Oliver Daumke

Host cell interactome of tyrosine-phosphorylated bacterial proteins

Selective interactions between tyrosine-phosphorylated proteins and their cognate, SH2-domain containing ligands play key roles in mammalian signal transduction. Several bacterial pathogens use secretion systems to inject tyrosine kinase substrates into host cells. Upon phosphorylation, these effector proteins recruit cellular binding partners to manipulate host cell functions. So far, only a few interaction partners have been identified. Here we report the results of a proteomic screen to systematically identify binding partners of all known tyrosine-phosphorylated bacterial effectors by high-resolution mass spectrometry. We identified 39 host interactions, all mediated by SH2 domains, including four of the five already known interaction partners. Individual phosphorylation sites recruited a surprisingly high number of cellular interaction partners suggesting that individual phosphorylation sites can interfere with multiple cellular signaling pathways. Collectively, our results indicate that tyrosine-phosphorylation sites of bacterial effector proteins have evolved as versatile interaction modules that can recruit a rich repertoire of cellular SH2 domains.

Membrane fission by dynamin: what we know and what we need to know.

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.

BAR Domain Scaffolds in Dynamin-Mediated Membrane Fission

Biological membranes undergo constant remodeling by membrane fission and fusion to change their shape and to exchange material between subcellular compartments. During clathrin-mediated endocytosis, the dynamic assembly and disassembly of protein scaffolds comprising members of the bin-amphiphysin-rvs (BAR) domain protein superfamily constrain the membrane into distinct shapes as the pathway progresses toward fission by the GTPase dynamin. In this Review, we discuss how BAR domain protein assembly and disassembly are controlled in space and time and which structural and biochemical features allow the tight regulation of their shape and function to enable dynamin-mediated membrane fission.

EHD2 regulates caveolar dynamics via ATP-driven targeting and oligomerization.

EH domain-containing 2 (EHD2) specifically and stably associates with caveolae at the plasma membrane and interacts with pacsin2 and cavin1. A loop in the nucleotide-binding domain, together with ATP binding, is required for caveolar localization. EHD2 stabilizes caveolae at the surface to control their dynamics.

Sarcolemmal repair is a slow process and includes EHD2.

Skeletal muscle is continually subjected to microinjuries that must be repaired to maintain structure and function. Fluorescent dye influx after laser injury of muscle fibers is a commonly used assay to study membrane repair. This approach reveals that initial resealing only takes a few seconds. However, by this method the process of membrane repair can only be studied in part and is therefore poorly understood. We investigated membrane repair by visualizing endogenous and GFP‐tagged repair proteins after laser wounding. We demonstrate that membrane repair and remodeling after injury is not a quick event but requires more than 20 min. The endogenous repair protein dysferlin becomes visible at the injury site after 20 seconds but accumulates further for at least 30 min. Annexin A1 and F‐actin are also enriched at the wounding area. We identified a new participant in the membrane repair process, the ATPase EHD2. We show, that EHD2, but not EHD1 or mutant EHD2, accumulates at the site of injury in human myotubes and at a peculiar structure that develops during membrane remodeling, the repair dome. In conclusion, we established an approach to visualize membrane repair that allows a new understanding of the spatial and temporal events involved.

Invited review: Mechanisms of GTP hydrolysis and conformational transitions in the dynamin superfamily

Dynamin superfamily proteins are multidomain mechano‐chemical GTPases which are implicated in nucleotide‐dependent membrane remodeling events. A prominent feature of these proteins is their assembly‐ stimulated mechanism of GTP hydrolysis. The molecular basis for this reaction has been initially clarified for the dynamin‐related guanylate binding protein 1 (GBP1) and involves the transient dimerization of the GTPase domains in a parallel head‐to‐head fashion. A catalytic arginine finger from the phosphate binding (P‐) loop is repositioned toward the nucleotide of the same molecule to stabilize the transition state of GTP hydrolysis. Dynamin uses a related dimerization‐dependent mechanism, but instead of the catalytic arginine, a monovalent cation is involved in catalysis. Still another variation of the GTP hydrolysis mechanism has been revealed for the dynamin‐like Irga6 which bears a glycine at the corresponding position in the P‐loop. Here, we highlight conserved and divergent features of GTP hydrolysis in dynamin superfamily proteins and show how nucleotide binding and hydrolysis are converted into mechano‐chemical movements. We also describe models how the energy of GTP hydrolysis can be harnessed for diverse membrane remodeling events, such as membrane fission or fusion.

Structural insights into RNA encapsidation and helical assembly of the Toscana virus nucleoprotein

Toscana virus is an emerging bunyavirus in Mediterranean Europe where it accounts for 80% of pediatric meningitis cases during the summer. The negative-strand ribonucleic acid (RNA) genome of the virus is wrapped around the virally encoded nucleoprotein N to form the ribonucleoprotein complex (RNP). We determined crystal structures of hexameric N alone (apo) and in complex with a nonameric single-stranded RNA. RNA is sequestered in a sequence-independent fashion in a deep groove inside the hexamer. At the junction between two adjacent copies of Ns, RNA binding induced an inter-subunit rotation, which opened the RNA-binding tunnel and created a new assembly interface at the outside of the hexamer. Based on these findings, we suggest a structural model for how binding of RNA to N promotes the formation of helical RNPs, which are a characteristic hallmark of many negative-strand RNA viruses.

Role of Nucleotide Binding and GTPase Domain Dimerization in Dynamin-like Myxovirus Resistance Protein A for GTPase Activation and Antiviral Activity

Background: Human myxovirus resistance protein A (MxA) is an antiviral dynamin-related GTPase. Results: Dimerization of MxA via a GTPase domain interface is required for GTP hydrolysis and antiviral activity. Conclusion: GTP binding allows GTPase domain dimerization and membrane-associated assembly of MxA, but it is not sufficient to induce a sustained antiviral effect. Significance: New mechanistic insights into the antiviral action of MxA are provided. Myxovirus resistance (Mx) GTPases are induced by interferon and inhibit multiple viruses, including influenza and human immunodeficiency viruses. They have the characteristic domain architecture of dynamin-related proteins with an N-terminal GTPase (G) domain, a bundle signaling element, and a C-terminal stalk responsible for self-assembly and effector functions. Human MxA (also called MX1) is expressed in the cytoplasm and is partly associated with membranes of the smooth endoplasmic reticulum. It shows a protein concentration-dependent increase in GTPase activity, indicating regulation of GTP hydrolysis via G domain dimerization. Here, we characterized a panel of G domain mutants in MxA to clarify the role of GTP binding and the importance of the G domain interface for the catalytic and antiviral function of MxA. Residues in the catalytic center of MxA and the nucleotide itself were essential for G domain dimerization and catalytic activation. In pulldown experiments, MxA recognized Thogoto virus nucleocapsid proteins independently of nucleotide binding. However, both nucleotide binding and hydrolysis were required for the antiviral activity against Thogoto, influenza, and La Crosse viruses. We further demonstrate that GTP binding facilitates formation of stable MxA assemblies associated with endoplasmic reticulum membranes, whereas nucleotide hydrolysis promotes dynamic redistribution of MxA from cellular membranes to viral targets. Our study highlights the role of nucleotide binding and hydrolysis for the intracellular dynamics of MxA during its antiviral action.

Functional mapping of human dynamin-1-like GTPase domain based on X-ray structure analyses

Human dynamin-1-like protein (DNM1L) is a GTP-driven molecular machine that segregates mitochondria and peroxisomes. To obtain insights into its catalytic mechanism, we determined crystal structures of a construct comprising the GTPase domain and the bundle signaling element (BSE) in the nucleotide-free and GTP-analogue-bound states. The GTPase domain of DNM1L is structurally related to that of dynamin and binds the nucleotide 5′-Guanylyl-imidodiphosphate (GMP-PNP) via five highly conserved motifs, whereas the BSE folds into a pocket at the opposite side. Based on these structures, the GTPase center was systematically mapped by alanine mutagenesis and kinetic measurements. Thus, residues essential for the GTPase reaction were characterized, among them Lys38, Ser39 and Ser40 in the phosphate binding loop, Thr59 from switch I, Asp146 and Gly149 from switch II, Lys216 and Asp218 in the G4 element, as well as Asn246 in the G5 element. Also, mutated Glu81 and Glu82 in the unique 16-residue insertion of DNM1L influence the activity significantly. Mutations of Gln34, Ser35, and Asp190 in the predicted assembly interface interfered with dimerization of the GTPase domain induced by a transition state analogue and led to a loss of the lipid-stimulated GTPase activity. Our data point to related catalytic mechanisms of DNM1L and dynamin involving dimerization of their GTPase domains.

Structural Insights into Membrane Interaction and Caveolar Targeting of Dynamin-like EHD2

The dynamin-related Eps15-homology domain-containingxa0protein 2 (EHD2) is a membrane-remodeling ATPase that regulates the dynamics of caveolae. Here, we established an electron paramagnetic resonance (EPR) approach to characterize structural features of membrane-bound EHD2. We show that residues at the tip of the helical domain can insert into the membrane and may create membrane curvature by a wedging mechanism. Using EPR and X-ray crystallography, we found that the N terminus is folded into a hydrophobic pocket of the GTPase domain in solution and can be released into the membrane. Cryoelectron microscopy demonstrated that the N terminus is not essential for oligomerization of EHD2 into a membrane-anchored scaffold. Instead, we found a function of the N terminus in regulating targeting and stable association of EHD2 to caveolae. Our data uncover an unexpected, membrane-induced regulatory switch in EHD2 and demonstrate the versatility of EPR to study structure and function of dynamin superfamily proteins.