Marilyn Leonard

G domain dimerization controls dynamin's assembly-stimulated GTPase activity

Dynamin is an atypical GTPase that catalyses membrane fission during clathrin-mediated endocytosis. The mechanisms of dynamin’s basal and assembly-stimulated GTP hydrolysis are unknown, though both are indirectly influenced by the GTPase effector domain (GED). Here we present the 2.0 Å resolution crystal structure of a human dynamin 1-derived minimal GTPase–GED fusion protein, which was dimeric in the presence of the transition state mimic GDP.AlF4-.The structure reveals dynamin’s catalytic machinery and explains how assembly-stimulated GTP hydrolysis is achieved through G domain dimerization. A sodium ion present in the active site suggests that dynamin uses a cation to compensate for the developing negative charge in the transition state in the absence of an arginine finger. Structural comparison to the rat dynamin G domain reveals key conformational changes that promote G domain dimerization and stimulated hydrolysis. The structure of the GTPase–GED fusion protein dimer provides insight into the mechanisms underlying dynamin-catalysed membrane fission.

A Pseudoatomic Model of the Dynamin Polymer Identifies a Hydrolysis-Dependent Powerstroke

The GTPase dynamin catalyzes membrane fission by forming a collar around the necks of clathrin-coated pits, but the specific structural interactions and conformational changes that drive this process remain a mystery. We present the GMPPCP-bound structures of the truncated human dynamin 1 helical polymer at 12.2 Å and a fusion protein, GG, linking human dynamin 1s catalytic G domain to its GTPase effector domain (GED) at 2.2 Å. The structures reveal the position and connectivity of dynamin fragments in the assembled structure, showing that G domain dimers only form between tetramers in sequential rungs of the dynamin helix. Using chemical crosslinking, we demonstrate that dynamin tetramers are made of two dimers, in which the G domain of one molecule interacts in trans with the GED of another. Structural comparison of GG(GMPPCP) to the GG transition-state complex identifies a hydrolysis-dependent powerstroke that may play a role in membrane-remodeling events necessary for fission.

Clusters of bioactive compounds target dynamic endomembrane networks in vivo

Endomembrane trafficking relies on the coordination of a highly complex, dynamic network of intracellular vesicles. Understanding the network will require a dissection of cargo and vesicle dynamics at the cellular level in vivo. This is also a key to establishing a link between vesicular networks and their functional roles in development. We used a high-content intracellular screen to discover small molecules targeting endomembrane trafficking in vivo in a complex eukaryote, Arabidopsis thaliana. Tens of thousands of molecules were prescreened and a selected subset was interrogated against a panel of plasma membrane (PM) and other endomembrane compartment markers to identify molecules that altered vesicle trafficking. The extensive image dataset was transformed by a flexible algorithm into a marker-by-phenotype-by-treatment time matrix and revealed groups of molecules that induced similar subcellular fingerprints (clusters). This matrix provides a platform for a systems view of trafficking. Molecules from distinct clusters presented avenues and enabled an entry point to dissect recycling at the PM, vacuolar sorting, and cell-plate maturation. Bioactivity in human cells indicated the value of the approach to identifying small molecules that are active in diverse organisms for biology and drug discovery.

SNX9 Activities are Regulated by Multiple Phosphoinositides Through both PX and BAR Domains

Sorting nexin 9 (SNX9) functions at the interface between membrane remodeling and the actin cytoskeleton. In particular, SNX9 links membrane binding to potentiation of N‐WASP and dynamin GTPase activities. SNX9 is one of a growing number of proteins that contain two lipid‐binding domains, a phox homology (PX) and a Bin1/Amphiphysin/RVS167 (BAR) domain, and localizes to diverse membranes that are enriched in different phosphoinositides. Here, we investigate the mechanism by which SNX9 functions at these varied membrane environments. We show that SNX9 has low‐lipid‐binding affinity and harnesses a broad range of phosphoinositides to synergistically enhance both dynamin and N‐WASP activities. We introduced point mutations in either the PX domain, BAR domain or both that are predicted to disrupt their functions and examined their respective roles in lipid‐binding, and dynamin and N‐WASP activation. We show that the broad lipid specificity of SNX9 is not because of independent and additive contributions by individual domains. Rather, the two domains appear to function in concert to confer lipid‐binding and SNX9’s membrane active properties. We also demonstrate that the two domains are differentially required for full SNX9 activity in N‐WASP and dynamin regulation, and for localization of SNX9 to clathrin‐coated pits and dorsal ruffles. In total, our results suggest that SNX9 can integrate signals from varied lipids through two domains to direct membrane remodeling events at multiple cellular locations.

Membrane Insertion of the Pleckstrin Homology Domain Variable Loop 1 Is Critical for Dynamin-catalyzed Vesicle Scission

The GTPase dynamin catalyzes the scission of deeply invaginated clathrin-coated pits at the plasma membrane, but the mechanisms governing dynamin-mediated membrane fission remain poorly understood. Through mutagenesis, we have altered the hydrophobic nature of the membrane-inserting variable loop 1 (VL1) of the pleckstrin homology (PH) domain of dynamin-1 and demonstrate that its stable insertion into the lipid bilayer is critical for high membrane curvature generation and subsequent membrane fission. Dynamin PH domain mutants defective in curvature generation regain function when assayed on precurved membrane templates in vitro, but they remain defective in the scission of clathrin-coated pits in vivo. These results demonstrate that, in concert with dynamin self-assembly, PH domain membrane insertion is essential for fission and vesicle release in vitro and for clathrin-mediated endocytosis in vivo.

An Intramolecular Signaling Element that Modulates Dynamin Function In Vitro and In Vivo

Dynamin exhibits a high basal rate of GTP hydrolysis that is enhanced by self-assembly on a lipid template. Dynamins GTPase effector domain (GED) is required for this stimulation, though its mechanism of action is poorly understood. Recent structural work has suggested that GED may physically dock with the GTPase domain to exert its stimulatory effects. To examine how these interactions activate dynamin, we engineered a minimal GTPase-GED fusion protein (GG) that reconstitutes dynamins basal GTPase activity and utilized it to define the structural framework that mediates GEDs association with the GTPase domain. Chemical cross-linking of GG and mutagenesis of full-length dynamin establishes that the GTPase-GED interface is comprised of the N- and C-terminal helices of the GTPase domain and the C-terminus of GED. We further show that this interface is essential for structural stability in full-length dynamin. Finally, we identify mutations in this interface that disrupt assembly-stimulated GTP hydrolysis and dynamin-catalyzed membrane fission in vitro and impair the late stages of clathrin-mediated endocytosis in vivo. These data suggest that the components of the GTPase-GED interface act as an intramolecular signaling module, which we term the bundle signaling element, that can modulate dynamin function in vitro and in vivo.

Robust colorimetric assays for dynamin's basal and stimulated GTPase activities.

Dynamin, unlike many GTPase superfamily members, exhibits a relatively rapid basal rate of GTP hydrolysis that is not rate-limited by GTP binding or GDP dissociation. Also unique to dynamin GTPase family members is their ability to self-assemble into rings and helical stacks of rings either in solution or onto lipid templates. Self-assembly stimulates dynamins GTPase activity by >100-fold. Given these robust rates of GTP hydrolysis compared to most GTPases, GTP hydrolysis by dynamin can be easily measured using a simple colorimetic assay to detect released phosphate. We describe this assay and report variations in assay conditions that have contributed to the wide range of reported values for dynamins basal and assembly-stimulated rates of GTP hydrolysis.

An internal GAP domain negatively regulates presynaptic dynamin in vivo: a two-step model for dynamin function

The mechanism by which the self-assembling GTPase dynamin functions in vesicle formation remains controversial. Point mutations in shibire, the Drosophila dynamin, cause temperature-sensitive (ts) defects in endocytosis. We show that the ts2 mutation, which occurs in the switch 2 region of dynamins GTPase domain, compromises GTP binding affinity. Three second-site suppressor mutations, one in the switch 1 region of the GTPase domain and two in the GTPase effector domain (GED), dynamins putative GAP, fully rescue the shits2 defects in synaptic vesicle recycling. The functional rescue in vivo correlates with a reduction in both the basal and assembly-stimulated GTPase activity in vitro. These findings demonstrate that GED is indeed an internal dynamin GAP and establish that, as for other GTPase superfamily members, dynamins function in vivo is negatively regulated by its GAP activity. Based on these and other observations, we propose a two-step model for dynamin during vesicle formation in which an early regulatory GTPase-like function precedes late, assembly-dependent steps during which GTP hydrolysis is required for vesicle release.

Structure and function of the yeast listerin (Ltn1) conserved N-terminal domain in binding to stalled 60S ribosomal subunits

Significance The listerin (Ltn1) E3 ubiquitin ligase ubiquitylates and promotes degradation of aberrant nascent chains that become stalled on ribosomal 60S subunits. Ltn1-dependent nascent chain ubiquitylation was reconstituted in vitro using extracts of genetically manipulated Neurospora strains. Such extracts, supplemented or not with recombinant factors (such as Ltn1 from Saccharomyces cerevisiae), represent a new system to study ribosome-associated protein quality control. Utilizing this system, we show that mutations in Ltn1’s conserved N-terminal domain result in defective 60S binding and nascent chain ubiquitylation, without affecting Ltn1’s intrinsic E3 activity. Furthermore, we have solved the crystal structure of Ltn1’s N-terminal domain, which provides detailed information and insights into how Ltn1 interacts with stalled 60S subunits. Our observations shed light on how cells handle protein quality control substrates. The Ltn1 E3 ligase (listerin in mammals) has emerged as a paradigm for understanding ribosome-associated ubiquitylation. Ltn1 binds to 60S ribosomal subunits to ubiquitylate nascent polypeptides that become stalled during synthesis; among Ltn1’s substrates are aberrant products of mRNA lacking stop codons [nonstop translation products (NSPs)]. Here, we report the reconstitution of NSP ubiquitylation in Neurospora crassa cell extracts. Upon translation in vitro, ribosome-stalled NSPs were ubiquitylated in an Ltn1-dependent manner, while still ribosome-associated. Furthermore, we provide biochemical evidence that the conserved N-terminal domain (NTD) plays a significant role in the binding of Ltn1 to 60S ribosomal subunits and that NTD mutations causing defective 60S binding also lead to defective NSP ubiquitylation, without affecting Ltn1’s intrinsic E3 ligase activity. Finally, we report the crystal structure of the Ltn1 NTD at 2.4-Å resolution. The structure, combined with additional mutational studies, provides insight to NTD’s role in binding stalled 60S subunits. Our findings show that Neurospora extracts can be used as a tool to dissect mechanisms underlying ribosome-associated protein quality control and are consistent with a model in which Ltn1 uses 60S subunits as adapters, at least in part via its NTD, to target stalled NSPs for ubiquitylation.

An Improved Model for Dynamin Assembly Revealed by Cryo-EM

Dynamin is a multidomain GTPase that assembles into collar-like structures at the necks of deeply invaginated coated pits during the final stages of clathrin-mediated endocytosis (CME) and catalyzes membrane scission. Assembly of purified dynamin tetramers in vitro yields helical structures comparable to those observed in vivo. The formation of these oligomers stimulates dynamins basal GTP hydrolysis >100-fold. Mutational analysis indicates that dynamins stimulated GTP hydrolysis is required for CME; however, mounting evidence suggests that this activity causes disassembly of the dynamin collar rather than direct membrane severing. Despite recent structural studies showing that stimulated hydrolysis arises from the transition-dependent dimerization of dynamins catalytic G domains, little is known about the conformational changes that precede and/or result from this interaction in the context of the polymer. Specifically, it is unclear how the G domains are properly oriented, which subunits associate, and how catalysis triggers dissociation of the pleckstrin homology (PH) domain at the membrane surface. Much of this ambiguity can be attributed to the low resolution (>20A) of previous dynamin polymer models and the absence of a complete dynamin tetramer crystal structure. To clarify these issues, we have used cryo-EM and iterative helical real space refinement to generate an 11A reconstruction of a truncated form of dynamin (ΔPRD) in the assembled, GMPPCP-bound state. This map reveals new structural characteristics including a twisted, interlacing interaction that stabilizes the middle/GED stalk and a previously uncharacterized density feature adjacent to the exterior GTPase head. Computational docking of crystallized dynamin fragments reveals the location and connectivity of different domains within the assembled polymer. Chemical crosslinking experiments also provide new insights into the architecture and organization of dynamin tetramer. These data have important implications regarding the conformational changes associated with dynamin catalyzed GTP hydrolysis and membrane fission.