Structural insights into the activation mechanism of antimicrobial GBP1.

The dynamin-related human guanylate-binding protein 1 (GBP1) mediates host defenses against microbial pathogens. Upon GTP binding and hydrolysis, auto-inhibited GBP1 monomers dimerize and assemble into soluble and membrane-bound oligomers, which are crucial for innate immune responses. How higher-order GBP1 oligomers are built from dimers, and how assembly is coordinated with nucleotide-dependent conformational changes, has remained elusive. Here, we present cryo-electron microscopy-based structural data of soluble and membrane-bound GBP1 oligomers, which show that GBP1 assembles in an outstretched dimeric conformation. We identify a surface-exposed helix in the large GTPase domain that contributes to the oligomerization interface, and we probe its nucleotide- and dimerization-dependent movements that facilitate the formation of an antimicrobial protein coat on a gram-negative bacterial pathogen. Our results reveal a sophisticated activation mechanism for GBP1, in which nucleotide-dependent structural changes coordinate dimerization, oligomerization, and membrane binding to allow encapsulation of pathogens within an antimicrobial protein coat.

Not Quite Lost in Translation: Mistranslation Alters Adaptive Landscape Topography and the Dynamics of Evolution.

Mistranslation-the erroneous incorporation of amino acids into nascent proteins-is a source of protein variation that is orders of magnitude more frequent than DNA mutation. Like other sources of nongenetic variation, it can affect adaptive evolution. We study the evolutionary consequences of mistranslation with experimental data on mistranslation rates applied to three empirical adaptive landscapes. We find that mistranslation generally flattens adaptive landscapes by reducing the fitness of high fitness genotypes and increasing that of low fitness genotypes, but it does not affect all genotypes equally. Most importantly, it increases genetic variation available to selection by rendering many neutral DNA mutations nonneutral. Mistranslation also renders some beneficial mutations deleterious and vice versa. It increases the probability of fixation of 3-8% of beneficial mutations. Even though mistranslation increases the incidence of epistasis, it also allows populations evolving on a rugged landscape to evolve modestly higher fitness. Our observations show that mistranslation is an important source of nongenetic variation that can affect adaptive evolution on fitness landscapes in multiple ways.

A Novel Group of Dynamin-Related Proteins Shared by Eukaryotes and Giant Viruses Is Able to Remodel Mitochondria From Within the Matrix.

The diverse GTPases of the dynamin superfamily play various roles in the cell, as exemplified by the dynamin-related proteins (DRPs) Mgm1 and Opa1, which remodel the mitochondrial inner membrane in fungi and metazoans, respectively. Via an exhaustive search of genomic and metagenomic databases, we found previously unknown DRP types occurring in diverse eukaryotes and giant viruses (phylum Nucleocytoviricota). One novel DRP clade, termed MidX, combined hitherto uncharacterized proteins from giant viruses and six distantly related eukaryote taxa (Stramenopiles, Telonemia, Picozoa, Amoebozoa, Apusomonadida, and Choanoflagellata). MidX stood out because it was not only predicted to be mitochondria-targeted but also to assume a tertiary structure not observed in other DRPs before. To understand how MidX affects mitochondria, we exogenously expressed MidX from Hyperionvirus in the kinetoplastid Trypanosoma brucei, which lacks Mgm1 or Opa1 orthologs. MidX massively affected mitochondrial morphology from inside the matrix, where it closely associates with the inner membrane. This unprecedented mode of action contrasts to those of Mgm1 and Opa1, which mediate inner membrane remodeling in the intermembrane space. We speculate that MidX was acquired in Nucleocytoviricota evolution by horizontal gene transfer from eukaryotes and is used by giant viruses to remodel host mitochondria during infection. MidX's unique structure may be an adaptation for reshaping mitochondria from the inside. Finally, Mgm1 forms a sister group to MidX and not Opa1 in our phylogenetic analysis, throwing into question the long-presumed homology of these DRPs with similar roles in sister lineages.

Reconstituting membrane fission using a high content and throughput assay.

Protein-mediated membrane fission has been analyzed both in bulk and at the single event resolution. Studies on membrane fission in vitro using tethers have provided fundamental insights into the process but are low in throughput. In recent years, supported membrane template (SMrT) have emerged as a facile and convenient assay system for membrane fission. SMrTs provide useful information on intermediates in the pathway to fission and are therefore high in content. They are also high in throughput because numerous fission events can be monitored in a single experiment. This review discusses the utility of SMrTs in providing insights into fission pathways and its adaptation to annotate membrane fission functions in proteins.

Structural insights into the activation mechanism of dynamin-like EHD ATPases.

Eps15 (epidermal growth factor receptor pathway substrate 15)-homology domain containing proteins (EHDs) comprise a family of dynamin-related mechano-chemical ATPases involved in cellular membrane trafficking. Previous studies have revealed the structure of the EHD2 dimer, but the molecular mechanisms of membrane recruitment and assembly have remained obscure. Here, we determined the crystal structure of an amino-terminally truncated EHD4 dimer. Compared with the EHD2 structure, the helical domains are 50° rotated relative to the GTPase domain. Using electron paramagnetic spin resonance (EPR), we show that this rotation aligns the two membrane-binding regions in the helical domain toward the lipid bilayer, allowing membrane interaction. A loop rearrangement in GTPase domain creates a new interface for oligomer formation. Our results suggest that the EHD4 structure represents the active EHD conformation, whereas the EHD2 structure is autoinhibited, and reveal a complex series of domain rearrangements accompanying activation. A comparison with other peripheral membrane proteins elucidates common and specific features of this activation mechanism.

A dynamin homolog promotes the transition from hemifusion to content mixing in intracellular membrane fusion.

The convergence of the antagonistic reactions of membrane fusion and fission at the hemifusion/hemifission intermediate has generated a captivating enigma of whether Soluble N-ethylmaleimide sensitive factor Attachment Protein Receptor (SNAREs) and dynamin have unusual counter-functions in fission and fusion, respectively. SNARE-mediated fusion and dynamin-driven fission are fundamental membrane flux reactions known to occur during ubiquitous cellular communication events such as exocytosis, endocytosis and vesicle transport. Here we demonstrate the influence of the dynamin homolog Vps1 (Vacuolar protein sorting 1) on lipid mixing and content mixing properties of yeast vacuoles, and on the incorporation of SNAREs into fusogenic complexes. We propose a novel concept that Vps1, through its oligomerization and SNARE domain binding, promotes the hemifusion-content mixing transition in yeast vacuole fusion by increasing the number of trans-SNAREs.

Dynamin Superfamily at Pre- and Postsynapses: Master Regulators of Synaptic Transmission and Plasticity in Health and Disease.

Dynamin superfamily proteins (DSPs) comprise a large group of GTP-ases that orchestrate membrane fusion and fission, and cytoskeleton remodeling in different cell-types. At the central nervous system, they regulate synaptic vesicle recycling and signaling-receptor turnover, allowing the maintenance of synaptic transmission. In the presynapses, these GTP-ases control the recycling of synaptic vesicles influencing the size of the ready-releasable pool and the release of neurotransmitters from nerve terminals, whereas in the postsynapses, they are involved in AMPA-receptor trafficking to and from postsynaptic densities, supporting excitatory synaptic plasticity, and consequently learning and memory formation. In agreement with these relevant roles, an important number of neurological disorders are associated with mutations and/or dysfunction of these GTP-ases. Along the present review we discuss the importance of DSPs at synapses and their implication in different neuropathological contexts.

Mitochondrial Fusion: The Machineries In and Out.

Mitochondria are highly dynamic organelles that constantly undergo fission and fusion. Disruption of mitochondrial dynamics undermines their function and causes several human diseases. The fusion of the outer (OMM) and inner mitochondrial membranes (IMM) is mediated by two classes of dynamin-like protein (DLP): mitofusin (MFN)/fuzzy onions 1 (Fzo1) and optic atrophy 1/mitochondria genome maintenance 1 (OPA1/Mgm1). Given the lack of structural information on these fusogens, the molecular mechanisms underlying mitochondrial fusion remain unclear, even after 20 years. Here, we review recent advances in structural studies of the mitochondrial fusion machinery, discuss their implication for DLPs, and summarize the pathogenic mechanisms of disease-causing mutations in mitochondrial fusion DLPs.

Cellular functions and intrinsic attributes of the ATP-binding Eps15 homology domain-containing proteins.

Several cellular processes rely on a cohort of dedicated proteins that manage tubulation, fission, and fusion of membranes. A notably large number of them belong to the dynamin superfamily of proteins. Among them is the evolutionarily conserved group of ATP-binding Eps15-homology domain-containing proteins (EHDs). In the two decades since their discovery, EHDs have been linked to a range of cellular processes that require remodeling or maintenance of specific membrane shapes such as during endocytic recycling, caveolar biogenesis, ciliogenesis, formation of T-tubules in skeletal muscles, and membrane resealing after rupture. Recent work has shed light on their structure and the unique attributes they possess in linking ATP hydrolysis to membrane remodeling. This review summarizes some of these recent developments and reconciles intrinsic protein functions to their cellular roles.

Assay to Measure Interactions between Purified Drp1 and Synthetic Liposomes.

A mitochondrion is a dynamic intracellular organelle that actively divides and fuses to control its size, number and shape in cells. A regulated balance between mitochondrial division and fusion is fundamental to the function, distribution and turnover of mitochondria (Roy et al., 2015). Mitochondrial division is mediated by dynamin-related protein 1 (Drp1), a mechano-chemical GTPase that constricts mitochondrial membranes (Tamura et al., 2011). Mitochondrial membrane lipids such as phosphatidic acid and cardiolipin bind Drp1, and Drp1-phospholipid interactions provide key regulatory mechanisms for mitochondrial division (Montessuit et al., 2010; Bustillo-Zabalbeitia et al., 2014; Macdonald et al., 2014; Stepanyants et al., 2015; Adachi et al., 2016). Here, we describe biochemical experiments that quantitatively measure interactions of Drp1 with lipids using purified recombinant Drp1 and synthetic liposomes with a defined set of phospholipids. This assay makes it possible to define the specificity of protein-lipid interaction and the role of the head group and acyl chains.