Hui-Chun Cheng

Phase transitions in the assembly of multivalent signalling proteins

Cells are organized on length scales ranging from ångström to micrometres. However, the mechanisms by which ångström-scale molecular properties are translated to micrometre-scale macroscopic properties are not well understood. Here we show that interactions between diverse synthetic, multivalent macromolecules (including multi-domain proteins and RNA) produce sharp liquid–liquid-demixing phase separations, generating micrometre-sized liquid droplets in aqueous solution. This macroscopic transition corresponds to a molecular transition between small complexes and large, dynamic supramolecular polymers. The concentrations needed for phase transition are directly related to the valency of the interacting species. In the case of the actin-regulatory protein called neural Wiskott–Aldrich syndrome protein (N-WASP) interacting with its established biological partners NCK and phosphorylated nephrin, the phase transition corresponds to a sharp increase in activity towards an actin nucleation factor, the Arp2/3 complex. The transition is governed by the degree of phosphorylation of nephrin, explaining how this property of the system can be controlled to regulatory effect by kinases. The widespread occurrence of multivalent systems suggests that phase transitions may be used to spatially organize and biochemically regulate information throughout biology.

Hierarchical Regulation of WASP/WAVE Proteins

Members of the Wiskott-Aldrich syndrome protein (WASP) family control actin dynamics in eukaryotic cells by stimulating the actin nucleating activity of the Arp2/3 complex. The prevailing paradigm for WASP regulation invokes allosteric relief of autoinhibition by diverse upstream activators. Here we demonstrate an additional level of regulation that is superimposed upon allostery: dimerization increases the affinity of active WASP species for Arp2/3 complex by up to 180-fold, greatly enhancing actin assembly by this system. This finding explains a large and apparently disparate set of observations under a common mechanistic framework. These include WASP activation by the bacterial effector EspFu and a large number of SH3 domain proteins, the effects on WASP of membrane localization/clustering and assembly into large complexes, and cooperativity between different family members. Allostery and dimerization act in hierarchical fashion, enabling WASP/WAVE proteins to integrate different classes of inputs to produce a wide range of cellular actin responses.

Arp2/3-independent assembly of actin by Vibrio type III effector VopL

Microbial pathogens use a variety of mechanisms to disrupt the actin cytoskeleton during infection. Vibrio parahaemolyticus (V. para) is a Gram-negative bacterium that causes gastroenteritis, and new pandemic strains are emerging throughout the world. Analysis of the V. para genome revealed a type III secretion system effector, VopL, encoding three Wiskott–Aldrich homology 2 domains that are interspersed with three proline-rich motifs. Infection of HeLa cells with V. para induces the formation of long actin fibers in a VopL-dependent manner. Transfection of VopL promotes the assembly of actin stress fibers. In vitro, recombinant VopL potently induces assembly of actin filaments that grow at their barbed ends, independent of eukaryotic factors. Vibrio VopL is predicted to be a bacterial virulence factor that disrupts actin homeostasis during an enteric infection of the host.

Insulin receptor tyrosine kinase substrate links the E. coli O157:H7 actin assembly effectors Tir and EspF(U) during pedestal formation

Enterohemorrhagic Escherichia coli O157:H7 translocates 2 effectors to trigger localized actin assembly in mammalian cells, resulting in filamentous actin “pedestals.” One effector, the translocated intimin receptor (Tir), is localized in the plasma membrane and clustered upon binding the bacterial outer membrane protein intimin. The second, the proline-rich effector EspFU (aka TccP) activates the actin nucleation-promoting factor WASP/N-WASP, and is recruited to sites of bacterial attachment by a mechanism dependent on an Asn-Pro-Tyr (NPY458) sequence in the Tir C-terminal cytoplasmic domain. Tir, EspFU, and N-WASP form a complex, but neither EspFU nor N-WASP bind Tir directly, suggesting involvement of another protein in complex formation. Screening of the mammalian SH3 proteome for the ability to bind EspFU identified the SH3 domain of insulin receptor tyrosine kinase substrate (IRTKS), a factor known to regulate the cytoskeleton. Derivatives of WASP, EspFU, and the IRTKS SH3 domain were capable of forming a ternary complex in vitro, and replacement of the C terminus of Tir with the IRTKS SH3 domain resulted in a fusion protein competent for actin assembly in vivo. A second domain of IRTKS, the IRSp53/MIM homology domain (IMD), bound to Tir in a manner dependent on the C-terminal NPY458 sequence, thereby recruiting IRTKS to sites of bacterial attachment. Ectopic expression of either the IRTKS SH3 domain or the IMD, or genetic depletion of IRTKS, blocked pedestal formation. Thus, enterohemorrhagic E. coli translocates 2 effectors that bind to distinct domains of a common host factor to promote the formation of a complex that triggers robust actin assembly at the plasma membrane.

Structural mechanism of WASP activation by the enterohaemorrhagic E. coli effector EspF(U).

During infection, enterohaemorrhagic Escherichia coli (EHEC) takes over the actin cytoskeleton of eukaryotic cells by injecting the EspFU protein into the host cytoplasm. EspFU controls actin by activating members of the Wiskott–Aldrich syndrome protein (WASP) family. Here we show that EspFU binds to the autoinhibitory GTPase binding domain (GBD) in WASP proteins and displaces it from the activity-bearing VCA domain (for verprolin homology, central hydrophobic and acidic regions). This interaction potently activates WASP and neural (N)-WASP in vitro and induces localized actin assembly in cells. In the solution structure of the GBD–EspFU complex, EspFU forms an amphipathic helix that binds the GBD, mimicking interactions of the VCA domain in autoinhibited WASP. Thus, EspFU activates WASP by competing directly for the VCA binding site on the GBD. This mechanism is distinct from that used by the eukaryotic activators Cdc42 and SH2 domains, which globally destabilize the GBD fold to release the VCA. Such diversity of mechanism in WASP proteins is distinct from other multimodular systems, and may result from the intrinsically unstructured nature of the isolated GBD and VCA elements. The structural incompatibility of the GBD complexes with EspFU and Cdc42/SH2, plus high-affinity EspFU binding, enable EHEC to hijack the eukaryotic cytoskeletal machinery effectively.

Lipid binding in rice nonspecific lipid transfer protein‐1 complexes from Oryza sativa

Nonspecific lipid transfer proteins (nsLTPs) facilitate the transfer of phospholipids, glycolipids, fatty acids and steroids between membranes, with wide‐ranging binding affinities. Three crystal structures of rice nsLTP1 from Oryza sativa, complexed with myristic (MYR), palmitic (PAL) or stearic acid (STE) were determined. The overall structures of the rice nsLTP1 complexes belong to the four‐helix bundle folding with a long C‐terminal loop. The nsLTP1–MYR and the nsLTP1–STE complexes bind a single fatty acid while the nsLTP1–PAL complex binds two molecules of fatty acids. The C‐terminal loop region is elastic in order to accommodate a diverse range of lipid molecules. The lipid molecules interact with the nsLTP1‐binding cavity mainly with hydrophobic interactions. Significant conformational changes were observed in the binding cavity and the C‐terminal loop of the rice nsLTP1 upon lipid binding.

Mechanism of actin filament nucleation by the bacterial effector VopL

Vibrio parahaemolyticus protein L (VopL) is an actin nucleation factor that induces stress fibers when injected into eukaryotic host cells. VopL contains three N-terminal Wiskott-Aldrich homology 2 (WH2) motifs and a unique VopL C-terminal domain (VCD). We describe crystallographic and biochemical analyses of filament nucleation by VopL. The WH2 element of VopL does not nucleate on its own and requires the VCD for activity. The VCD forms a U-shaped dimer in the crystal, stabilized by a terminal coiled coil. Dimerization of the WH2 motifs contributes strongly to nucleation activity, as do contacts of the VCD to actin. Our data lead to a model in which VopL stabilizes primarily lateral (short-pitch) contacts between actin monomers to create the base of a two-stranded filament. Stabilization of lateral contacts may be a common feature of actin filament nucleation by WH2-based factors.

Repetitive N-WASP–Binding Elements of the Enterohemorrhagic Escherichia coli Effector EspFU Synergistically Activate Actin Assembly

Enterohemorrhagic Escherichia coli (EHEC) generate F-actin–rich adhesion pedestals by delivering effector proteins into mammalian cells. These effectors include the translocated receptor Tir, along with EspFU, a protein that associates indirectly with Tir and contains multiple peptide repeats that stimulate actin polymerization. In vitro, the EspFU repeat region is capable of binding and activating recombinant derivatives of N-WASP, a host actin nucleation-promoting factor. In spite of the identification of these important bacterial and host factors, the underlying mechanisms of how EHEC so potently exploits the native actin assembly machinery have not been clearly defined. Here we show that Tir and EspFU are sufficient for actin pedestal formation in cultured cells. Experimental clustering of Tir-EspFU fusion proteins indicates that the central role of the cytoplasmic portion of Tir is to promote clustering of the repeat region of EspFU. Whereas clustering of a single EspFU repeat is sufficient to bind N-WASP and generate pedestals on cultured cells, multi-repeat EspFU derivatives promote actin assembly more efficiently. Moreover, the EspFU repeats activate a protein complex containing N-WASP and the actin-binding protein WIP in a synergistic fashion in vitro, further suggesting that the repeats cooperate to stimulate actin polymerization in vivo. One explanation for repeat synergy is that simultaneous engagement of multiple N-WASP molecules can enhance its ability to interact with the actin nucleating Arp2/3 complex. These findings define the minimal set of bacterial effectors required for pedestal formation and the elements within those effectors that contribute to actin assembly via N-WASP-Arp2/3–mediated signaling pathways.

Crystal structure of HP0242, a hypothetical protein from Helicobacter pylori with a novel fold.

Introduction. Helicobacter pylori is a spiral-shaped, gram-negative microorganism that was found in 1979 and isolated in 1982. In 1994, the International Agency for Cancer Research declared H. pylori to be a carcinogen of human. Two complete genome sequences of H. pylori, strain 26695 and strain J99, have been determined by the whole-genome random sequencing method. About 33% protein sequences in the whole genome are annotated as “hypothetical proteins” whose functions and three-dimensional structures have never been identified. The better understanding of these proteins’ cellular processes could provide the basis for discoveries of potential antibacterial drug targets. Therefore, the determination of the structural and functional relationship of these proteins has thus drawn much research attention. HP0242 is a hypothetical protein encoded from H. pylori strain 26695. The genomic microarray analysis reveals that HP0242 is an acid-adaptive protein. It indicates that HP0242 may have an important function in bacteriological physiology when H. pylori colonize in the highly acidic environment of the human stomach. Although HP0242 has no significant sequence similarity with other functional proteins, a PSI-BLAST search with HP0242 shows four homology proteins (Fig. 1). The HP0242 gene is located next to the napA gene (HP0243/HP-NAP), which upstream contains a ferric-uptake regulator binding site. This operon governs coordinated expression of seven proteins totally. There are five functional proteins (TIGR: http://www.tigr.org/): HP0243 (neutrophil activating protein), HP0240 (octaprenyl-diphosphate synthase), HP0239 (glutamyl-tRNA reductase), HP0238 (prolyl-tRNA synthetase), and HP0237 (porphobilinogen deaminase), and two hypothetical proteins, HP0242 and HP0241. The biological functions of HP0243, HP0239, and HP0237 have been determined to be related to the iron storage and heme biosynthesis. We have determined the three-dimensional structure of HP0242 by multiwavelength anomalous dispersion (MAD) phasing from a selenomethinoine (Se-HP0242) protein. The novel folding of HP0242 will be discussed and the possible functional regions will be proposed from the detailed structure analysis.