I am a structural biologist working in the Signalling and Structural Biology Lab in the Francis Crick Institute in London, UK.
I have previously worked for Professor Erhard Hohenester at Imperial College on the Laminin family of proteins, and on glycosaminoglycan biosynthesis. Before that, I worked for Professor Tony Day at the University of Manchester, investigating the role of the serum proteoglycan Inter-alpha-inhibitor, in matrix remodelling in Arthritis and Ovulation. Prior to that I worked for Prof Neil McDonald at Cancer Research UK, and before that I obtained my PhD in Protein Crystallography working for Prof David Moss at Birkbeck College, London.
My research interests focus on human extracellular proteins, specifically on protein:protein and protein:sugar interactions within the extracellular matrix Supervisors: Prof Neil McDonald
Dystroglycan is a highly glycosylated extracellular matrix receptor with essential functions in s... more Dystroglycan is a highly glycosylated extracellular matrix receptor with essential functions in skeletal muscle and the nervous system. Reduced matrix binding by α-dystroglycan (α-DG) due to perturbed glycosylation is a pathological feature of several forms of muscular dystrophy. Like-acetylglucosaminyltransferase (LARGE) synthesizes the matrix-binding heteropolysaccharide [-glucuronic acid-β1,3-xylose-α1,3-]n. Using a dual exoglycosidase digestion, we confirm that this polysaccharide is present on native α-DG from skeletal muscle. The atomic details of matrix binding were revealed by a high-resolution crystal structure of laminin-G-like (LG) domains 4 and 5 (LG4 and LG5) of laminin-α2 bound to a LARGE-synthesized oligosaccharide. A single glucuronic acid-β1,3-xylose disaccharide repeat straddles a Ca2+ ion in the LG4 domain, with oxygen atoms from both sugars replacing Ca2+-bound water molecules. The chelating binding mode accounts for the high affinity of this protein–carbohydrate interaction. These results reveal a previously uncharacterized mechanism of carbohydrate recognition and provide a structural framework for elucidating the mechanisms underlying muscular dystrophy.
Laminins are a major constituent of all basement membranes. The polymerisation of laminins at the... more Laminins are a major constituent of all basement membranes. The polymerisation of laminins at the cell surface is mediated by the three short arms of the cross-shaped laminin heterotrimer. The short arms contain repeats of laminin-type epidermal growth factor-like (LE) domains, interspersed with globular domains of unknown function. A single LF domain is inserted between LE5 and LE6 of the laminin β1 and β2 chains. We report the crystal structure at 1.85Å resolution of the laminin β2 LE5-LF-LE6 region. The LF domain consists of a β-sandwich related to bacterial family 35 carbohydrate binding modules, and more distantly to the L4 domains present in the short arms of laminin α and γ chains. An α-helical region mediates the extensive interaction of the LF domain with LE5. The relative arrangement of LE5 and LE6 is very similar to that of consecutive LE domains in uninterrupted LE tandems. Fitting atomic models to a low-resolution structure of the first eight domains of the laminin β1 chain determined by small-angle X-ray scattering suggests a deviation from the regular LE array at the LE4-LE5 junction. These results advance our understanding of laminin structure.
The C-type mannose receptor and its homolog Endo180 (or uPARAP, for urokinase plasminogen activat... more The C-type mannose receptor and its homolog Endo180 (or uPARAP, for urokinase plasminogen activator receptor-associated protein) mediate the endocytic uptake of collagen by macrophages and fibroblasts. This process is required for normal tissue remodeling, but also facilitates the growth and dissemination of tumors. We have determined the crystal structure at 2.5 Å resolution of the N-terminal region of Endo180, consisting of a ricin-like domain, a fibronectin type II (FN2) domain, and two C-type lectin (CTL) domains. The L-shaped arrangement of these domains creates a shallow trench spanning the FN2 and CTL1 domains, which was shown by mutagenesis to bind triple-helical and denatured collagen. Small-angle X-ray scattering showed that the L-shaped structure is maintained in solution at neutral and acidic pH, irrespective of calcium ion loading. Collagen binding was equally unaffected by acidic pH, suggesting that collagen release in endosomes is not regulated by changes within the Endo180 N-terminal region.
Background: TSG-6 (TNF-stimulated gene-6)-dependent transfer of heavy chains from inter-α-inhibit... more Background: TSG-6 (TNF-stimulated gene-6)-dependent transfer of heavy chains from inter-α-inhibitor onto hyaluronan is critical for ovulation.
Results: A calcium ion and chelating glutamate within TSG-6 mediate formation of the covalent heavy chain-TSG-6 intermediate.
Conclusion: TSG-6 transferase activity rather than hyaluronan binding drives cumulus expansion.
Significance: The role of metal ions in hyaluronan-heavy chain formation has been determined.
Background: The proteins pentraxin 3 (PTX3) and TNF-stimulated gene-6 (TSG-6) and the proteoglyca... more Background: The proteins pentraxin 3 (PTX3) and TNF-stimulated gene-6 (TSG-6) and the proteoglycan inter-α-inhibitor (IαI) are known to be involved in the stabilization of hyaluronan (HA)-rich extracellular matrices.
Results: PTX3 incorporation into HA matrices is tightly regulated.
Conclusion: PTX3, TSG-6, and IαI are sufficient to cross-link HA matrices.
Significance: The results provide mechanistic insights into the regulation of HA-protein interactions.
Background: The polysaccharide hyaluronan is organized through interactions with the protein TSG-... more Background: The polysaccharide hyaluronan is organized through interactions with the protein TSG-6 during inflammation and ovulation. Results: NMR spectroscopy on TSG-6 in the presence of defined sugars provided restraints that allowed modeling of a refined hyaluronan/TSG-6 complex. Conclusion: TSG-6 binding causes bending of hyaluronan that explains its condensation of this polysaccharide.
Background: Inflammation/ovulation-associated protein TSG-6 performs multiple functions in hyalur... more Background: Inflammation/ovulation-associated protein TSG-6 performs multiple functions in hyaluronan (HA)-rich extracellular matrices. Results: Inter-α-inhibitor (IαI) affects HA-TSG-6 interactions and enhancement of cell adhesion while promoting covalent complex formation between IαI heavy chains and HA.
Clostridium perfringens enterotoxin (CPE) is a major cause of food poisoning and antibiotic-assoc... more Clostridium perfringens enterotoxin (CPE) is a major cause of food poisoning and antibiotic-associated diarrhea. Upon its release from C. perfringens spores, CPE binds to its receptor, claudin, at the tight junctions between the epithelial cells of the gut wall and subsequently forms pores in the cell membranes. A number of different complexes between CPE and claudin have been observed, and the process of pore formation has not been fully elucidated. We have determined the three-dimensional structure of the soluble form of CPE in two crystal forms by X-ray crystallography, to a resolution of 2.7 and 4.0 Å, respectively, and found that the N-terminal domain shows structural homology with the aerolysin-like β-pore-forming family of proteins. We show that CPE forms a trimer in both crystal forms and that this trimer is likely to be biologically relevant but is not the active pore form. We use these data to discuss models of pore formation.
Tumor necrosis factor-stimulated gene-6 (TSG-6) is a hyaluronan (HA)-binding protein that plays i... more Tumor necrosis factor-stimulated gene-6 (TSG-6) is a hyaluronan (HA)-binding protein that plays important roles in inflammation and ovulation. TSG-6 mediated cross-linking of HA has been proposed as a functional mechanism (e.g. for regulating leukocyte adhesion), but direct evidence for cross-linking is lacking, and we know very little about its impact on HA ultrastructure. Here we used films of polymeric and oligomeric HA chains, end-grafted to a solid support, and a combination of surface-sensitive biophysical techniques, to quantify the binding of TSG-6 into HA films, and to correlate binding to morphological changes. We find that full length TSG-6 binds with pronounced positive cooperativity, and demonstrate that it can cross-link HA at physiologically relevant concentrations. Our data indicates that cooperative binding of full length TSG-6 arises from HA-induced protein oligomerization, and that the TSG-6 oligomers act as cross-linkers. In contrast, the HA-binding domain of TSG-6 (the Link module) alone binds without positive cooperativity, and weaker than the full-length protein. Both Link module and full length TSG-6 condensed and rigidified HA films, and the degree of condensation scaled with the affinity between the TSG-6 constructs and HA. We propose that condensation is the result of protein-mediated HA cross-linking. Our findings firmly establish that TSG-6 is a potent HA cross-linking agent and might hence have important implications for the mechanistic understanding of the biological function of TSG-6, e.g. in inflammation.
Actin related proteins (Arps) are a highly conserved family of proteins that have extensive seque... more Actin related proteins (Arps) are a highly conserved family of proteins that have extensive sequence and structural similarity to actin. All characterized Arps are components of large multimeric complexes associated with chromatin or the cytoskeleton. In addition, the human genome encodes five conserved but largely uncharacterized ″orphan″ Arps, which appear to be mostly testis specific. Here we show that Arp7A, which has 43% sequence identity with β-actin, forms a complex with the cytoskeletal proteins Tes and Mena in the subacrosomal layer of round spermatids. The N-terminal 65 residue extension to the actin-like fold of Arp7A interacts directly with Tes. The crystal structure of the 1-65Arp7A:LIM2-3Tes:EVH1Mena complex reveals that residues 28 - 49 of Arp7A contact the LIM2-3 domains of Tes. Two alanine residues from Arp7A that occupy equivalent apolar pockets in both LIM domains as well as an intervening GPAK linker that binds the LIM2-3 junction, are critical for the Arp7A:Tes interaction. Equivalent occupied apolar pockets are also seen in the tandem LIM domain structures of LMO4 and Lhx3 bound to unrelated ligands. Our results indicate that apolar pocket interactions are a common feature of tandem LIM domain interactions, but ligand specificity is principally determined by the linker sequence.
Clostridium perfringens is a Gram-positive anaerobic species of bacterium that is notable for its... more Clostridium perfringens is a Gram-positive anaerobic species of bacterium that is notable for its ability to produce a plethora of toxins, including membrane-active toxins (alpha-toxins), pore-forming toxins (-toxins) and binary toxins (iota-toxins). Here, the crystallization of the full-length wild-type C. perfringens enterotoxin is reported, which is the causative agent of the second most prevalent food-borne illness in the United States and has been implicated in many other gastrointestinal pathologies. Several crystal forms were obtained. However, only two of these optimized crystal forms (I and II) were useable for X-ray diffraction data collection. The form I crystals diffracted to d(min) = 2.7 A and belonged to space group C2, while the form II crystals diffracted to d(min) = 4 A and belonged to space group P2(1)3.
The inflammation-associated long pentraxin PTX3 plays key roles in innate immunity, female fertil... more The inflammation-associated long pentraxin PTX3 plays key roles in innate immunity, female fertility and vascular biology; e.g. it inhibits fibroblast growth factor 2 (FGF2)-mediated angiogenesis. PTX3 is composed of multiple protomers, each comprised of distinct N- and C-terminal domains, however, it is not known how these are organized or contribute to its functional properties. Here, biophysical analyses reveal that PTX3 is composed of eight identical protomers, associated through disulfide bonds, forming an elongated, and asymmetric, molecule with two differently sized domains interconnected by a stalk. The N-terminal region of the protomer provides the main structural determinant underlying this quaternary organization, supporting formation of a disulfide-linked tetramer and a dimer of dimers (a non-covalent tetramer) giving rise to the asymmetry of the molecule. Furthermore, the PTX3 octamer is shown to contain two FGF2 binding sites, where it is the tetramers that act as the functional units in ligand recognition. Thus, these studies provide a unifying model of the PTX3 oligomer explaining both its quaternary organization and how this is required for its anti-angiogenic function.
Reactive oxygen species trigger cellular responses by activation of stress-responsive mitogen-act... more Reactive oxygen species trigger cellular responses by activation of stress-responsive mitogen-activated protein kinase (MAPK) signalling pathways. Reversal of MAPK activation requires the transcriptional induction of specialized cysteine-based phosphatases that mediate MAPK dephosphorylation. Paradoxically, oxidative stresses generally inactivate cysteine-based phosphatases by thiol modification and thus could lead to sustained or uncontrolled MAPK activation. Here we describe how the stress-inducible MAPK phosphatase, Sdp1, presents an unusual solution to this apparent paradox by acquiring enhanced catalytic activity under oxidative conditions. Structural and biochemical evidence reveals that Sdp1 employs an intramolecular disulphide bridge and an invariant histidine side chain to selectively recognize a tyrosine-phosphorylated MAPK substrate. Optimal activity critically requires the disulphide bridge, and thus, to the best of our knowledge, Sdp1 is the first example of a cysteine-dependent phosphatase that couples oxidative stress with substrate recognition. We show that Sdp1, and its paralogue Msg5, have similar properties and belong to a new group of phosphatases unique to yeast and fungal taxa.
The intracellular targeting of Ena/VASP family members is achieved via the interaction of their E... more The intracellular targeting of Ena/VASP family members is achieved via the interaction of their EVH1 domain with FPPPP sequence motifs found in a variety of cytoskeletal proteins, including lamellipodin, vinculin, and zyxin. Here we show that the LIM3 domain of Tes, which lacks the FPPPP motif, binds to the EVH1 domain of Mena, but not to those of VASP or Evl. The structure of the LIM3:EVH1 complex reveals that Tes occludes the FPPPP-binding site and competes with FPPPP-containing proteins for EVH1 binding. Structure-based gain-of-function experiments define the molecular basis for the specificity of the Tes-Mena interaction. Consistent with in vitro observations, the LIM3 domain displaces Mena, but not VASP, from the leading edge and focal adhesions. It also regulates cell migration through a Mena-dependent mechanism. Our observations identify Tes as an atypical EVH1 binding partner and a regulator specific to a single Ena/VASP family member.
The staphylococcal superantigen-like proteins (SSLs) are a family of polymorphic paralogs encoded... more The staphylococcal superantigen-like proteins (SSLs) are a family of polymorphic paralogs encoded in the Staphylococcus aureus genome whose function is unknown. The crystal structure of SSL7 was determined and compared to that of SSL5 and that of a classical superantigen, streptococcal pyrogenic exotoxin. Although the overall architecture of the superantigen family is retained in both SSL7 and SSL5, there are significant differences in the structures which suggest that the characteristic major histocompatibility complex binding site of superantigens has been lost. To complement these data, the abilities of SSL7 and a closely related paralog, SSL9, to interact with cells of the immune system were investigated. In populations of human white blood cells, both SSLs interacted selectively with monocytes via specific saturable but separate binding sites, which led to rapid uptake of the SSLs. In addition, SSLs were rapidly taken up by dendritic cells, but not by macrophages, into the same endosomal compartment as dextran. The ability of these secreted proteins to target antigen-presenting cells may enhance a misplaced antibody response against the proteins, which may facilitate bacterial colonization rather than contribute to host protection. Like classical superantigens, therefore, SSLs may distract the host's immune system, but they may do so via entirely different molecular mechanisms.
Clostridium absonum phospholipase C (Caa) is a 42.7 kDa protein, which shows 60% amino acid seque... more Clostridium absonum phospholipase C (Caa) is a 42.7 kDa protein, which shows 60% amino acid sequence identity with the Clostridium perfringens phospholipase C, or alpha-toxin (Cpa), and has been isolated from patients suffering from gas gangrene. We report the cloning and sequencing, purification, characterisation and crystal structure of the Caa enzyme. Caa had twice the phospholipid-hydrolysing (lecithinase) activity, 1.5 times the haemolytic activity and over seven times the activity towards phosphatidylcholine-based liposomes when compared with Cpa. However, the Caa enzyme had a lower activity than Cpa to the free (i.e. not in lipid bilayer) substrate para-nitrophenylphosphorylcholine, towards sphingomyelin-based liposomes and showed half the cytotoxicity. The lethal dose (LD(50)) of Caa in mice was approximately twice that of Cpa. The crystal structure of Caa shows that the 72-93 residue loop is in a conformation different from those of previously determined open-form alpha-toxin structures. This conformational change suggests a role for W84 in membrane binding and a possible route of entry into the active site along a hydrophobic channel created by the re-arrangement of this loop. Overall, the properties of Caa are compatible with a role as a virulence-determinant in gas gangrene caused by C.absonum.
Dystroglycan is a highly glycosylated extracellular matrix receptor with essential functions in s... more Dystroglycan is a highly glycosylated extracellular matrix receptor with essential functions in skeletal muscle and the nervous system. Reduced matrix binding by α-dystroglycan (α-DG) due to perturbed glycosylation is a pathological feature of several forms of muscular dystrophy. Like-acetylglucosaminyltransferase (LARGE) synthesizes the matrix-binding heteropolysaccharide [-glucuronic acid-β1,3-xylose-α1,3-]n. Using a dual exoglycosidase digestion, we confirm that this polysaccharide is present on native α-DG from skeletal muscle. The atomic details of matrix binding were revealed by a high-resolution crystal structure of laminin-G-like (LG) domains 4 and 5 (LG4 and LG5) of laminin-α2 bound to a LARGE-synthesized oligosaccharide. A single glucuronic acid-β1,3-xylose disaccharide repeat straddles a Ca2+ ion in the LG4 domain, with oxygen atoms from both sugars replacing Ca2+-bound water molecules. The chelating binding mode accounts for the high affinity of this protein–carbohydrate interaction. These results reveal a previously uncharacterized mechanism of carbohydrate recognition and provide a structural framework for elucidating the mechanisms underlying muscular dystrophy.
Laminins are a major constituent of all basement membranes. The polymerisation of laminins at the... more Laminins are a major constituent of all basement membranes. The polymerisation of laminins at the cell surface is mediated by the three short arms of the cross-shaped laminin heterotrimer. The short arms contain repeats of laminin-type epidermal growth factor-like (LE) domains, interspersed with globular domains of unknown function. A single LF domain is inserted between LE5 and LE6 of the laminin β1 and β2 chains. We report the crystal structure at 1.85Å resolution of the laminin β2 LE5-LF-LE6 region. The LF domain consists of a β-sandwich related to bacterial family 35 carbohydrate binding modules, and more distantly to the L4 domains present in the short arms of laminin α and γ chains. An α-helical region mediates the extensive interaction of the LF domain with LE5. The relative arrangement of LE5 and LE6 is very similar to that of consecutive LE domains in uninterrupted LE tandems. Fitting atomic models to a low-resolution structure of the first eight domains of the laminin β1 chain determined by small-angle X-ray scattering suggests a deviation from the regular LE array at the LE4-LE5 junction. These results advance our understanding of laminin structure.
The C-type mannose receptor and its homolog Endo180 (or uPARAP, for urokinase plasminogen activat... more The C-type mannose receptor and its homolog Endo180 (or uPARAP, for urokinase plasminogen activator receptor-associated protein) mediate the endocytic uptake of collagen by macrophages and fibroblasts. This process is required for normal tissue remodeling, but also facilitates the growth and dissemination of tumors. We have determined the crystal structure at 2.5 Å resolution of the N-terminal region of Endo180, consisting of a ricin-like domain, a fibronectin type II (FN2) domain, and two C-type lectin (CTL) domains. The L-shaped arrangement of these domains creates a shallow trench spanning the FN2 and CTL1 domains, which was shown by mutagenesis to bind triple-helical and denatured collagen. Small-angle X-ray scattering showed that the L-shaped structure is maintained in solution at neutral and acidic pH, irrespective of calcium ion loading. Collagen binding was equally unaffected by acidic pH, suggesting that collagen release in endosomes is not regulated by changes within the Endo180 N-terminal region.
Background: TSG-6 (TNF-stimulated gene-6)-dependent transfer of heavy chains from inter-α-inhibit... more Background: TSG-6 (TNF-stimulated gene-6)-dependent transfer of heavy chains from inter-α-inhibitor onto hyaluronan is critical for ovulation.
Results: A calcium ion and chelating glutamate within TSG-6 mediate formation of the covalent heavy chain-TSG-6 intermediate.
Conclusion: TSG-6 transferase activity rather than hyaluronan binding drives cumulus expansion.
Significance: The role of metal ions in hyaluronan-heavy chain formation has been determined.
Background: The proteins pentraxin 3 (PTX3) and TNF-stimulated gene-6 (TSG-6) and the proteoglyca... more Background: The proteins pentraxin 3 (PTX3) and TNF-stimulated gene-6 (TSG-6) and the proteoglycan inter-α-inhibitor (IαI) are known to be involved in the stabilization of hyaluronan (HA)-rich extracellular matrices.
Results: PTX3 incorporation into HA matrices is tightly regulated.
Conclusion: PTX3, TSG-6, and IαI are sufficient to cross-link HA matrices.
Significance: The results provide mechanistic insights into the regulation of HA-protein interactions.
Background: The polysaccharide hyaluronan is organized through interactions with the protein TSG-... more Background: The polysaccharide hyaluronan is organized through interactions with the protein TSG-6 during inflammation and ovulation. Results: NMR spectroscopy on TSG-6 in the presence of defined sugars provided restraints that allowed modeling of a refined hyaluronan/TSG-6 complex. Conclusion: TSG-6 binding causes bending of hyaluronan that explains its condensation of this polysaccharide.
Background: Inflammation/ovulation-associated protein TSG-6 performs multiple functions in hyalur... more Background: Inflammation/ovulation-associated protein TSG-6 performs multiple functions in hyaluronan (HA)-rich extracellular matrices. Results: Inter-α-inhibitor (IαI) affects HA-TSG-6 interactions and enhancement of cell adhesion while promoting covalent complex formation between IαI heavy chains and HA.
Clostridium perfringens enterotoxin (CPE) is a major cause of food poisoning and antibiotic-assoc... more Clostridium perfringens enterotoxin (CPE) is a major cause of food poisoning and antibiotic-associated diarrhea. Upon its release from C. perfringens spores, CPE binds to its receptor, claudin, at the tight junctions between the epithelial cells of the gut wall and subsequently forms pores in the cell membranes. A number of different complexes between CPE and claudin have been observed, and the process of pore formation has not been fully elucidated. We have determined the three-dimensional structure of the soluble form of CPE in two crystal forms by X-ray crystallography, to a resolution of 2.7 and 4.0 Å, respectively, and found that the N-terminal domain shows structural homology with the aerolysin-like β-pore-forming family of proteins. We show that CPE forms a trimer in both crystal forms and that this trimer is likely to be biologically relevant but is not the active pore form. We use these data to discuss models of pore formation.
Tumor necrosis factor-stimulated gene-6 (TSG-6) is a hyaluronan (HA)-binding protein that plays i... more Tumor necrosis factor-stimulated gene-6 (TSG-6) is a hyaluronan (HA)-binding protein that plays important roles in inflammation and ovulation. TSG-6 mediated cross-linking of HA has been proposed as a functional mechanism (e.g. for regulating leukocyte adhesion), but direct evidence for cross-linking is lacking, and we know very little about its impact on HA ultrastructure. Here we used films of polymeric and oligomeric HA chains, end-grafted to a solid support, and a combination of surface-sensitive biophysical techniques, to quantify the binding of TSG-6 into HA films, and to correlate binding to morphological changes. We find that full length TSG-6 binds with pronounced positive cooperativity, and demonstrate that it can cross-link HA at physiologically relevant concentrations. Our data indicates that cooperative binding of full length TSG-6 arises from HA-induced protein oligomerization, and that the TSG-6 oligomers act as cross-linkers. In contrast, the HA-binding domain of TSG-6 (the Link module) alone binds without positive cooperativity, and weaker than the full-length protein. Both Link module and full length TSG-6 condensed and rigidified HA films, and the degree of condensation scaled with the affinity between the TSG-6 constructs and HA. We propose that condensation is the result of protein-mediated HA cross-linking. Our findings firmly establish that TSG-6 is a potent HA cross-linking agent and might hence have important implications for the mechanistic understanding of the biological function of TSG-6, e.g. in inflammation.
Actin related proteins (Arps) are a highly conserved family of proteins that have extensive seque... more Actin related proteins (Arps) are a highly conserved family of proteins that have extensive sequence and structural similarity to actin. All characterized Arps are components of large multimeric complexes associated with chromatin or the cytoskeleton. In addition, the human genome encodes five conserved but largely uncharacterized ″orphan″ Arps, which appear to be mostly testis specific. Here we show that Arp7A, which has 43% sequence identity with β-actin, forms a complex with the cytoskeletal proteins Tes and Mena in the subacrosomal layer of round spermatids. The N-terminal 65 residue extension to the actin-like fold of Arp7A interacts directly with Tes. The crystal structure of the 1-65Arp7A:LIM2-3Tes:EVH1Mena complex reveals that residues 28 - 49 of Arp7A contact the LIM2-3 domains of Tes. Two alanine residues from Arp7A that occupy equivalent apolar pockets in both LIM domains as well as an intervening GPAK linker that binds the LIM2-3 junction, are critical for the Arp7A:Tes interaction. Equivalent occupied apolar pockets are also seen in the tandem LIM domain structures of LMO4 and Lhx3 bound to unrelated ligands. Our results indicate that apolar pocket interactions are a common feature of tandem LIM domain interactions, but ligand specificity is principally determined by the linker sequence.
Clostridium perfringens is a Gram-positive anaerobic species of bacterium that is notable for its... more Clostridium perfringens is a Gram-positive anaerobic species of bacterium that is notable for its ability to produce a plethora of toxins, including membrane-active toxins (alpha-toxins), pore-forming toxins (-toxins) and binary toxins (iota-toxins). Here, the crystallization of the full-length wild-type C. perfringens enterotoxin is reported, which is the causative agent of the second most prevalent food-borne illness in the United States and has been implicated in many other gastrointestinal pathologies. Several crystal forms were obtained. However, only two of these optimized crystal forms (I and II) were useable for X-ray diffraction data collection. The form I crystals diffracted to d(min) = 2.7 A and belonged to space group C2, while the form II crystals diffracted to d(min) = 4 A and belonged to space group P2(1)3.
The inflammation-associated long pentraxin PTX3 plays key roles in innate immunity, female fertil... more The inflammation-associated long pentraxin PTX3 plays key roles in innate immunity, female fertility and vascular biology; e.g. it inhibits fibroblast growth factor 2 (FGF2)-mediated angiogenesis. PTX3 is composed of multiple protomers, each comprised of distinct N- and C-terminal domains, however, it is not known how these are organized or contribute to its functional properties. Here, biophysical analyses reveal that PTX3 is composed of eight identical protomers, associated through disulfide bonds, forming an elongated, and asymmetric, molecule with two differently sized domains interconnected by a stalk. The N-terminal region of the protomer provides the main structural determinant underlying this quaternary organization, supporting formation of a disulfide-linked tetramer and a dimer of dimers (a non-covalent tetramer) giving rise to the asymmetry of the molecule. Furthermore, the PTX3 octamer is shown to contain two FGF2 binding sites, where it is the tetramers that act as the functional units in ligand recognition. Thus, these studies provide a unifying model of the PTX3 oligomer explaining both its quaternary organization and how this is required for its anti-angiogenic function.
Reactive oxygen species trigger cellular responses by activation of stress-responsive mitogen-act... more Reactive oxygen species trigger cellular responses by activation of stress-responsive mitogen-activated protein kinase (MAPK) signalling pathways. Reversal of MAPK activation requires the transcriptional induction of specialized cysteine-based phosphatases that mediate MAPK dephosphorylation. Paradoxically, oxidative stresses generally inactivate cysteine-based phosphatases by thiol modification and thus could lead to sustained or uncontrolled MAPK activation. Here we describe how the stress-inducible MAPK phosphatase, Sdp1, presents an unusual solution to this apparent paradox by acquiring enhanced catalytic activity under oxidative conditions. Structural and biochemical evidence reveals that Sdp1 employs an intramolecular disulphide bridge and an invariant histidine side chain to selectively recognize a tyrosine-phosphorylated MAPK substrate. Optimal activity critically requires the disulphide bridge, and thus, to the best of our knowledge, Sdp1 is the first example of a cysteine-dependent phosphatase that couples oxidative stress with substrate recognition. We show that Sdp1, and its paralogue Msg5, have similar properties and belong to a new group of phosphatases unique to yeast and fungal taxa.
The intracellular targeting of Ena/VASP family members is achieved via the interaction of their E... more The intracellular targeting of Ena/VASP family members is achieved via the interaction of their EVH1 domain with FPPPP sequence motifs found in a variety of cytoskeletal proteins, including lamellipodin, vinculin, and zyxin. Here we show that the LIM3 domain of Tes, which lacks the FPPPP motif, binds to the EVH1 domain of Mena, but not to those of VASP or Evl. The structure of the LIM3:EVH1 complex reveals that Tes occludes the FPPPP-binding site and competes with FPPPP-containing proteins for EVH1 binding. Structure-based gain-of-function experiments define the molecular basis for the specificity of the Tes-Mena interaction. Consistent with in vitro observations, the LIM3 domain displaces Mena, but not VASP, from the leading edge and focal adhesions. It also regulates cell migration through a Mena-dependent mechanism. Our observations identify Tes as an atypical EVH1 binding partner and a regulator specific to a single Ena/VASP family member.
The staphylococcal superantigen-like proteins (SSLs) are a family of polymorphic paralogs encoded... more The staphylococcal superantigen-like proteins (SSLs) are a family of polymorphic paralogs encoded in the Staphylococcus aureus genome whose function is unknown. The crystal structure of SSL7 was determined and compared to that of SSL5 and that of a classical superantigen, streptococcal pyrogenic exotoxin. Although the overall architecture of the superantigen family is retained in both SSL7 and SSL5, there are significant differences in the structures which suggest that the characteristic major histocompatibility complex binding site of superantigens has been lost. To complement these data, the abilities of SSL7 and a closely related paralog, SSL9, to interact with cells of the immune system were investigated. In populations of human white blood cells, both SSLs interacted selectively with monocytes via specific saturable but separate binding sites, which led to rapid uptake of the SSLs. In addition, SSLs were rapidly taken up by dendritic cells, but not by macrophages, into the same endosomal compartment as dextran. The ability of these secreted proteins to target antigen-presenting cells may enhance a misplaced antibody response against the proteins, which may facilitate bacterial colonization rather than contribute to host protection. Like classical superantigens, therefore, SSLs may distract the host's immune system, but they may do so via entirely different molecular mechanisms.
Clostridium absonum phospholipase C (Caa) is a 42.7 kDa protein, which shows 60% amino acid seque... more Clostridium absonum phospholipase C (Caa) is a 42.7 kDa protein, which shows 60% amino acid sequence identity with the Clostridium perfringens phospholipase C, or alpha-toxin (Cpa), and has been isolated from patients suffering from gas gangrene. We report the cloning and sequencing, purification, characterisation and crystal structure of the Caa enzyme. Caa had twice the phospholipid-hydrolysing (lecithinase) activity, 1.5 times the haemolytic activity and over seven times the activity towards phosphatidylcholine-based liposomes when compared with Cpa. However, the Caa enzyme had a lower activity than Cpa to the free (i.e. not in lipid bilayer) substrate para-nitrophenylphosphorylcholine, towards sphingomyelin-based liposomes and showed half the cytotoxicity. The lethal dose (LD(50)) of Caa in mice was approximately twice that of Cpa. The crystal structure of Caa shows that the 72-93 residue loop is in a conformation different from those of previously determined open-form alpha-toxin structures. This conformational change suggests a role for W84 in membrane binding and a possible route of entry into the active site along a hydrophobic channel created by the re-arrangement of this loop. Overall, the properties of Caa are compatible with a role as a virulence-determinant in gas gangrene caused by C.absonum.
Uploads
Results: A calcium ion and chelating glutamate within TSG-6 mediate formation of the covalent heavy chain-TSG-6 intermediate.
Conclusion: TSG-6 transferase activity rather than hyaluronan binding drives cumulus expansion.
Significance: The role of metal ions in hyaluronan-heavy chain formation has been determined.
Results: PTX3 incorporation into HA matrices is tightly regulated.
Conclusion: PTX3, TSG-6, and IαI are sufficient to cross-link HA matrices.
Significance: The results provide mechanistic insights into the regulation of HA-protein interactions.
Results: Inter-α-inhibitor (IαI) affects HA-TSG-6 interactions and enhancement of cell adhesion while promoting covalent complex formation between IαI heavy chains and HA.
Results: A calcium ion and chelating glutamate within TSG-6 mediate formation of the covalent heavy chain-TSG-6 intermediate.
Conclusion: TSG-6 transferase activity rather than hyaluronan binding drives cumulus expansion.
Significance: The role of metal ions in hyaluronan-heavy chain formation has been determined.
Results: PTX3 incorporation into HA matrices is tightly regulated.
Conclusion: PTX3, TSG-6, and IαI are sufficient to cross-link HA matrices.
Significance: The results provide mechanistic insights into the regulation of HA-protein interactions.
Results: Inter-α-inhibitor (IαI) affects HA-TSG-6 interactions and enhancement of cell adhesion while promoting covalent complex formation between IαI heavy chains and HA.