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Biomedical subjects

Ian A Wilson

Publications and source records attributed to Ian A Wilson.

At least 73 records · Page 4Linked to original sources
PubMed2005

Design, synthesis, and biological evaluation of 10-methanesulfonyl-DDACTHF, 10-methanesulfonyl-5-DACTHF, and 10-methylthio-DDACTHF as potent inhibitors of GAR Tfase and the de novo purine biosynthetic pathway.

The synthesis and evaluation of 10-methanesulfonyl-DDACTHF (1), 10-methanesulfonyl-5-DACTHF (2), and 10-methylthio-DDACTHF (3) as potential inhibitors of glycinamide ribonucleotide transformylase (GAR Tfase) and aminoimidazole carboxamide ribonucleotide transformylase (AICAR Tfase) are reported. The compounds 10-methanesulfonyl-DDACTHF (1, K(i) = 0.23 microM), 10-methanesulfonyl-5-DACTHF (2, K(i) = 0.58 microM), and 10-methylthio-DDACTHF (3, K(i) = 0.25 microM) were found to be selective and potent inhibitors of recombinant human GAR Tfase. Of these, 3 exhibited exceptionally potent, purine sensitive growth inhibition activity (3, IC50 = 100 nM) against the CCRF-CEM cell line being 3-fold more potent than Lometrexol and 30-fold more potent than the parent, unsubstituted DDACTHF, whereas 1 and 2 exhibited more modest growth inhibition activity (1, IC50 = 1.0 microM and 2, IC50 = 2.0 microM).

Antineoplastic Agents↗
PubMed2005

Kinetic stabilization of an oligomeric protein by a single ligand binding event.

Protein native state stabilization imposed by small molecule binding is an attractive strategy to prevent the misfolding and misassembly processes associated with amyloid diseases. Transthyretin (TTR) amyloidogenesis requires rate-limiting tetramer dissociation before misassembly of a partially denatured monomer ensues. Selective stabilization of the native TTR tetramer over the dissociative transition state by small molecule binding to both thyroxine binding sites raises the kinetic barrier of tetramer dissociation, preventing amyloidogenesis. Assessing the amyloidogenicity of a TTR tetramer having only one amyloidogenesis inhibitor (I) bound is challenging because the two small molecule binding constants are generally not distinct enough to allow for the exclusive formation of TTR.I in solution to the exclusion of TTR.I(2) and unliganded TTR. Herein, we report a method to tether one fibril formation inhibitor to TTR by disulfide bond formation. Occupancy of only one of the two thyroxine binding sites is sufficient to inhibit tetramer dissociation in 6.0 M urea and amyloidogenesis under acidic conditions by imposing kinetic stabilization on the entire tetramer. The sufficiency of single occupancy for stabilizing the native state of TTR provides the incentive to search for compounds displaying striking negative binding cooperativity (e.g., K(d1) in nanomolar range and K(d2) in the micromolar to millimolar range), enabling lower doses of inhibitor to be employed in the clinic, mitigating potential side effects.

Amyloid↗
PubMed2005

Kinetic stabilization of the native state by protein engineering: implications for inhibition of transthyretin amyloidogenesis.

The amyloidogenic homotetrameric protein transthyretin (TTR) must undergo rate-limiting dissociation to partially denatured monomers in order to aggregate. TTR contains two distinct quaternary interfaces, one of which defines the binding sites for thyroxine and small-molecule amyloidogenesis inhibitors. Kinetic stabilization of the tetramer can be accomplished either by the binding of amyloidogenesis inhibitors selectively to the native state over the dissociative transition state or by the introduction of trans-suppressor subunits (T119M) into heterotetramers to destabilize the dissociative transition state. In each case, increasing the dissociation activation barrier prevents tetramer dissociation. Herein, we demonstrate that tethering two subunits whose quaternary interface defines the thyroxine binding site also dramatically increases the barrier for tetramer dissociation, apparently by destabilization of the dissociative transition state. The tethered construct (TTR-L-TTR)2 is structurally and functionally equivalent to wild-type TTR. Urea is unable to denature (TTR-L-TTR)2, yet it is able to maintain the denatured state once denaturation is achieved by GdnHCl treatment, suggesting that (TTR-L-TTR)2 is kinetically rather than thermodynamically stabilized, consistent with the identical wild-type TTR and (TTR-L-TTR)2 GdnHCl denaturation curves. Studies focused on a construct containing a single TTR-L-TTR chain and two normal monomer subunits establish that alteration of only one quaternary structural interface is sufficient to impose kinetic stabilization on the entire quaternary structure.

Amyloid↗
PubMed2005

Structural origins of efficient proton abstraction from carbon by a catalytic antibody.

Antibody 34E4 catalyzes the conversion of benzisoxazoles to salicylonitriles with high rates and multiple turnovers. The crystal structure of its complex with the benzimidazolium hapten at 2.5-angstroms resolution shows that a combination of hydrogen bonding, pi stacking, and van der Waals interactions is exploited to position both the base, Glu(H50), and the substrate for efficient proton transfer. Suboptimal placement of the catalytic carboxylate, as observed in the 2.8-angstroms structure of the Glu(H50)Asp variant, results in substantially reduced catalytic efficiency. In addition to imposing high positional order on the transition state, the antibody pocket provides a highly structured microenvironment for the reaction in which the carboxylate base is activated through partial desolvation, and the highly polarizable transition state is stabilized by dispersion interactions with the aromatic residue Trp(L91) and solvation of the leaving group oxygen by external water. The enzyme-like efficiency of general base catalysis in this system directly reflects the original hapten design, in which a charged guanidinium moiety was strategically used to elicit an accurately positioned functional group in an appropriate reaction environment and suggests that even larger catalytic effects may be achievable by extending this approach to the induction of acid-base pairs capable of bifunctional catalysis.

Amino Acid Substitution↗
PubMed2005

NMR screening and crystal quality of bacterially expressed prokaryotic and eukaryotic proteins in a structural genomics pipeline.

In the Joint Center for Structural Genomics, one-dimensional (1D) 1H NMR spectroscopy is routinely used to characterize the folded state of protein targets and, thus, serves to guide subsequent crystallization efforts and to identify proteins for NMR structure determination. Here, we describe 1D 1H NMR screening of a group of 79 mouse homologue proteins, which correlates the NMR data with the outcome of subsequent crystallization experiments and crystallographic structure determination. Based on the 1D 1H NMR spectra, the proteins are classified into four groups, "A" to "D." A-type proteins are candidates for structure determination by NMR or crystallography; "B"-type are earmarked for crystallography; "C" indicates folded globular proteins with broadened line shapes; and "D" are nonglobular, "unfolded" polypeptides. The results obtained from coarse- and fine-screen crystallization trials imply that only A- and B-type proteins should be used for extensive crystallization trials in the future, with C and D proteins subjected only to coarse-screen crystallization trials. Of the presently studied 79 soluble protein targets, 63% yielded A- or B-quality 1D 1H NMR spectra. Although similar yields of crystallization hits were obtained for all four groups, A to D, crystals from A- and B-type proteins diffracted on average to significantly higher resolution than crystals produced from C- or D-type proteins. Furthermore, the output of refined crystal structures from this test set of proteins was 4-fold higher for A- and B-type than for C- and D-type proteins.

Animals↗
PubMed2005

Protein production and crystallization at the joint center for structural genomics.

By definition, structural genomics centers must be able to address a large number of diverse protein targets. The methods developed should permit parallel and cost-effective processing while allowing for the diverse nature of proteins. Our approach to this problem is a multi-tiered effort where targets are characterized and categorized by behavior and processed in parallel by appropriate methods. The Joint Center for Structural Genomics (JCSG) has applied this tactic to create a fully integrated and scaleable structure determination pipeline. Highlights of the development of the current pipeline for protein production and crystallization are presented here.

Crystallization↗
PubMed2005

CD1 assembly and the formation of CD1-antigen complexes.

The CD1 antigen presentation system presents lipid antigens to effector T cells, which have diverse roles in antimicrobial responses, antitumor immunity and in regulating the balance between tolerance and autoimmunity. The trafficking of CD1 molecules and lipid antigens facilitates their intersection and binding in specific intracellular compartments. Recent studies have now identified unexpected accessory molecules that are critical to CD1 assembly and lipid loading. The atomic structures of CD1-antigen complexes have defined both the orientation of polar headgroups between the alpha1 and alpha2 helices of CD1 and the manner in which distinct CD1 isoforms bind a range of lipids that have different lengths and numbers of hydrocarbon chains.

Animals↗
PubMed2005

T-cell activation by lipopeptide antigens.

The discovery of the CD1 antigen-presenting system reveals that T cells survey the lipid content of target cells via T-cell receptor (TCR) contact with CD1 bound to lipids, glycolipids and small molecules. Recently, CD1 proteins have been found to present mycobacterial lipopeptides that are involved in scavenging iron from infected cells. The mechanism of lipopeptide antigen presentation by CD1 involves the anchoring of antigens in the hydrophobic binding groove, resulting in exposure of the peptide moiety for TCR contact. These findings expand the range of known antigens for T cells and raise the intriguing possibility that CD1, similar to MHC class I and II molecules, enables T cells to discriminate among peptide sequences.

Animals↗
PubMed2005

Molecular mechanism of lipopeptide presentation by CD1a.

CD1a is expressed on Langerhans cells (LCs) and dendritic cells (DCs), where it mediates T cell recognition of glycolipid and lipopeptide antigens that contain either one or two alkyl chains. We demonstrate here that CD1a-restricted T cells can discriminate the peptide component of didehydroxymycobactin lipopeptides. Structure analysis of CD1a cocrystallized with a synthetic mycobactin lipopeptide at 2.8 A resolution further reveals that the single alkyl chain is inserted deep within the A' pocket of the groove, whereas its two peptidic branches protrude along the F' pocket to the outer, alpha-helical surface of CD1a for recognition by the TCR. Remarkably, the cyclized lysine branch of the peptide moiety lies in the shallow F' pocket in a conformation that closely mimics that of the alkyl chain in the CD1a-sulfatide structure. Thus, this structural study illustrates how a single chain lipid can be presented by CD1 and that the peptide moiety of the lipopeptide is recognized by the TCR.

Antigen Presentation↗
PubMed2005

Broadly neutralizing anti-HIV antibody 4E10 recognizes a helical conformation of a highly conserved fusion-associated motif in gp41.

Broadly neutralizing monoclonal antibodies to HIV-1 are rare but invaluable for vaccine design. 4E10 is the broadest neutralizing antibody known and recognizes a contiguous and highly conserved epitope in the membrane-proximal region of gp41. The crystal structure of Fab 4E10 was determined at 2.2 A resolution in complex with a 13-residue peptide containing the gp41 core epitope (NWFDIT). The bound peptide adopts a helical conformation in which the key contact residues, TrpP672, PheP673, IleP675, and ThrP676, map to one face of the helix. The peptide binds in a hydrophobic pocket that may emulate its potential interaction with the host cell membrane. The long CDR H3 of the antibody extends beyond the bound peptide in an orientation that suggests that its apex could contact the viral membrane when 4E10 is bound to its membrane-proximal epitope. These structural insights should assist in the design of immunogens to elicit 4E10-like neutralizing responses.

Amino Acid Motifs↗
PubMed2005

Anatomy of CD1-lipid antigen complexes.

CD1 proteins bind lipids to form antigen complexes that contact T-cell receptors and activate T cells. Recent crystal structures of CD1 proteins show that their antigen-binding grooves are composed of up to four pockets (A', C', F' and T') and two antigen portals (C' and F'). Although certain structural features are conserved among CD1 proteins, the grooves of CD1a, CD1b and CD1d differ in the number, shape and connectivity of their antigen-binding pockets. Here, we outline how the portals and pockets of CD1 antigen-binding grooves influence ligand specificity and facilitate the presentation of a surprisingly diverse set of antigenic lipids, glycolipids, lipopeptides and even small, non-lipidic molecules.

Antigens, CD1↗
PubMed2005

Crystal structures of apo wild-type M. jannaschii tyrosyl-tRNA synthetase (TyrRS) and an engineered TyrRS specific for O-methyl-L-tyrosine.

The Methanococcus jannaschii tRNA(Tyr)/TyrRS pair has been engineered to incorporate unnatural amino acids into proteins in E. coli. To reveal the structural basis for the altered specificity of mutant TyrRS for O-methyl-L-tyrosine (OMeTyr), the crystal structures for the apo wild-type and mutant M. jannaschii TyrRS were determined at 2.66 and 3.0 A, respectively, for comparison with the published structure of TyrRS complexed with tRNA(Tyr) and substrate tyrosine. A large conformational change was found for the anticodon recognition loop 257-263 of wild-type TyrRS upon tRNA binding in order to facilitate recognition of G34 of the anticodon loop through pi-stacking and hydrogen bonding interactions. Loop 133-143, which is close to the tRNA acceptor stem-binding site, also appears to be stabilized by interaction with the tRNA(Tyr). Binding of the substrate tyrosine results in subtle and cooperative movements of the side chains within the tyrosine-binding pocket. In the OMeTyr-specific mutant synthetase structure, the signature motif KMSKS loop and acceptor stem-binding loop 133-143 were surprisingly ordered in the absence of bound ATP and tRNA. The active-site mutations result in altered hydrogen bonding and steric interactions which favor binding of OMeTyr over L-tyrosine. The structure of the mutant and wild-type TyrRS now provide a basis for generating new active-site libraries to evolve synthetases specific for other unnatural amino acids.

Crystallography, X-Ray↗
PubMed2005

A single amino acid substitution in 1918 influenza virus hemagglutinin changes receptor binding specificity.

The receptor binding specificity of influenza viruses may be important for host restriction of human and avian viruses. Here, we show that the hemagglutinin (HA) of the virus that caused the 1918 influenza pandemic has strain-specific differences in its receptor binding specificity. The A/South Carolina/1/18 HA preferentially binds the alpha2,6 sialic acid (human) cellular receptor, whereas the A/New York/1/18 HA, which differs by only one amino acid, binds both the alpha2,6 and the alpha2,3 sialic acid (avian) cellular receptors. Compared to the conserved consensus sequence in the receptor binding site of avian HAs, only a single amino acid at position 190 was changed in the A/New York/1/18 HA. Mutation of this single amino acid back to the avian consensus resulted in a preference for the avian receptor.

Amino Acid Substitution↗