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GTPase is a key mediator of cell-autonomous innate immunityHis-tagged full-length human MxA (Fig. 1a) was recombinantly expressed in bacteria and purified to homogeneity (Methods, Supp. Fig. 1). In crystallization trials, small needle-shaped protein crystals were obtained which represented proteolytic cleavage products of the MD and GED (Supp. Fig. 2). We solved the phase problem by a single anomalous dispersion protocol and could build and refine a model containing two molecules in the asymmetric unit (Methods, Supp. Table 1 and 2). Each monomer spans nearly the complete MD and the amino(N-)-terminal part of the GED (amino acids 366-633) which together fold into an elongated anti-parallel fourhelical bundle where the MD contributes three helices and the GED one (Fig. 1b, Supp. Fig. 3). This segment corresponds to the stalk region of dynamin 7 , and we refer to it as stalk of MxA. The first visible amino acid, Glu366, is 15 amino acids downstream of the last visible residue of the corresponding G-domain structure in rat dynamin (Supp. Fig. 3) 8 . It marks the start of helix α1 in the MxA stalk which is divided in α1 N and α1 C by a 10 amino acid long loop, L1, introducing a 30° kink. A putative loop L2 (amino acids 438-447) opposite of the deduced position of the G-domain is not visible in our structure. L2 was previously demonstrated to be the target of a functionally neutralising monoclonal antibody 9,10 . Helix α2 runs anti-parallel to α1 back to the G-domain. It ends in a short loop L3 and is followed by helix α3 that extends in parallel to α1. The 40 amino acid long loop L4 (residues 532-572) is at the equivalent sequence position as the PH domain of dynamin (Fig. 1a, Supp. Fig. 3) and is absent in our model. L4 is predicted to be unstructured and was previously shown to be proteinase K sensitive 11 .At the C-terminus, the GED supplies 44 residues to helix α4 which proceeds in parallel to helix α2 back to the G-domain. It is followed by a short helix α5 which directs the polypeptide chain towards the N-terminus of the MD. The carboxy(C-)-terminal 30 highly conserved residues of the GED known to be involved in antiviral specificity 12 are missing in our model. In dynamin, the corresponding residues were shown to directly interact with the G-domain 13 . The stalk of MxA is divergent from the corresponding structures of other dynamin superfamily members, such as GBP1 14 , EHD2 15 and BDLP 16 although some features are shared (Supp. Fig. 4). 4 In the crystal lattice, each MxA stalk is assembled in a criss-cross pattern resulting in a linear oligomer, where each stalk contributes three distinct interfaces (Fig. 1c). Such an arrangement of the stalks is plausible for the Mx oligomer since all G-domains would be located at one side of the oligomer whereas the putative substrate-binding site in L2 and L4 would be located at the opposite side (Fig. 1b, c).The hydrophobic interface-1 covering 1300 Å 2 is conserved among Mx proteins and dynamins and has a two-fold symmetry between the associating monomers (Fig....

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.

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.

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.

A spectrum of membrane curvatures exists within cells, and proteins have evolved different modules to detect, create, and maintain these curvatures. Here we present the crystal structure of one such module found within human FCHo2. This F-BAR (extended FCH) module consists of two F-BAR domains, forming an intrinsically curved all-helical antiparallel dimer with a Kd of 2.5 microM. The module binds liposomes via a concave face, deforming them into tubules with variable diameters of up to 130 nm. Pulse EPR studies showed the membrane-bound dimer is the same as the crystal dimer, although the N-terminal helix changed conformation on membrane binding. Mutation of a phenylalanine on this helix partially attenuated narrow tubule formation, and resulted in a gain of curvature sensitivity. This structure shows a distant relationship to curvature-sensing BAR modules, and suggests how similar coiled-coil architectures in the BAR superfamily have evolved to expand the repertoire of membrane-sculpting possibilities.

Human 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.