Shenggen Yao

Bax Crystal Structures Reveal How Bh3 Domains Activate Bax and Nucleate its Oligomerization to Induce Apoptosis.

In stressed cells, apoptosis ensues when Bcl-2 family members Bax or Bak oligomerize and permeabilize the mitochondrial outer membrane. Certain BH3-only relatives can directly activate them to mediate this pivotal, poorly understood step. To clarify the conformational changes that induce Bax oligomerization, we determined crystal structures of BaxΔC21 treated with detergents and BH3 peptides. The peptides bound the Bax canonical surface groove but, unlike their complexes with prosurvival relatives, dissociated Bax into two domains. The structures define the sequence signature of activator BH3 domains and reveal how they can activate Bax via its groove by favoring release of its BH3 domain. Furthermore, Bax helices α2-α5 alone adopted a symmetric homodimer structure, supporting the proposal that two Bax molecules insert their BH3 domain into each others surface groove to nucleate oligomerization. A planar lipophilic surface on this homodimer may engage the membrane. Our results thus define critical Bax transitions toward apoptosis.

Aromatase-deficient (ArKO) mice accumulate excess adipose tissue.

Aromatase is the enzyme which catalyses the conversion of C19 steroids into C18 estrogens. We have generated a mouse model wherein the Cyp19 gene, which encodes aromatase, has been disrupted, and hence, the aromatase knockout (ArKO) mouse cannot synthesise endogenous estrogens. We examined the consequences of estrogen deficiency on accumulation of adipose depots in male and female ArKO mice, observing that these animals progressively accrue significantly more intra-abdominal adipose tissue than their wildtype (WT) litter mates, reflected in increased adipocyte volume and number. This increased adiposity was not due to hyperphagia or reduced resting energy expenditure, but was associated with reduced spontaneous physical activity levels, reduced glucose oxidation, and a decrease in lean body mass. Elevated circulating levels of leptin and cholesterol were present in 1-year-old ArKO mice compared to WT controls, as were elevated insulin levels, although blood glucose was unchanged. Associated with these changes, the livers of ArKO animals were characterised by a striking accumulation of lipid droplets. Our findings demonstrate an important role for estrogen in the maintenance of lipid homeostasis in both males and females.

The SPRY domain of SSB-2 adopts a novel fold that presents conserved Par-4-binding residues

The four mammalian SPRY domain–containing SOCS box proteins (SSB-1 to SSB-4) are characterized by a C-terminal SOCS box and a central SPRY domain. We have determined the first SPRY-domain structure, as part of SSB-2, by NMR. This domain adopts a novel fold consisting of a β-sandwich structure formed by two four-stranded antiparallel β-sheets with a unique topology. We demonstrate that SSB-1, SSB-2 and SSB-4, but not SSB-3, bind prostate apoptosis response protein-4 (Par-4). Mutational analysis of SSB-2 loop regions identified conserved structural determinants for its interaction with Par-4 and the hepatocyte growth factor receptor, c-Met. Mutations in analogous loop regions of pyrin and midline-1 SPRY domains have been shown to cause Mediterranean fever and Opitz syndrome, respectively. Our findings provide a template for SPRY-domain structure and an insight into the mechanism of SPRY-protein interaction.

The SOCS box domain of SOCS3: structure and interaction with the elonginBC-cullin5 ubiquitin ligase.

Suppressor of cytokine signalling 3 (SOCS3) is responsible for regulating the cellular response to a variety of cytokines, including interleukin 6 and leukaemia inhibitory factor. Identification of the SOCS box domain led to the hypothesis that SOCS3 can associate with functional E3 ubiquitin ligases and thereby induce the degradation of bound signalling proteins. This model relies upon an interaction between the SOCS box, elonginBC and a cullin protein that forms the E3 ligase scaffold. We have investigated this interaction in vitro using purified components and show that SOCS3 binds to elonginBC and cullin5 with high affinity. The SOCS3-elonginBC interaction was further characterised by determining the solution structure of the SOCS box-elonginBC ternary complex and by deletion and alanine scanning mutagenesis of the SOCS box. These studies revealed that conformational flexibility is a key feature of the SOCS-elonginBC interaction. In particular, the SOCS box is disordered in isolation and only becomes structured upon elonginBC association. The interaction depends upon the first 12 residues of the SOCS box domain and particularly on a deeply buried, conserved leucine. The SOCS box, when bound to elonginBC, binds tightly to cullin5 with 100 nM affinity. Domains upstream of the SOCS box are not required for elonginBC or cullin5 association, indicating that the SOCS box acts as an independent binding domain capable of recruiting elonginBC and cullin5 to promote E3 ligase formation.

Antibodies specifically targeting a locally misfolded region of tumor associated EGFR

Epidermal Growth Factor Receptor (EGFR) is involved in stimulating the growth of many human tumors, but the success of therapeutic agents has been limited in part by interference from the EGFR on normal tissues. Previously, we reported an antibody (mab806) against a truncated form of EGFR found commonly in gliomas. Remarkably, it also recognizes full-length EGFR on tumor cells but not on normal cells. However, the mechanism for this activity was unclear. Crystallographic structures for Fab:EGFR287–302 complexes of mAb806 (and a second, related antibody, mAb175) show that this peptide epitope adopts conformations similar to those found in the wtEGFR. However, in both conformations observed for wtEGFR, tethered and untethered, antibody binding would be prohibited by significant steric clashes with the CR1 domain. Thus, these antibodies must recognize a cryptic epitope in EGFR. Structurally, it appeared that breaking the disulfide bond preceding the epitope might allow the CR1 domain to open up sufficiently for antibody binding. The EGFRC271A/C283A mutant not only binds mAb806, but binds with 1:1 stoichiometry, which is significantly greater than wtEGFR binding. Although mAb806 and mAb175 decrease tumor growth in xenografts displaying mutant, overexpressed, or autocrine stimulated EGFR, neither antibody inhibits the in vitro growth of cells expressing wtEGFR. In contrast, mAb806 completely inhibits the ligand-associated stimulation of cells expressing EGFRC271A/C283A. Clearly, the binding of mAb806 and mAb175 to the wtEGFR requires the epitope to be exposed either during receptor activation, mutation, or overexpression. This mechanism suggests the possibility of generating antibodies to target other wild-type receptors on tumor cells.

Non-invasive observation of flow profiles and polarisation layers in hollow fibre membrane filtration modules using NMR micro-imaging

Abstract Nuclear magnetic resonance (NMR) micro-imaging techniques have been applied to the non-invasive study of flow and polarisation effects in hollow fibre membrane filtration modules. Flow distributions for both feedstock (shell side) and permeate (in the inner lumens of the hollow fibre membranes) have been mapped and the corresponding flow rates measured under different operating conditions. Measured flow velocities, when integrated over the cross section of the modules, yielded volume flow rates in good agreement with directly measured values, confirming the accuracy and reliability of the flow imaging methods employed. The results show evidence for ‘channelling’ of flow in regions of low membrane fibre packing density. Chemical shift selective imaging techniques have been used to visualise the development of oil polarisation layers in the outer surfaces of the fibres during filtration of an oil/water emulsion. Preliminary results are consistent with previous, more invasive, studies of gel polarisation phenomena in membrane filtration modules. They confirm that the hydraulic resistance of the oil layer is a major factor in controlling permeate flux. Attempts to detect flow of the oil layers themselves were unsuccessful, giving an upper limit to the flow rate of the polarisation layers of less than 2 mm/min.

Structure, dynamics and selectivity of the sodium channel blocker mu-conotoxin SIIIA

mu-SIIIA, a novel mu-conotoxin from Conus striatus, appeared to be a selective blocker of tetrodotoxin-resistant sodium channels in frog preparations. It also exhibited potent analgesic activity in mice, although its selectivity profile against mammalian sodium channels remains unknown. We have determined the structure of mu-SIIIA in aqueous solution and characterized its backbone dynamics by NMR and its functional properties electrophysiologically. Consistent with the absence of hydroxyprolines, mu-SIIIA adopts a single conformation with all peptide bonds in the trans conformation. The C-terminal region contains a well-defined helix encompassing residues 11-16, while residues 3-5 in the N-terminal region form a helix-like turn resembling 3 10-helix. The Trp12 and His16 side chains are close together, as in the related conotoxin mu-SmIIIA, but Asn2 is more distant. Dynamics measurements show that the N-terminus and Ser9 have larger-magnitude motions on the subnanosecond time scale, while the C-terminus is more rigid. Cys4, Trp12, and Cys13 undergo significant conformational exchange on microsecond to millisecond time scales. mu-SIIIA is a potent, nearly irreversible blocker of Na V1.2 but also blocks Na V1.4 and Na V1.6 with submicromolar potency. The selectivity profile of mu-SIIIA, including poor activity against the cardiac sodium channel, Na V1.5, is similar to that of the closely related mu-KIIIA, suggesting that the C-terminal regions of both are critical for blocking neuronal Na V1.2. The structural and functional characterization described in this paper of an analgesic mu-conotoxin that targets neuronal subtypes of mammalian sodium channels provides a basis for the design of novel analogues with an improved selectivity profile.

Solution Conformation, Backbone Dynamics and Lipid Interactions of the Intrinsically Unstructured Malaria Surface Protein MSP2

Merozoite surface protein 2 (MSP2), one of the most abundant proteins on the surface of the merozoite stage of Plasmodium falciparum, is a potential component of a malaria vaccine, having shown some efficacy in a clinical trial in Papua New Guinea. MSP2 is a GPI-anchored protein consisting of conserved N- and C-terminal domains and a variable central region. Previous studies have shown that it is an intrinsically unstructured protein with a high propensity for fibril formation, in which the conserved N-terminal domain has a key role. Secondary structure predictions suggest that MSP2 contains long stretches of random coil with very little alpha-helix or beta-strand. Circular dichroism spectroscopy confirms this prediction under physiological conditions (pH 7.4) and in more acidic solutions (pH 6.2 and 3.4). Pulsed field gradient NMR diffusion measurements showed that MSP2 under physiological conditions has a large effective hydrodynamic radius consistent with an intrinsic pre-molten globule state, as defined by Uversky. This was supported by sedimentation velocity studies in the analytical ultracentrifuge. NMR resonance assignments have been obtained for FC27 MSP2, allowing the residual secondary structure and backbone dynamics to be defined. There is some motional restriction in the conserved C-terminal region in the vicinity of an intramolecular disulfide bond. Two other regions show motional restrictions, both of which display helical structure propensities. One of these helical regions is within the conserved N-terminal domain, which adopts essentially the same conformation in full-length MSP2 as in corresponding peptide fragments. We see no evidence of long-range interactions in the full-length protein. MSP2 associates with lipid micelles, but predominantly through the N-terminal region rather than the C terminus, which is GPI-anchored to the membrane in the parasite.

An investigation of concentration polarization phenomena in membrane filtration of colloidal silica suspensions by NMR micro-imaging

Nuclear magnetic resonance (NMR) micro-imaging has been used to investigate concentration polarization phenomena in membrane filtration of colloidal silica suspensions using a single tubular microfiltration membrane, with the feedstock fed to the inner lumen of the membrane and the filtrate removed from the (outer) shell side. H NMR images, in which the signal intensity is weighted by the longitudinal relaxation time (T) of the solvent (water) protons, clearly exhibit details of the formation and dissipation of the silica particle concentration polarization layers at the surface of the membrane in response to changes in trans-membrane pressure difference and feedstock crossflow rate. The images were used to map the spatial distribution of the silica polarization layer as a function of time, distance from the filter inlet, and applied trans-membrane pressure difference. In each case the polarization layer was observed to be highly asymmetric, being much thicker at the bottom of the module than at the top. The performance of the filter was compared for different orientations of the filter module. The permeate flux rate was shown to be highly dependent on the orientation of the filter axis with respect to the vertical. This is consistent with the fact that the observed asymmetry in the layer is caused by flow of the polarization layer over the surface of the membrane due to gravitational effects. Phase sensitive NMR flow imaging was used to map the 1D distribution of the feedstock crossflow on the lumen side of the membrane as well as measuring the axial flow profile within the concentration polarization layer itself. The axial component of flow of the polarization layers is driven, not by gravity, but by the shear induced by the feedstock crossflow, The flow profile of the polarization layer presented in this paper therefore provides direct experimental evidence for fluidity and motion of concentration polarization layers, an assumption which has been invoked for the development of some theoretical models but which has not previously been confirmed experimentally.Nuclear magnetic resonance (NMR) micro-imaging has been used to investigate concentration polarization phenomena in membrane filtration of colloidal silica suspensions using a single tubular microfiltration membrane. H NMR images were used to map the spatial distribution of the silica polarization layer as a function of time, distance from the filter inlet, and applied trans-membrane pressure difference. The performance of the filter was compared for different orientations of the filter module. Further, phase sensitive NMR flow imaging was used to map the 1D distribution of the feedstock crossflow on the lumen side of the membrane as well as measuring the axial flow profile within the concentration polarization layer itself.

Secondary structure assignment of mouse SOCS3 by NMR defines the domain boundaries and identifies an unstructured insertion in the SH2 domain

SOCS3 is a negative regulator of cytokine signalling that inhibits Janus kinase‐signal transduction and activator of transcription (JAK‐STAT) mediated signal tranduction by binding to phosphorylated tyrosine residues on intracellular subunits of various cytokine receptors, as well as possibly the JAK proteins. SOCS3 consists of a short N‐terminal sequence followed by a kinase inhibitory region, an extended SH2 domain and a C‐terminal suppressor of cytokine signalling (SOCS) box. SOCS3 and the related protein, cytokine‐inducible SH2‐containing protein, are unique among the SOCS family of proteins in containing a region of mostly low complexity sequence, between the SH2 domain and the C‐terminal SOCS box. Using NMR, we assigned and determined the secondary structure of a murine SOCS3 construct. The SH2 domain, unusually, consists of 140 residues, including an unstructured insertion of 35 residues. This insertion fits the criteria for a PEST sequence and is not required for phosphotyrosine binding, as shown by isothermal titration calorimetry. Instead, we propose that the PEST sequence has a functional role unrelated to phosphotyrosine binding, possibly mediating efficient proteolytic degradation of the protein. The latter half of the kinase inhibitory region and the entire extended SH2 subdomain form a single α‐helix. The mapping of the true SH2 domain, and the location of its C terminus more than 50 residues further downstream than predicted by sequence homology, explains a number of previously unexpected results that have shown the importance of residues close to the SOCS box for phosphotyrosine binding.