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A K Downing

Publications and source records attributed to A K Downing.

At least 19 recordsLinked to original sources
PubMed1999

EGF-like domain calcium affinity modulated by N-terminal domain linkage in human fibrillin-1.

Calcium binding epidermal growth factor-like domains (cbEGFs) are present in many extracellular proteins, including fibrillin-1, Notch-3, protein S, factor IX and the low density lipoprotein (LDL) receptor, which perform a diverse range of functions. Genetic mutations that cause amino acid changes within these proteins have been linked to the Marfan syndrome (MFS), CADASIL, protein S deficiency, haemophilia B and familial hypercholesterolaemia, respectively. A number of these mutations disrupt calcium binding to cbEGFs, emphasising the critical functional role of calcium in these proteins. We have determined the calcium binding affinity of two sites within a cbEGF pair (cbEGF12-13) from human fibrillin-1 using two-dimensional nuclear magnetic resonance (NMR) and fluorescence techniques. Fibrillin-1 is a mosaic protein containing 43 cbEGF domains, mainly arranged as tandem repeats. Our results show that the cbEGF13 site in the cbEGF12-13 pair possesses the highest calcium affinity of any cbEGF investigated from fibrillin-1. A comparative analysis of these and previously reported calcium binding data from fibrillin-1 demonstrate that the affinity of cbEGF13 is enhanced more than 70-fold by the linkage of an N-terminal cbEGF domain. In contrast, comparison of calcium binding by cbEGF32 in isolation relative to when linked to a transforming growth factor beta-binding protein-like domain (TB6-cbEGF32) reveals that the same enhancement is not observed for this heterologous domain pair. Taken together, these results indicate that fibrillin-1 cbEGF Ca2+ affinity can be significantly modulated by the type of domain which is linked to its N terminus. The cbEGF12-13 pair is located within the longest contiguous section of cbEGFs in fibrillin-1, and a number of mutations in this region are associated with the most severe neonatal form of MFS. The affinities of cbEGF domains 13 and 14 in this region are substantially higher than in the C-terminal region of fibrillin-1. This increased affinity may be important for fibrillin assembly into 10-12 nm connective tissue microfibrils and/or may contribute to the biomechanical properties of the microfibrillar network.

Binding Sites↗
PubMed1999

Defective calcium binding to fibrillin-1: consequence of an N2144S change for fibrillin-1 structure and function.

Fibrillin-1 is a major structural component of 10-12 nm connective tissue microfibrils and has a modular organisation that includes 43 calcium binding epidermal growth factor-like (cbEGF) domains and seven transforming growth factor beta-binding protein-like (TB) domains. Mutations in the fibrillin-1 (FBN1) gene cause the Marfan syndrome (MFS) and related connective tissue disorders. We have previously investigated an N2144S change, identified in a MFS patient, which removes one of the key calcium binding ligands within cbEGF domain 32. In this study the structural consequences of the N2144S amino acid change for the folding and calcium binding properties of mutant and wild-type TB6-cbEGF32 and cbEGF32-33 domain pairs have been analysed by nuclear magnetic resonance. The presence of an N2144S substitution does not alter the native fold of either the TB6 domain, or cbEGF domains 32 and 33. Comparison of calcium dissociation constants measured for the wild-type and mutant pairs shows that: (i) the affinity of cbEGF32 is weakly enhanced by N-terminal linkage of TB6 relative to cbEGF32 in isolation; (ii) the affinity of cbEGF32 is approximately ninefold decreased by the N2144S substitution in the TB-cbEGF pair; and (iii) reduced affinity of cbEGF32 does not result in lower affinity of cbEGF33 for calcium. Together, these data suggest that the TB6-cbEGF32 linkage is flexible and the structural effect of the mutation is localised to the interdomain linkage. We have also investigated the effect of defective calcium binding to cbEGF32 on fibrillin-1 produced by N2144S MFS fibroblasts. 35S-pulse-chase analysis shows that the N2144S substitution does not detectably affect fibrillin-1 biosynthesis, rate of secretion or processing. Deposition of reducible fibrillin-1 into the extracellular matrix was also unaffected. The implications of these results for the assembly and properties of the microfibril are discussed.

Actin Cytoskeleton↗
PubMed1998

A Gly --> Ser change causes defective folding in vitro of calcium-binding epidermal growth factor-like domains from factor IX and fibrillin-1.

The calcium-binding epidermal growth factor-like (cbEGF) domain is a common motif found in extracellular proteins. A mutation that changes a highly conserved Gly residue to Ser in this domain has been identified both in the factor IX (FIX) and fibrillin-1 genes, where it is associated with relatively mild variants of hemophilia B and Marfan syndrome, respectively. We have investigated the structural consequences in vitro of this amino acid change when introduced into single cbEGF domains from human FIX (G60S) and human fibrillin-1 (G1127S), and a covalently linked pair of cbEGF domains from fibrillin-1. High pressure liquid chromatography analysis, mass spectrometry, and 1H NMR analysis demonstrate that wild-type cbEGF domains purified in the reduced form and refolded in vitro adopt the native fold. In contrast, the Gly --> Ser change causes defective folding of FIX and fibrillin-1 cbEGF domains. However, in the case of the factor IX mutant domain, a Ca2+-dependent change in conformation, identified by NMR in a proportion of the refolded material, suggests that some material refolds to a native-like structure. This is consistent with enzyme-linked immunosorbent assay analysis of FIX G60S from a hemophilia B patient Oxford d2, which demonstrates that the mutant protein is partially recognized by a monoclonal antibody specific for this region of FIX. NMR analysis of a covalently linked pair of fibrillin cbEGF domains demonstrates that the C-terminal domain adopts the native epidermal growth factor fold, despite the fact that the adjacent mutant domain is misfolded. The implications of these results for disease pathogenesis are discussed.

Amino Acid Substitution↗
PubMed1998

NMR of modular proteins.

NMR studies of domains, dissected from large modular proteins, are described. Particular emphasis is placed on modules from the extracellular proteins fibrillin-1 and fibronectin.

Amino Acid Sequence↗
PubMed1998

NMR analysis of cbEGF domains gives new insights into the structural consequences of a P1148A substitution in fibrillin-1.

Fibrillin-1 is a modular glycoprotein and a major component of the 10-12 nm microfibrils of the extracellular matrix. Mutations in the fibrillin-1 (FBN 1) gene result in the connective tissue disease the Marfan syndrome (MFS) and related disorders. The calcium binding EGF-like (cbEGF) domain is the predominant structural motif of the protein and >70% of mutations leading to MFS disrupt this domain. A missense mutation which changes a proline to alanine (P1148A) in cbEGF domain 13 has been associated with a number of fibrillin disorders including MFS and Shprintzen-Goldberg syndrome. However, it has also been described as a polymorphism. In this study comparative NMR analyses on wild-type and mutant forms of covalently-linked fibrillin cbEGF domain pairs have been performed to investigate the structural consequences of this substitution. A comparison of the two-dimensional NOESY spectra of the wild-type and mutant forms of cbEGF domains 12 & 13 and cbEGF domains 13 & 14 indicated that the proline to alanine amino acid change does not introduce a significant structural defect into cbEGF domain 13 or the adjacent domains and most likely represents a polymorphism. These results demonstrate how, in the case of a protein with a well defined domain organisation such as fibrillin-1, comparative NMR analyses can be used to substantiate genetic evidence for the polymorphic status of an amino acid.

Alanine↗
PubMed1997

Solution structure of the transforming growth factor beta-binding protein-like module, a domain associated with matrix fibrils.

Here we describe the high resolution nuclear magnetic resonance (NMR) structure of a transforming growth factor beta (TGF-beta)-binding protein-like (TB) domain, which comes from human fibrillin-1, the protein defective in the Marfan syndrome (MFS). This domain is found in fibrillins and latent TGF-beta-binding proteins (LTBPs) which are localized to fibrillar structures in the extracellular matrix. The TB domain manifests a novel fold which is globular and comprises six antiparallel beta-strands and two alpha-helices. An unusual cysteine triplet conserved in the sequences of TB domains is localized to the hydrophobic core, at the C-terminus of an alpha-helix. The structure is stabilized by four disulfide bonds which pair in a 1-3, 2-6, 4-7, 5-8 pattern, two of which are solvent exposed. Analyses of MFS-causing mutations and the fibrillin-1 cell-binding RGD site provide the first clues to the surface specificity of TB domain interactions. Modelling of a homologous TB domain from LTBP-1 (residues 1018-1080) suggests that hydrophobic contacts may play a role in its interaction with the TGF-beta1 latency-associated peptide.

Amino Acid Sequence↗
PubMed1996

Solution structure of a pair of calcium-binding epidermal growth factor-like domains: implications for the Marfan syndrome and other genetic disorders.

The nuclear magnetic resonance structure of a covalently linked pair of calcium-binding (cb) epidermal growth factor-like (EGF) domains from human fibrillin-1, the protein defective in the Marfan syndrome, is described. The two domains are in a rigid, rod-like arrangement, stabilized by interdomain calcium binding and hydrophobic interactions. We propose a model for the arrangement of fibrillin monomers in microfibrils that reconciles structural and antibody binding data, and we describe a set of disease-causing mutations that provide the first clues to the specificity of cbEFG interactions. The residues involved in stabilizing the domain linkage are highly conserved in fibrillin, fibulin, thrombomodulin, and the low density lipoprotein receptor. We propose that the relative orientation of tandem cbEGF domains in these proteins is similar, but that in others, including Notch, pairs adopt a completely different conformation.

Calcium-Binding Proteins↗
PubMed1996

Calcium binding properties of an epidermal growth factor-like domain pair from human fibrillin-1.

Ca2+ binding epidermal growth factor-like (EGF-like) domains are found in a large number of extracellular proteins with diverse functions, including those involved in blood coagulation, determination of cell fate, cell adhesion and connective tissue architecture. Their importance is emphasised by the identification of mutations in these domains in patients with haemophilia B (defective in coagulation factor IX) and the Marfan syndrome (defective in the connective tissue protein fibrillin-1). The X-ray crystal structure of a single Ca2+ binding EGF-like domain from human coagulation factor IX has recently been solved. It shows that the Ca2+ ligands form a pentagonal bipyramid, where one ligand is provided by an adjacent (N-terminal) EGF-like domain in the crystal. The N and C termini of the neighbouring domains are only approximately 4 angstrum apart, hence the crystal packing has been proposed as a model for the association of contiguous EGF-like domains in proteins. Since the adjacent EGF-like domain in the crystal, although close, is not covalently linked to its neighbour, this model requires verification. In this study we have expressed and purified a Ca2+ binding EGF-like domain pair from human fibrillin-1 and used an in vitro refolding system to obtain protein with the correct EGF fold. The Ca2+ binding properties of the protein have been investigated by two-dimensional NMR. The affinity of the C-terminal domain for Ca2+ is approximately 25-fold higher than that of the N-terminal domain, consistent with the two Ca2+ binding sites having different local environments. In addition, these data provide the first direct experimental evidence that Ca2+ plays a major role in defining the interdomain linkage in multiple repeats of Ca2+ binding EGF-like domains.

Amino Acid Sequence↗
PubMed1995

The solution structure and backbone dynamics of the fibronectin type I and epidermal growth factor-like pair of modules of tissue-type plasminogen activator.

BACKGROUND: The thrombolytic serine protease tissue-type plasminogen activator (t-PA) is a classical modular protein consisting of three types of domain in addition to the serine protease domain: F1 (homologous to fibronectin type I); G (epidermal growth factor-like) and kringle. Biochemical data suggest that the F1 and G modules play a major role in the binding of t-PA to fibrin and to receptors on hepatocytes. RESULTS: We have derived the solution structure of the F1 and G pair of modules from t-PA by two- and three-dimensional NMR techniques, in combination with dynamical simulated annealing calculations. We have also obtained information about the molecule's backbone dynamics through measurement of amide 15N relaxation parameters. CONCLUSIONS: Although the F1 and G modules each adopt their expected tertiary structure, the modules interact intimately to bury a hydrophobic core, and the inter-module linker makes up the third strand of the G module's major beta-sheet. The new structural results allow the interpretation of earlier mutational data relevant to fibrin-binding and hepatocyte-receptor binding.

Amino Acid Sequence↗
PubMed1995

The effects of variable glycosylation on the functional activities of ribonuclease, plasminogen and tissue plasminogen activator.

The relatively large size and dynamics of oligosaccharides can result in substantial shielding of functionally important areas of proteins to which they are attached, modulate the interactions of glycoconjugates with other molecules and affect the rate of processes which involve conformational changes. This review focuses on the occupancy of N-linked glycosylation sites on three enzymes, ribonuclease, plasminogen and tissue plasminogen activator. Each of these proteins occurs naturally as two populations of molecules, distinguished from each other only by the presence or absence of an oligosaccharide at one glycosylation site. The presence of an oligomannose sugar on ribonuclease (at Asn-34) alters its overall dynamics, increases its stability towards proteinases and decreases its functional activity towards double-stranded RNA. The N-linked sugar on plasminogen (at Asn-288) within kringle 3 reduces the rate of the beta- to alpha-conformational change, modulates the transport of plasminogen into the extravascular compartment, decreases plasminogen binding to U937 cells and downregulates the activation of plasminogen by both urokinase and tissue plasminogen activator. Additionally, in fibrinolysis, within a ternary complex of fibrin, plasminogen and tissue plasminogen activator, the N-linked sugar of plasminogen hinders the initial interaction with tissue plasminogen activator (i.e., it alters Km). The presence of an N-linked glycan (at Asn-184) in the kringle 2 domain of tissue plasminogen activator hinders the rearrangement of this ternary complex, decreasing the turnover rate (Kcat).

Animals↗
PubMed1995

The calcium binding properties and molecular organization of epidermal growth factor-like domains in human fibrillin-1.

Human fibrillin-1 is a 350-kDa glycoprotein found in 10-nm connective tissue microfibrils. Mutations in the gene encoding this protein cause the Marfan syndrome, a disease characterized by cardiovascular, ocular, and skeletal abnormalities. Fibrillin-1 has a modular structure that includes 47 epidermal growth factor-like (EGF-like) domains, 43 of which contain a consensus sequence associated with calcium binding. A mutation causing an Asn-2144 --> Ser amino acid change in one of the potential calcium binding residues has been described in a patient with the Marfan syndrome. We have chemically synthesized a wild-type EGF-like domain (residues 2126-2165 of human fibrillin-1) and a mutant EGF-like domain containing the Asn-2144 --> Ser amino acid change and measured calcium binding to each using 1H-NMR spectroscopy. The wild-type domain binds calcium with a similar affinity to isolated EGF-like domains from coagulation factors IX and X; however, the mutant domain exhibits > 5-fold reduction in affinity. Rotary shadowing of fibrillin-containing microfibrils, isolated from dermal fibroblast cultures obtained from the Marfan patient, shows that the mutation does not prevent assembly of fibrillin into microfibrils but does alter the appearance of the interbead region. We have modeled a region of fibrillin-1 (residues 2126-2331) encompassing five calcium binding EGF-like domains, using data derived from the recently determined crystal structure of a calcium binding EGF-like domain from human factor IX. Our model suggests that these fibrillin-1 EGF-like domains adopt a helical arrangement stabilized by calcium and that defective calcium binding to a single EGF-like domain results in distortion of the helix. We propose a mechanism for the interaction of contiguous arrays of calcium binding EGF-like domains within the microfibril.

Amino Acid Sequence↗
PubMed1995

The activation of type 1 and type 2 plasminogen by type I and type II tissue plasminogen activator.

Tissue plasminogen activator (tPA) was fractionated using lysine-Sepharose affinity chromatography. Type I, type II, and a minor peak with high affinity for lysine (designated type D) tPA were recovered. In an indirect amidolytic assay involving native human Glu-plasminogen and fibrin, type II tPA showed a 2-fold higher activity than type I. To explore the combinatorial effect of the variable glycosylation status of both tPA and plasminogen, kinetic constants for fibrin-dependent plasminogen activation were determined for combinations of type I, II, and D tPA with type 1 and 2 plasminogen. Within a 4-fold range, the fastest rate was achieved from the combination of type D (type II + D) tPA and type 2 plasminogen. N-Glycosylation of plasminogen increased the Km value for activation by all tPA variants; N-glycosylation of type I tPA at Asn184 decreased the kcat (turnover) values for the fibrin-dependent activation of plasminogen over type II tPA, while type D tPA showed the highest turnover rate. In the presence of fibrinogen fragments, N-glycosylation of plasminogen at site 289 modulates the kinetics of association of enzyme and substrate, while N-glycosylation at site 184 on tPA modulates the turnover rate of the enzyme.

Amino Acid Sequence↗
PubMed1994

Three-dimensional solution structure of the pleckstrin homology domain from dynamin.

BACKGROUND: The pleckstrin homology (PH) domain is a region of approximately 100 amino acids, defined by sequence similarity, that has been found in about 60 proteins, many of which are involved in signal transduction downstream of cell surface receptors; the function of PH domains is unknown. The only clue to the function of PH domains is the circumstantial evidence that they may link beta gamma subunits of G proteins to second messenger systems. Knowledge of the three-dimensional structures of PH domains should help to elucidate the roles they play in the proteins that contain them. RESULTS: Using homonuclear and heteronuclear magnetic resonance spectroscopy, we have determined the solution structure of the PH domain of the GTPase dynamin, one of a number of proteins that have PH domains and interact with GTP. The fold of the dynamin PH domain is composed of two antiparallel beta-sheets, which pack face-to-face at an angle of approximately 60 degrees. The first beta-sheet comprises four strands (residues 13-58) from the amino-terminal half of the protein sequence; the second beta-sheet contains three strands (residues 63-99). A single alpha-helix (residues 102-116) flanks one edge of the interface between the two sheets, parallel in orientation to the second sheet, in an alpha/beta roll motif similar to that of the B oligomer of verotoxin-1 from Escherichia coli. CONCLUSIONS: The structure of the dynamin PH domain is very similar to the recently reported structures of the pleckstrin and spectrin PH domains. This shows that, despite the low level of sequence similarity between different PH domains, they do have a characteristic polypeptide fold. On the basis of our structure, the suggestion that PH domains engage in coiled-coil interactions with G protein beta gamma subunits seems unlikely and should be re-evaluated.

Amino Acid Sequence↗
PubMed1994

Secondary structure of fibronectin type 1 and epidermal growth factor modules from tissue-type plasminogen activator by nuclear magnetic resonance.

A segment of human tissue-type plasminogen activator (t-PA) corresponding to the fibronectin type 1 (F1) and epidermal growth factor-like (G) pair of modules, residues 1-91, has been produced as a recombinant protein in Saccharomyces cerevisiae, with a single conservative Cys to Ser substitution. The sequence-specific assignment of the 1H and 15N nuclear magnetic resonances from the pair of modules has been completed using 2D 1H nuclear magnetic resonance (NMR) spectra in conjunction with 3D, 15N-edited, 1H and 2D 15N-1H NMR spectra. Slowly exchanging amide protons have been identified, and estimates of a number of backbone 3JNH-C alpha H coupling constants were obtained by line-shape-fitting. The secondary structure of the F1 module in the pair closely matches that previously determined for the isolated F1 module from t-PA, and that of the G module conforms to the "consensus" G module structure determined previously from several isolated G modules. In the module pair, the residues linking the two modules appear to form an extended beta-strand, the carboxy-terminal end of which makes up a third strand of the major beta-sheet of the G module. The intermodule interface is defined by NOEs between residues in the ranges 22-24 in the F1 module and 65-72 in the G module. The NMR data indicate that there is little or no reorientation of the two modules with respect to one another but rather that they combine with a fixed hydrophobic contact dominated by the side chain of leucine-22.

Amino Acid Sequence↗
PubMed1994

Building protein structure and function from modular units.

Many proteins in multicellular organisms are made from combinations of several, clearly identifiable, autonomously folding domains or modules. The structures of many of the constituent modules and some module pairs are now known. This review briefly describes some of the recent X-ray crystallographic and nuclear magnetic resonance (NMR) structural work on modules 'dissected' from proteins that are often large, membrane-bound and glycosylated. These include important proteins involved in cell adhesion, clotting, fibrinolysis and signalling. The structure and function of the intact proteins is discussed in the light of the recent structural work.

Animals↗
PubMed1993

Solution structure and ligand-binding site of the SH3 domain of the p85 alpha subunit of phosphatidylinositol 3-kinase.

SH3 domains are found in proteins associated with receptor tyrosine kinase signal transduction complexes. The solution structure of the SH3 domain of the 85 kd regulatory subunit of phosphatidylinositol 3-kinase is shown to be a compact beta barrel consisting of five beta strands arranged in two beta sheets of three and two strands. The structure is similar to that of chicken brain alpha spectrin but represents a distinct class of SH3 domain, with an insertion between the second and third beta strands that may influence binding specificity. 1H chemical shift changes induced by complex formation with a synthetic peptide derived from the SH3-binding protein dynamin, together with amino acid sequence comparisons, suggest that the ligand-binding site consists of a hydrophobic surface flanked by two charged loops.

Amino Acid Sequence↗
PubMed1992

Structure of an SH2 domain of the p85 alpha subunit of phosphatidylinositol-3-OH kinase.

Receptor protein-tyrosine kinases, through phosphorylation of specific tyrosine residues, generate high-affinity binding sites which direct assembly of multienzyme signalling complexes. Many of these signalling proteins, including phospholipase C gamma, GTPase-activating protein and phosphatidylinositol-3-OH kinase, contain src-homology 2 (SH2) domains, which bind with high affinity and specificity to tyrosine-phosphorylated sequences. The critical role played by SH2 domains in signalling has been highlighted by recent studies showing that mutation of specific phosphorylation sites on the platelet-derived growth factor receptor impair its association with phosphatidylinositol-3-OH kinase, preventing growth factor-induced mitogenesis. Here we report the solution structure of an isolated SH2 domain from the 85K regulatory subunit of phosphatidylinositol-3-OH kinase, determined using multidimensional nuclear magnetic resonance spectroscopy. The structure is characterized by a central region of beta-sheet flanked by two alpha-helices, with a highly flexible loop close to functionally important residues previously identified by site-directed mutagenesis.

Animals↗