Biomedical subjects

A K Downing

Publications and source records attributed to A K Downing.

25 records · Page 2Linked to original sources

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↗

PubMed1992

Solution structure of the fibrin binding finger domain of tissue-type plasminogen activator determined by 1H nuclear magnetic resonance.

The amino acid sequence of the first domain of tissue-type plasminogen activator (t-PA) includes eight residues that are highly conserved in the type 1 finger domains found in human fibronectin. A construct comprising 50 residues from this finger domain of t-PA has been expressed and its solution structure has been determined by two-dimensional nuclear magnetic resonance spectroscopy. A total of 782 experimental restraints consisting of 723 interproton distances derived from nuclear Overhauser effect measurements, 43 torsion angles, and 16 hydrogen bond restraints were used as the input for dynamical simulated annealing structure calculations. Twenty-eight structures were obtained that satisfied the experimental data with no single distance violation greater than 0.3 A. The average atomic root-mean-square distribution for the backbone atoms of the final structures was 0.41 (+/- 0.13) A for the well defined part of the structure (residues 4 to 47). The overall fold of the t-PA finger domain shows a striking similarity to that of the seventh type 1 repeat of human fibronectin with the side-chains of conserved residues lying in similar conformations. One significant difference between the two molecules is that hydrophobic residues cover the exposed surface of the principal beta-sheet region in the t-PA finger domain. It is suggested that one face of this region may interact with parts of the complete t-PA protein.

Amino Acid Sequence↗