts to repurpose them for non-native functions. Here, we comprehensively investigate proteins involved in the assembly of the 1,2-propanediol utilization bacterial microcompartment from Salmonella enterica serovar Typhimurium LT2, one of the most widely studied microcompartment systems. We first demonstrate that two shell proteins, PduA and PduJ, have a high propensity for self-assembly upon overexpression, and we provide a novel method for self-assembly quantification. Using genomic knock-outs and knock-ins, we systematically show that these two proteins play an essential and redundant role in bacterial microcompartment assembly that cannot be compensated by other shell proteins. At least one of the two proteins PduA and PduJ must be present for the bacterial microcompartment shell to assemble. We also demonstrate that assembly-deficient variants of these proteins are unable to rescue microcompartment formation, highlighting the importance of this assembly property. Our work provides insight into the assembly mechanism of these bacterial organelles and will aid downstream engineering efforts.

Isolation and Analysis of Mitochondrial Fission Enzyme DNM1 from Saccharomyces cerevisiae.

Mitochondrial fission, an essential process for mitochondrial and cellular homeostasis, is accomplished by evolutionarily conserved members of the dynamin superfamily of large GTPases. These enzymes couple the hydrolysis of guanosine triphosphate to the mechanical work of membrane remodeling that ultimately leads to membrane scission. The importance of mitochondrial dynamins is exemplified by mutations in the human family member that causes neonatal lethality. In this chapter, we describe the subcloning, purification, and preliminary characterization of the budding yeast mitochondrial dynamin, DNM1, from Saccharomyces cerevisiae, which is the first mitochondrial dynamin isolated from native sources. The yeast-purified enzyme exhibits assembly-stimulated hydrolysis of GTP similar to other fission dynamins, but differs from the enzyme isolated from non-native sources.

Apparent size and morphology of bacterial microcompartments varies with technique.

Bacterial microcompartments (MCPs) are protein-based organelles that encapsulate metabolic pathways. Metabolic engineers have recently sought to repurpose MCPs to encapsulate heterologous pathways to increase flux through pathways of interest. As MCP engineering becomes more common, standardized methods for analyzing changes to MCPs and interpreting results across studies will become increasingly important. In this study, we demonstrate that different imaging techniques yield variations in the apparent size of purified MCPs from Salmonella enterica serovar Typhimurium LT2, likely due to variations in sample preparation methods. We provide guidelines for preparing samples for MCP imaging and outline expected variations in apparent size and morphology between methods. With this report we aim to establish an aid for comparing results across studies.

Cargo encapsulation in bacterial microcompartments: Methods and analysis.

Metabolic engineers seek to produce high-value products from inexpensive starting materials in a sustainable and cost-effective manner by using microbes as cellular factories. However, pathway development and optimization can be arduous tasks, complicated by pathway bottlenecks and toxicity. Pathway organization has emerged as a potential solution to these issues, and the use of protein- or DNA-based scaffolds has successfully increased the production of several industrially relevant compounds. These efforts demonstrate the usefulness of pathway colocalization and spatial organization for metabolic engineering applications. In particular, scaffolding within an enclosed, subcellular compartment shows great promise for pathway optimization, offering benefits such as increased local enzyme and substrate concentrations, sequestration of toxic or volatile intermediates, and alleviation of cofactor and resource competition with the host. Here, we describe the 1,2-propanediol utilization (Pdu) bacterial microcompartment (MCP) as an enclosed scaffold for pathway sequestration and organization. We first describe methods for controlling Pdu MCP formation, expressing and encapsulating heterologous cargo, and tuning cargo loading levels. We further describe assays for analyzing Pdu MCPs and assessing encapsulation levels. These methods will enable the repurposing of MCPs as tunable nanobioreactors for heterologous pathway encapsulation.

Development and Validation of 2D Difference Intensity Analysis for Chemical Library Screening by Protein-Detected NMR Spectroscopy.

An academic chemical screening approach was developed by using 2D protein-detected NMR, and a 352-chemical fragment library was screened against three different protein targets. The approach was optimized against two protein targets with known ligands: CXCL12 and BRD4. Principal component analysis reliably identified compounds that induced nonspecific NMR crosspeak broadening but did not unambiguously identify ligands with specific affinity (hits). For improved hit detection, a novel scoring metric-difference intensity analysis (DIA)-was devised that sums all positive and negative intensities from 2D difference spectra. Applying DIA quickly discriminated potential ligands from compounds inducing nonspecific NMR crosspeak broadening and other nonspecific effects. Subsequent NMR titrations validated chemotypes important for binding to CXCL12 and BRD4. A novel target, mitochondrial fission protein Fis1, was screened, and six hits were identified by using DIA. Screening these diverse protein targets identified quinones and catechols that induced nonspecific NMR crosspeak broadening, hampering NMR analyses, but are currently not computationally identified as pan-assay interference compounds. The results established a streamlined screening workflow that can easily be scaled and adapted as part of a larger screening pipeline to identify fragment hits and assess relative binding affinities in the range of 0.3-1.6 mm. DIA could prove useful in library screening and other applications in which NMR chemical shift perturbations are measured.

Molecular Motor Dnm1 Synergistically Induces Membrane Curvature To Facilitate Mitochondrial Fission.

Dnm1 and Fis1 are prototypical proteins that regulate yeast mitochondrial morphology by controlling fission, the dysregulation of which can result in developmental disorders and neurodegenerative diseases in humans. Loss of Dnm1 blocks the formation of fission complexes and leads to elongated mitochondria in the form of interconnected networks, while overproduction of Dnm1 results in excessive mitochondrial fragmentation. In the current model, Dnm1 is essentially a GTP hydrolysis-driven molecular motor that self-assembles into ring-like oligomeric structures that encircle and pinch the outer mitochondrial membrane at sites of fission. In this work, we use machine learning and synchrotron small-angle X-ray scattering (SAXS) to investigate whether the motor Dnm1 can synergistically facilitate mitochondrial fission by membrane remodeling. A support vector machine (SVM)-based classifier trained to detect sequences with membrane-restructuring activity identifies a helical Dnm1 domain capable of generating negative Gaussian curvature (NGC), the type of saddle-shaped local surface curvature found on scission necks during fission events. Furthermore, this domain is highly conserved in Dnm1 homologues with fission activity. Synchrotron SAXS measurements reveal that Dnm1 restructures membranes into phases rich in NGC, and is capable of inducing a fission neck with a diameter of 12.6 nm. Through in silico mutational analysis, we find that the helical Dnm1 domain is locally optimized for membrane curvature generation, and phylogenetic analysis suggests that dynamin superfamily proteins that are close relatives of human dynamin Dyn1 have evolved the capacity to restructure membranes via the induction of curvature mitochondrial fission. In addition, we observe that Fis1, an adaptor protein, is able to inhibit the pro-fission membrane activity of Dnm1, which points to the antagonistic roles of the two proteins in the regulation of mitochondrial fission.

The Putative Drp1 Inhibitor mdivi-1 Is a Reversible Mitochondrial Complex I Inhibitor that Modulates Reactive Oxygen Species.

Mitochondrial fission mediated by the GTPase dynamin-related protein 1 (Drp1) is an attractive drug target in numerous maladies that range from heart disease to neurodegenerative disorders. The compound mdivi-1 is widely reported to inhibit Drp1-dependent fission, elongate mitochondria, and mitigate brain injury. Here, we show that mdivi-1 reversibly inhibits mitochondrial complex I-dependent O2 consumption and reverse electron transfer-mediated reactive oxygen species (ROS) production at concentrations (e.g., 50 µM) used to target mitochondrial fission. Respiratory inhibition is rescued by bypassing complex I using yeast NADH dehydrogenase Ndi1. Unexpectedly, respiratory impairment by mdivi-1 occurs without mitochondrial elongation, is not mimicked by Drp1 deletion, and is observed in Drp1-deficient fibroblasts. In addition, mdivi-1 poorly inhibits recombinant Drp1 GTPase activity (Ki > 1.2 mM). Overall, these results suggest that mdivi-1 is not a specific Drp1 inhibitor. The ability of mdivi-1 to reversibly inhibit complex I and modify mitochondrial ROS production may contribute to effects observed in disease models.

Resistance of Dynamin-related Protein 1 Oligomers to Disassembly Impairs Mitophagy, Resulting in Myocardial Inflammation and Heart Failure.

We have reported previously that a missense mutation in the mitochondrial fission gene Dynamin-related protein 1 (Drp1) underlies the Python mouse model of monogenic dilated cardiomyopathy. The aim of this study was to investigate the consequences of the C452F mutation on Drp1 protein function and to define the cellular sequelae leading to heart failure in the Python monogenic dilated cardiomyopathy model. We found that the C452F mutation increased Drp1 GTPase activity. The mutation also conferred resistance to oligomer disassembly by guanine nucleotides and high ionic strength solutions. In a mouse embryonic fibroblast model, Drp1 C452F cells exhibited abnormal mitochondrial morphology and defective mitophagy. Mitochondria in C452F mouse embryonic fibroblasts were depolarized and had reduced calcium uptake with impaired ATP production by oxidative phosphorylation. In the Python heart, we found a corresponding progressive decline in oxidative phosphorylation with age and activation of sterile inflammation. As a corollary, enhancing autophagy by exposure to a prolonged low-protein diet improved cardiac function in Python mice. In conclusion, failure of Drp1 disassembly impairs mitophagy, leading to a downstream cascade of mitochondrial depolarization, aberrant calcium handling, impaired ATP synthesis, and activation of sterile myocardial inflammation, resulting in heart failure.