A. Kristina Downing

Solution structure and ligand-binding site of the SH3 domain of the p85α 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.

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.

Solution Structure of the LDL Receptor EGF-AB Pair: A Paradigm for the Assembly of Tandem Calcium Binding EGF Domains

BACKGROUND From the observed structure and sequence of a pair of calcium binding (cb) epidermal growth factor-like (EGF) domains from human fibrillin-1, we proposed that many tandem cbEGF domains adopt a conserved relative conformation. The low-density lipoprotein receptor (LDLR), which is functionally unrelated to fibrillin-1, contains a single pair of EGF domains that was chosen for study in the validation of this hypothesis. The LDLR is the protein that is defective in familial hypercholesterolaemia, a common genetic disorder that predisposes individuals to cardiovascular complications and premature death. RESULTS Here, we present the solution structure of the first two EGF domains from the LDL receptor, determined using conventional NMR restraints and residual dipolar couplings. The cbEGF domains have an elongated, rod-like arrangement, as predicted. The new structure allows a detailed assessment of the consequences of mutations associated with familial hypercholesterolaemia to be made. CONCLUSIONS The validation of the conserved arrangement of EGF domains in functionally distinct proteins has important implications for structural genomics, since multiple tandem cbEGF pairs have been identified in many essential proteins that are implicated in human disease. Our results provide the means to use homology modeling to probe structure-function relationships in this diverse family of proteins and may hold the potential for the design of novel diagnostics and therapies in the future.

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 molecules 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 modules major beta-sheet. The new structural results allow the interpretation of earlier mutational data relevant to fibrin-binding and hepatocyte-receptor binding.

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.

Electrostatic and Functional Analysis of the Seven-Bladed WD β-Propellers

β-propeller domains composed of WD repeats are highly ubiquitous and typically used as multi-site docking platforms to coordinate and integrate the activities of groups of proteins. Here, we have used extensive homology modelling of the WD40–repeat family of seven-bladed β-propellers coupled with subsequent structural classification and clustering of these models to define subfamilies of β-propellers with common structural, and probable, functional characteristics. We show that it is possible to assign seven-bladed WD β-propeller proteins into functionally different groups based on the information gained from homology modelling. We examine general structural diversity within the WD40-repeat family of seven-bladed β-propellers and demonstrate that seven-bladed β-propellers composed of WD-repeats are structurally distinct from other seven-bladed β-propellers. We further provide some insights into the multifunctional diversity of the seven-bladed WD β-propeller surfaces. This report once again reinforces the importance of structural data and the usefulness of homology models in functional classification.