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Balanced fusion and fission are key for the proper function and physiology of mitochondria1,2. Remodelling of the mitochondrial inner membrane is mediated by the dynamin-like protein mitochondrial genome maintenance 1 (Mgm1) in fungi or the related protein optic atrophy 1 (OPA1) in animals3–5. Mgm1 is required for the preservation of mitochondrial DNA in yeast6, whereas mutations in the OPA1 gene in humans are a common cause of autosomal dominant optic atrophy—a genetic disorder that affects the optic nerve7,8. Mgm1 and OPA1 are present in mitochondria as a membrane-integral long form and a short form that is soluble in the intermembrane space. Yeast strains that express temperature-sensitive mutants of Mgm19,10 or mammalian cells that lack OPA1 display fragmented mitochondria11,12, which suggests that Mgm1 and OPA1 have an important role in inner-membrane fusion. Consistently, only the mitochondrial outer membrane—not the inner membrane—fuses in the absence of functional Mgm113. Mgm1 and OPA1 have also been shown to maintain proper cristae architecture10,14; for example, OPA1 prevents the release of pro-apoptotic factors by tightening crista junctions15. Finally, the short form of OPA1 localizes to mitochondrial constriction sites, where it presumably promotes mitochondrial fission16. How Mgm1 and OPA1 perform their diverse functions in membrane fusion, scission and cristae organization is at present unknown. Here we present crystal and electron cryo-tomography structures of Mgm1 from Chaetomium thermophilum. Mgm1 consists of a GTPase (G) domain, a bundle signalling element domain, a stalk, and a paddle domain that contains a membrane-binding site. Biochemical and cell-based experiments demonstrate that the Mgm1 stalk mediates the assembly of bent tetramers into helical filaments. Electron cryo-tomography studies of Mgm1-decorated lipid tubes and fluorescence microscopy experiments on reconstituted membrane tubes indicate how the tetramers assemble on positively or negatively curved membranes. Our findings convey how Mgm1 and OPA1 filaments dynamically remodel the mitochondrial inner membrane. Crystal and electron cryo-tomography structures of Mgm1 from Chaetomium thermophilum reveal that Mgm1 forms bent tetramers, which further assemble into helical filaments on both positively and negatively curved membranes.

The GTPase dynamin is a molecular machine that assembles at the neck of clathrin-coated pits and catalyzes the scission of the vesicle neck in a GTPase-dependent fashion [1]. Recent structural work, in combination with biochemical and cell-based experiments, have led to a molecular model of how dynamin functions.

Ionotropic glutamate receptors are postsynaptic tetrameric ligand-gated channels whose activity mediates fast excitatory transmission. Glutamate binding to clamshell-shaped ligand binding domains (LBDs) triggers opening of the integral ion channel, but how the four LBDs orchestrate receptor activation is unknown. Here, we present a high-resolution x-ray crystal structure displaying two tetrameric LBD arrangements fully bound to glutamate. Using a series of engineered metal ion trapping mutants, we showed that the more compact of the two assemblies corresponds to an arrangement populated during activation of full-length receptors. State-dependent cross-linking of the mutants identified zinc bridges between the canonical active LBD dimers that formed when the tetramer was either fully or partially bound by glutamate. These bridges also stabilized the resting state, consistent with the recently published full-length apo structure. Our results provide insight into the activation mechanism of glutamate receptors and the complex conformational space that the LBD layer can sample.

Dynamin 1-like protein (DNM1L) mediates fission of mitochondria and peroxisomes, and dysfunction of DNM1L has been implicated in several neurological disorders. To study the molecular basis of mitochondrial remodelling, we determined the crystal structure of DNM1L that is comprised of a G domain, a bundle signalling element and a stalk. DNM1L assembled via a central stalk interface, and mutations in this interface disrupted dimerization and interfered with membrane binding and mitochondrial targeting. Two sequence stretches at the tip of the stalk were shown to be required for ordered assembly of DNM1L on membranes and its function in mitochondrial fission. In the crystals, DNM1L dimers further assembled via a second, previously undescribed, stalk interface to form a linear filament. Mutations in this interface interfered with liposome tubulation and mitochondrial remodelling. Based on these results and electron microscopy reconstructions, we propose an oligomerization mode for DNM1L which differs from that of dynamin and might be adapted to the remodelling of mitochondria.

The large GTPase dynamin is the first protein shown to catalyze membrane fission. Dynamin and its related proteins are essential to many cell functions, from endocytosis to organelle division and fusion, and it plays a critical role in many physiological functions such as synaptic transmission and muscle contraction. Research of the past three decades has focused on understanding how dynamin works. In this review, we present the basis for an emerging consensus on how dynamin functions. Three properties of dynamin are strongly supported by experimental data: first, dynamin oligomerizes into a helical polymer; second, dynamin oligomer constricts in the presence of GTP; and third, dynamin catalyzes membrane fission upon GTP hydrolysis. We present the two current models for fission, essentially diverging in how GTP energy is spent. We further discuss how future research might solve the remaining open questions presently under discussion.

A-kinase anchoring proteins (AKAPs) interact with the dimerization/docking (D/D) domains of regulatory subunits of the ubiquitous protein kinase A (PKA). AKAPs tether PKA to defined cellular compartments establishing distinct pools to increase the specificity of PKA signalling. Here, we elucidated the structure of an extended PKA-binding domain of AKAP18{beta} bound to the D/D domain of the regulatory RII{alpha} subunits of PKA. We identified three hydrophilic anchor points in AKAP18{beta} outside the core PKA-binding domain, which mediate contacts with the D/D domain. Such anchor points are conserved within AKAPs that bind regulatory RII subunits of PKA. We derived a different set of anchor points in AKAPs binding regulatory RI subunits of PKA. In vitro and cell-based experiments confirm the relevance of these sites for the interaction of RII subunits with AKAP18 and of RI subunits with the RI-specific smAKAP. Thus we report a novel mechanism governing interactions of AKAPs with PKA. The sequence specificity of each AKAP around the anchor points and the requirement of these points for the tight binding of PKA allow the development of selective inhibitors to unequivocally ascribe cellular functions to the AKAP18-PKA and other AKAP-PKA interactions.

SummaryHuman myxovirus resistance protein 1 (MxA) is an interferon-induced dynamin-like GTPase that acts as a cell-autonomous host restriction factor against many viral pathogens including influenza viruses. To study the molecular principles of its antiviral activity, we determined the crystal structure of nucleotide-free MxA, which showed an extended three-domain architecture. The central bundle signaling element (BSE) connected the amino-terminal GTPase domain with the stalk via two hinge regions. MxA oligomerized in the crystal via the stalk and the BSE, which in turn interacted with the stalk of the neighboring monomer. We demonstrated that the intra- and intermolecular domain interplay between the BSE and stalk was essential for oligomerization and the antiviral function of MxA. Based on these results, we propose a structural model for the mechano-chemical coupling in ring-like MxA oligomers as the principle mechanism for this unique antiviral effector protein.

SummaryThe yeast suppressor of myosin 2 protein (Smy2) interacts with mRNA-processing proteins through recognition of proline-rich sequences (PRS). Here, we describe the crystal structure of the GYF domain of Smy2 in association with a PRS from the yeast branch point binding protein (BBP/ScSF1). Complex formation requires that the β-hairpin of the central PPGL motif of the ligand is accommodated by an extended hydrophobic cleft in the domain—a specificity feature that is maintained in the human protein GIGYF2. SILAC/MS experiments in combination with PRS site inhibition show that Smy2 associates with the Ccr4-NOT deadenylase complex, whereas GIGYF2 interacts not only with mRNA surveillance factors, but also with vesicular transport proteins and Atrophin-1. GIGYF2 is shown to associate with COPII-vesicle proteins and localize to the ER and Golgi in resting cells, whereas environmental challenge drives GIGYF2 into stress granules. The current study highlights the structural basis for PRS recognition by Smy2-type GYF domains, and implicates Smy2 and GIGYF2 in both mRNA processing and the secretory pathway.

Interaction mapping is a powerful strategy to elucidate the biological function of protein assemblies and their regulators. Here, we report the generation of a quantitative interaction network, directly linking 14 human proteins to the AAA+ ATPase p97, an essential hexameric protein with multiple cellular functions. We show that the high-affinity interacting protein ASPL efficiently promotes p97 hexamer disassembly, resulting in the formation of stable p97:ASPL heterotetramers. High-resolution structural and biochemical studies indicate that an extended UBX domain (eUBX) in ASPL is critical for p97 hexamer disassembly and facilitates the assembly of p97:ASPL heterotetramers. This spontaneous process is accompanied by a reorientation of the D2 ATPase domain in p97 and a loss of its activity. Finally, we demonstrate that overproduction of ASPL disrupts p97 hexamer function in ERAD and that engineered eUBX polypeptides can induce cell death, providing a rationale for developing anti-cancer polypeptide inhibitors that may target p97 activity., The AAA+ ATPase p97 is an essential hexameric protein with multiple protein interaction partners and cellular functions. Here, the authors use interaction mapping to examine partner proteins of this large complex, and assess the effects of these proteins on the disassembly of the p97 complex.

Many MAS (magic angle spinning) solid-state NMR investigations of biologically relevant protein samples are hampered by poor resolution, particularly in the 15N chemical shift dimension. We show that dynamics in the nanosecond-microsecond time scale in solid-state samples can induce significant line broadening of 15N resonances in solid-state NMR experiments. Averaging of 15NH(alpha/beta) multiplet components due to 1H decoupling induces effective relaxation of the 15N coherence in case the N-H spin pair undergoes significant motion. High resolution solid-state NMR spectra can then only be recorded by application of TROSY (Transverse Relaxation Optimized Spectroscopy) type techniques which select the narrow component of the multiplet pattern. We speculate that this effect has been the major obstacle to the NMR spectroscopic characterization of many membrane proteins and fibrillar aggregates so far. Only in very favorable cases, where dynamics are either absent or very fast (picosecond), high-resolution spectra were obtained. We expect that this approach which requires intense deuteration will have a significant impact on the quality and the rate at which solid-state NMR spectroscopic investigations will emerge in the future.

Water molecules are a major determinant of protein stability and are important for understanding protein-protein interactions. We present two experiments which allow to measure first the effective T(2) decay rate of individual amide proton, and second the magnetization build-up rates for a selective transfer from H(2)O to H(N) using spin diffusion as a mixing element. The experiments are demonstrated for a uniformly (2)H, (15)N labeled sample of a microcrystalline SH3 domain in which exchangeable deuterons were back-substituted with protons. In order to evaluate the NMR experimental data, as X-ray structure of the protein was determined using the same crystallization protocol as for the solid-state NMR sample. The NMR experimental data are correlated with the dipolar couplings calculated from H(2)O-H(N) distances which were extracted from the X-ray structure of the protein. We find that the H(N) T(2) decay rates and H(2)O-H(N) build-up rates are sensitive to distance and dynamics of the detected water molecules with respect to the protein. We show that qualitative information about localization and dynamics of internal water molecules can be obtained in the solid-state by interpretation of the spin dynamics of a reporter amide proton.

The outstanding importance of the antigen-antibody recognition process for the survival and defence strategy of higher organisms is in sharp contrast to the limited high resolution structural data available on antibody-antigen pairs with antigenic proteins. The limitation is the most severe for structural data not restricted to the antigen-antibody complex but extending to the uncomplexed antigen and antibody. We report the crystal structure of the complex between tissue factor (TF) and the humanized Fab fragment D3h44 at a resolution of 1.85 A together with the structure of uncomplexed D3h44 at the same resolution. In conjunction with the previously reported 1.7 A crystal structure of uncomplexed TF, a unique opportunity is generated to explore details of the recognition process. The TF·D3h44 interface is characterised by a high number of polar interactions, including as may as 46 solvent molecules. Conformational changes upon complex formation are very small and almost exclusively limited to the reorientation of side-chains. The binding epitope is in complete agreement with earlier mutagenesis experiments. A revaluation of two other antibody-antigen pairs reported at similar resolutions, shows that all these complexes are very similar with respect to the solvation of the interface, the number of solvent positions conserved in the uncomplexed and complexed proteins and the number of water molecules expelled from the surface and replaced by hydrophilic atoms from the binding partner upon complex formation. A strategy is proposed on how to exploit this high resolution structural data to guide the affinity maturation of humanised antibodies.

Proteins of the dynamin superfamily are mechanochemical GTPases, which mediate nucleotide-dependent membrane remodeling events. The founding member dynamin is recruited to the neck of clathrin-coated endocytic vesicles where it oligomerizes into helical filaments. Nucleotide-hydrolysis-induced conformational changes in the oligomer catalyze scission of the vesicle neck. Here, we review recent insights into structure, function, and oligomerization of dynamin superfamily proteins and their roles in human diseases. We describe in detail the molecular mechanisms how dynamin oligomerizes at membranes and introduce a model how oligomerization is linked to membrane fission. Finally, we discuss molecular mechanisms how mutations in dynamin could lead to the congenital diseases, Centronuclear Myopathy and Charcot-Marie Tooth disease.

Proteins of the dynamin superfamily are mechanochemical GTPases, which mediate nucleotide-dependent membrane remodeling events. The founding member dynamin is recruited to the neck of clathrin-coated endocytic vesicles where it oligomerizes into helical filaments. Nucleotide-hydrolysis-induced conformational changes in the oligomer catalyze scission of the vesicle neck. Here, we review recent insights into structure, function, and oligomerization of dynamin superfamily proteins and their roles in human diseases. We describe in detail the molecular mechanisms how dynamin oligomerizes at membranes and introduce a model how oligomerization is linked to membrane fission. Finally, we discuss molecular mechanisms how mutations in dynamin could lead to the congenital diseases, Centronuclear Myopathy and Charcot-Marie Tooth disease.

Dynamin is a mechanochemical GTPase that oligomerizes around the neck of clathrin-coated pits and catalyses vesicle scission in a GTP-hydrolysis-dependent manner. The molecular details of oligomerization and the mechanism of the mechanochemical coupling are currently unknown. Here we present the crystal structure of human dynamin 1 in the nucleotide-free state with a four-domain architecture comprising the GTPase domain, the bundle signalling element, the stalk and the pleckstrin homology domain. Dynamin 1 oligomerized in the crystals via the stalks, which assemble in a criss-cross fashion. The stalks further interact via conserved surfaces with the pleckstrin homology domain and the bundle signalling element of the neighbouring dynamin molecule. This intricate domain interaction rationalizes a number of disease-related mutations in dynamin 2 and suggests a structural model for the mechanochemical coupling that reconciles previous models of dynamin function.

Heteronuclear correlation experiments employing perdeuterated proteins enable the observation of all hydroxyl protons in a microcrystalline protein by MAS solid-state NMR. Dipolar-based sequences allow magnetization transfers that are >50 times faster compared to scalar-coupling-based sequences, which significantly facilitates their assignment. Hydroxyl exchange rates were measured using EXSY-type experiments. We find a biexponential decay behavior for those hydroxyl groups that are involved in side chain-side chain C-O-H...O horizontal lineC hydrogen bonds. The quantification of the distances between the hydroxyl proton and the carbon atoms in the hydrogen-bonding donor as well as acceptor group is achieved via a REDOR experiment. In combination with X-ray data and isotropic proton chemical shifts, availability of (1)H,(13)C distance information can aid in the quantitative description of the geometry of these hydrogen bonds. Similarly, correlations between backbone amide proton and carbonyl atoms are observed, which will be useful in the analysis of the registry of beta-strand arrangement in amyloid fibrils.

We show in this manuscript that (2)H,(13)C correlation spectra in uniformly (2)H,(13)C isotopically enriched peptides and proteins can be recorded in MAS solid-state NMR with site specific resolution. A resolved deuterium dimension is obtained by evolving (2)H double-quantum coherences. Experimental (2)H line widths are obtained that are as small as 16 Hz (0.17 ppm at 600 MHz) in the double-quantum dimension. The unprecedented resolution in the deuterium dimension obtained for proteins opens new perspectives for correlation experiments and, in particular, for the characterization of dynamics of proteins in the solid-state.

We show in this communication that dynamic information for uniformly 2H,13C,15N isotopically enriched, crystalline proteins can be obtained by MAS solid-state NMR spectroscopy. The experiments make use of the deuterium quadrupolar tensor, which is the dominant interaction mechanism. Dynamic properties are accessed by measurement of the size of the quadrupolar coupling constant, Cq, and the value of the asymmetry parameter, eta, via evolution of the deuterium chemical shift, as well as by measurement of deuterium T1 relaxation times. Three-dimensional experiments are performed in order to obtain site-specific resolution. Due to proton dilution, no proton decoupling is required in the carbon evolution periods at MAS rotation frequencies of 10 kHz.

Ionotropic glutamate receptors are postsynaptic tetrameric channels whose activity mediates transmission of the synaptic signal. Binding of glutamate to their ligand binding domains (LBDs) shifts the receptors from the resting to active state followed by desensitization. Here we present two different tetrameric LBD arrangements of an AMPA receptor fully bound by glutamate at 1.3 A resolution. The novel inter-dimer interfaces presented by the crystal are probed through a series of engineered histidine mutants, which are then cross-linked by zinc in outside-out patches, with biochemical measurements on full-length receptors. The functional profiles of the His mutants indicate that the most compact tetrameric LBD arrangement captured by the crystal represents an active state of the receptor. The functional and structural data are further corroborated by computational modeling showing that most of the movement in agonist-bound LBDs is mediated by two subunits further away from the overall 2-fold axis of molecular symmetry (distal subunits). The results, thus, give the first insight into the open structure of the activated receptor at the level of the LBD tetramer.

Ionotropic glutamate receptors are tetrameric ion channels that are activated by the neurotransmitter glutamate at excitatory synapses. It is known that after the binding of glutamate, the AMPA-subtype of glutamate receptors transits through distinct functional states to become fully activated, however, the conformations sampled by the tetramer during activation remain unknown. We have subjected plausible models of the tetramer of ligand binding domains to a panel of trapping bridges combined with electrophysiology, biochemistry and crystallography. These experiments were designed to probe the geometry of the different states sampled by the ligand binding domains during receptor activation. We show that the mutant A665C is preferentially crosslinked in the presence of the partial agonist, kainate. In addition, the same crosslink trapped a much more stable conformation in the desensitized state. We also resolved fast disulfide trapping on the millisecond time scale in the resting state of the glutamate receptor, providing insight into dynamics of the receptor complex at rest. Finally, we present engineered metal trapping bridges that trap conformations distinct from those observed in the full-length resting state crystal structure (Sobolevsky et al, 2009 Nature). Overall, our results reinforce the idea that the ligand binding domains are highly flexible and sample a surprisingly large conformational space.

Dynamin is a multidomain mechanochemical guanine triphosphatase that catalyzes membrane scission, most notably of clathrin-coated endocytic vesicles. A number of recent publications have provided structural and mechanistic insights into the formation of helical dynamin filaments assembled by dynamic interactions of multiple domains within dynamin. As a prerequisite for membrane scission, this oligomer undergoes nucleotide-triggered large scale dynamic rearrangements. Here, we review these structural findings and discuss how the architecture of dynamin is poised for the assembly into right-handed helical filaments. Based on these data, we propose a structure-based model for dynamin-mediated scission of membranes.