Biomedical subjects

Peter N Lowe

Publications and source records attributed to Peter N Lowe.

7 recordsLinked to original sources

PubMed2005

Specificity determinants on Cdc42 for binding its effector protein ACK.

Cdc42 and Rac are highly homologous members of the Rho family of small G proteins that interact with several downstream effector proteins thereby causing cytoskeletal rearrangements, cell proliferation, and differentiation. While some effectors, such as the tyrosine kinase, ACK, and the scaffold protein, WASP, are unique to Cdc42, others, such as the serine-threonine kinase, PAK, are shared with Rac. Previous mutagenesis studies identified Val42 and Leu174 as residues that selectively affect binding of Cdc42 to ACK and WASP but not to PAK. However, it is unclear whether these discriminatory residues are sufficient determinants of specificity. In this study we sought to introduce "gain-of function" mutations into Rac to allow it to bind to ACK and WASP, thereby revealing all specificity determinants. Thirteen mutations were made changing Rac residues to those in Cdc42. Equilibrium binding constants of all mutant Rac proteins to ACK, WASP, and PAK were measured. A combination of seven mutations (S41A, A42V, N43T, D47G, N52T, W56F, and R174L) was determined to be necessary to change the binding affinity of Rac for ACK from negligible (K(d) < 1 microM) to a comparable affinity to Cdc42 (K(d) 25 nM). These mutations are not confined to interface residues. We interpret these data to indicate the importance of the structure of regions of the protein distinct from the contact residues. None of these mutant Rac proteins bound WASP with a similar affinity to Cdc42. Hence, residues as yet unidentified, outside the interface, must be necessary for binding WASP.

Amino Acid Sequence↗

PubMed2004

Evidence for the presence of three distinct binding sites for the thioflavin T class of Alzheimer's disease PET imaging agents on beta-amyloid peptide fibrils.

Imaging the progression of Alzheimer's disease would greatly facilitate the discovery of therapeutics, and a wide range of ligands are currently under development for the detection of beta-amyloid peptide (Abeta)-containing plaques by using positron emission tomography. Here we report an in-depth characterization of the binding of seven previously described ligands to in vitro generated Abeta-(1-40) polymers. All of the compounds were derived from the benzothiazole compound thioflavin T and include 2-[4'-(methylamino)phenyl]benzothiazole and 2-(4'-dimethylamino-)phenyl-imidazo[1,2-a]-pyridine derivatives, 2-[4'-(dimethylamino)phenyl]-6-iodobenzothiazole and 2-[4'-(4''-methylpiperazin-1-yl)phenyl]-6-iodobenzothiazole, and a benzofuran compound (5-bromo-2-(4-dimethylaminophenyl)benzofuran). By using a range of fluorescent and radioligand binding assays, we find that these compounds display a more complex binding pattern than described previously and are consistent with three classes of binding sites on the Abeta fibrils. All of the compounds bound with very high affinity (low nm K(d)) to a low capacity site (BS3) (1 ligand-binding site per approximately 300 Abeta-(1-40) monomers) consistent with the previously recognized binding site for these compounds on the fibrils. However, the compounds also bound with high affinity (K(d) approximately 100 nm) to either one of two additional binding sites on the Abeta-(1-40) polymer. The properties of these sites, BS1 and BS2, suggest they are adjacent or partially overlapping and have a higher capacity than BS3, occurring every approximately 35 or every approximately 4 monomers of Abeta-(1-40)-peptide, respectively. Compounds appear to display selectivity for BS2 based on the presence of a halogen substitution (2-[4'-(dimethylamino)phenyl]-6-iodobenzothiazole, 2-[4'-(4''-methylpiperazin-1-yl)phenyl]-6-iodobenzothiazole, and 5-bromo-2-(4-dimethylaminophenyl)benzofuran) on their aromatic ring system. The presence of additional ligand-binding sites presents potential new targets for ligand development and may allow a more complete modeling of the current positron emission tomography data.

Alzheimer Disease↗

PubMed2004

Fluorescence properties of green fluorescent protein FRET pairs concatenated with the small G protein, Rac, and its interacting domain of the kinase, p21-activated kinase.

Many diseases are caused by aberrant cell signalling controlled by intracellular protein-protein interactions. Inhibitors of such interactions thus have enormous potential as chemotherapeutic agents. It is advantageous to test for such inhibitors using cell-based screens in which modulation of the interaction gives a rapid response. Fluorescence resonance energy transfer (FRET) systems, based on interacting donor and acceptor green fluorescent proteins (GFPs), have potential in such screens. Here, we describe experiments aimed at using a FRET system to monitor the interaction between the small G protein Rac and a region of its binding partner, the Ser/Thr kinase, p21-activated kinase (PAK). Initial attempts to use a previously described construct, enhanced GFP-PAK-enhanced blue fluorescent protein, failed because of the difficulty of obtaining equal and high expression levels of both the fusion protein and Rac in mammalian cells. Here, three proteins in which Rac, PAK, and the two GFPs were concatenated in different combinations on a single protein were expressed and characterised. In each construct, however, intramolecular interaction of PAK and Rac was observed. As this was of extremely high affinity, presumably because of entropy effects from the interacting partners being tethered, these molecules were not suitable for detection of inhibitors of the interaction. Molecular modelling was used to investigate the way in which the concatenated constructs might form intramolecular interactions. As this explained key properties of these proteins, it is likely that this approach could be used to design constructs where the unwanted intramolecular protein-protein interactions are prevented, whilst allowing the desired intermolecular Rac/PAK interaction. This would provide constructs that are useable for drug discovery.

Animals↗

PubMed2003

Molecular dissection of the interaction between the small G proteins Rac1 and RhoA and protein kinase C-related kinase 1 (PRK1).

PRK1 is a serine/threonine kinase that belongs to the protein kinase C superfamily. It can be activated either by members of the Rho family of small G proteins, by proteolysis, or by interaction with lipids. Here we investigate the binding of PRK1 to RhoA and Rac1, two members of the Rho family. We demonstrate that PRK1 binds with a similar affinity to RhoA and Rac1. We present the solution structure of the second HR1 domain from the regulatory N-terminal region of PRK1, and we show that it forms an anti-parallel coiled-coil. In addition, we have used NMR to map the binding contacts of the HR1b domain with Rac1. These are compared with the contacts known to form between HR1a and RhoA. We have used mutagenesis to define the residues in Rac that are important for binding to HR1b. Surprisingly, as well as residues adjacent to Switch I, in Switch II, and in helix alpha5, it appears that the C-terminal stretch of basic amino acids in Rac is required for a high affinity interaction with HR1b.

Amino Acid Sequence↗

PubMed2003

Effect of detergent on "promiscuous" inhibitors.

The term "promiscuous" inhibitors has been coined for compounds whose inhibition mechanism involves the interaction of aggregates of many compound molecules with the target protein, rather than the binding of individual molecules. This paper demonstrates that promiscuous inhibitors can be differentiated from classical 1:1 inhibitors by the judicious use of detergents, making it possible to configure assays that significantly reduce this undesirable mechanism of inhibition without compromising assay performance.

Acetophenones↗

PubMed2002

MgF(3)(-) as a transition state analog of phosphoryl transfer.

The formation of complexes between small G proteins and certain of their effectors can be facilitated by aluminum fluorides. Solution studies suggest that magnesium may be able to replace aluminum in such complexes. We have determined the crystal structure of RhoA.GDP bound to RhoGAP in the presence of Mg(2+) and F(-) but without Al(3+). The metallofluoride adopts a trigonal planar arrangement instead of the square planar structure of AlF(4)(-). We have confirmed that these crystals contain magnesium and not aluminum by proton-induced X-ray emission spectroscopy. The structure adopted by GDP.MgF(-) possesses the stereochemistry and approximate charge expected for the transition state. We suggest that MgF3(-) may be the reagent of choice for studying phosphoryl transfer reactions.

Aluminum Compounds↗

PubMedDate not supplied

Measurement of dissociation constants of inhibitors binding to Src SH2 domain protein by non-covalent electrospray ionization mass spectrometry.

A mass spectrometric protocol for identifying ligands with a wide range of affinities (3-101 microM) and quantitative spectral analysis for non-covalent interactions have been developed using Src SH2 as a target. Dissociation constants of five compounds, three with a phospho moiety, one with a sulphonic acid group and one with carboxylic acid groups only, were determined using one-ligand one-binding-site, two-ligands one-binding site and one-ligand two-binding-sites models. The Kd values determined by ESI-MS of the three compounds containing the phospho moiety (3.2-7.9 microM) were comparable to those obtained from a solution equilibrium fluorescence polarization assay. The compound with a sulphonate group is a much weaker binding ligand (Kd=101 microM by ESI, >>300 microM by FP) towards the Src SH2 protein. Two complexes with different stoichiometric ratios 1:1 and 2:1 (ligand-protein) were observed by ESI-MS for the ligand GIXXX630X. Analysis of binding isotherms indicated the presence of two binding sites for the ligand with Kd values of 9.3 and 193 microM. These data confirmed that, for these polar compounds, non-covalent ESI-MS can measure affinity which very closely reflects the affinity measured under true solution equilibrium conditions. ESI-MS has several key advantages over many solution methods: it can identify the existence of and measure the affinity of complexes other than simple 1:1 ligand-enzyme complexes. Moreover, ESI-MS competition experiments can be readily performed to yield data on whether two ligands bind simultaneously or competitively at the same time as measuring the affinity of the ligand.

Binding Sites↗