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Biomedical subjects

I A Wilson

Publications and source records attributed to I A Wilson.

At least 19 recordsLinked to original sources
PubMed1996

Functional mimicry of a protein hormone by a peptide agonist: the EPO receptor complex at 2.8 A.

The functional mimicry of a protein by an unrelated small molecule has been a formidable challenge. Now, however, the biological activity of a 166-residue hematopoietic growth hormone, erythropoietin (EPO), with its class 1 cytokine receptor has been mimicked by a 20-residue cyclic peptide unrelated in sequence to the natural ligand. The crystal structure at 2.8 A resolution of a complex of this agonist peptide with the extracellular domain of EPO receptor reveals that a peptide dimer induces an almost perfect twofold dimerization of the receptor. The dimer assembly differs from that of the human growth hormone (hGH) receptor complex and suggests that more than one mode of dimerization may be able to induce signal transduction and cell proliferation. The EPO receptor binding site, defined by peptide interaction, corresponds to the smaller functional epitope identified for hGH receptor. Similarly, the EPO mimetic peptide ligand can be considered as a minimal hormone, and suggests the design of nonpeptidic small molecule mimetics for EPO and other cytokines may indeed be achievable.

Amino Acid Sequence↗
PubMed1996

Structure and mutational analysis of Rab GDP-dissociation inhibitor.

The crystal structure of the bovine alpha-isoform of Rab GDP-dissociation inhibitor (GDI), which functions in vesicle-membrane transport to recycle and regulate Rab GTPases, has been determined to a resolution of 1.81 A. GDI is constructed of two main structural units, a large complex multisheet domain I and a smaller alpha-helical domain II. The structural organization of domain I is surprisingly closely related to FAD-containing monooxygenases and oxidases. Sequence-conserved regions common to GDI and the choroideraemia gene product, which delivers Rab to catalytic subunits of Rab geranylgeranyltransferase II, are clustered on one face of the molecule. The two most sequence-conserved regions, which form a compact structure at the apex of GDI, are shown by site-directed mutagenesis to play a critical role in the binding of Rab proteins.

Adaptor Proteins, Signal Transducing↗
PubMed1996

Role of chain pairing for the production of functional soluble IA major histocompatibility complex class II molecules.

Structural studies of cellular receptor molecules involved in immune recognition require the production of large quantities of the extracellular domains of these glycoproteins. The murine major histocompatibility complex (MHC) class II-restricted response has been extensively studied by functional means, but the engineering and purification of the native, empty form of the most-studied murine MHC class II molecule, IA, has been difficult to achieve. IA molecules, which are the murine equivalent of human histocompatibility leukocyte antigen-DQ molecules, have a low efficiency of chain pairing, which results in poor transport to the cell surface and in the appearance of mixed isotype pairs. We have engineered soluble IA molecules whose pairing has been forced by the addition of leucine zipper peptide dimers at their COOH-terminus. The molecules are secreted "empty" into the extracellular medium and can be loaded with single peptide after purification. These IA molecules have been expressed in milligram quantity for crystallization as well as for activation of T cells and measurement of MHC class II-T cell receptor interactions.

Amino Acid Sequence↗
PubMed1995

CTL escape viral variants. I. Generation and molecular characterization.

Cytotoxic T lymphocytes (CTL) play a pivotal role in preventing persistent viral infections and aborting acute infections. H-2Db-restricted CTL optimally recognize a specific peptide of 9 to 11 amino acids (aa) derived from a viral protein and held in place (restricted) by a MHC class I glycoprotein on the surfaces of infected cells. Only three peptide sequences with the appropriate Db motif from lymphocytic choriomeningitis virus Armstrong strain (LCMV) are known to be presented to CTL by H-2Db molecules; they are from the glycoproteins (GP), residues 33-41 KAVYNFATC (GP1) and 276-286 SGVENPGGYCL (GP2), and the nucleoprotein (NP), 396-404 FQPQNGQFI. Incubation of virally infected H-2b cells with CTL clones that recognize only GP1, GP2, or NP leads to the selection of viral variants which upon infecting cells bearing H-2b molecules, escape recognition by CTL of the appropriate specificity. Nucleic acid sequencing showed a single mutation in GP1 (aa 38 F-->L), GP2 (aa 282 G-->D), or NP (aa 403 F-->L) in the variant viruses. When wild-type (wt) LCMV peptides and the three variant peptides (GP1, GP2, NP) were synthesized and subjected to a competitive inhibition binding assay, no differences in binding affinity for H-2Db were found between the wt and variant peptides. Uninfected cells coated with the wt peptide were recognized and lysed by the appropriate CTL clone or by in vivo-primed bulk CTL, but similar targets coated with the GP1, GP2, or NP variant peptides were not. This result, coupled with computer graphic analysis of these variant peptides with the recently solved three-dimensional structure for the Db MHC class I molecule, placed the side chain of the mutated residues on the outer surface of the MHC-peptide complex and accessible to the T cell receptor. Ala substitution at GP residue 38 or 282 or at NP 403 also abrogated CTL recognition and lysis. Inoculation of any one of the mutated viral variants into mice produced an effective CTL response to the other two nonmutated GP or NP peptides, suggesting that production of biologically relevant CTL escape virus variants in vivo requires selection of mutations in more than one and likely all the CTL epitopes, a low probability event.

Amino Acid Sequence↗
PubMed1995

Differential localization of the mRNA of the M and B isoforms of creatine kinase in myoblasts.

Creatine kinase (CK) plays an important role in buffering ATP and ADP levels in tissues which have intermittently high and fluctuating energy demands, such as skeletal muscle. This buffering function has a spatial, as well as a temporal aspect, which is dependent on the localization of different enzyme isoforms within the cell. We show here, by in situ hybridization, that the mRNAs for the cytoplasmic isoforms of CK are differentially localized in a mouse myoblast cell line (C2C12). The mRNA for the M form is localized at the cell periphery, while that for the B form is localized in the perinuclear region. Deletion of segments of the 3' untranslated regions of these mRNAs or swapping of these segments between the mRNAs for the two isoforms demonstrated that localization signals lie within these regions. Localization appears to be tissue-specific, since both the M and B mRNAs were distributed uniformly over the cytoplasm in a non-muscle cell line. These results, in conjunction with other studies which have shown that mRNA localization can lead to co-localization of the encoded protein, suggest that the localization of the mRNAs for the cytoplasmic isoforms of CK may be involved in the localization of the enzymes themselves.

Animals↗
PubMed1995

Towards structure-based drug design: crystal structure of a multisubstrate adduct complex of glycinamide ribonucleotide transformylase at 1.96 A resolution.

An inhibitor complex structure of glycinamide ribonucleotide transformylase (GAR-Tfase; EC 2.1.2.2) from Escherichia coli has been determined with a multisubstrate adduct BW1476U89 to an R-value of 19.1% at 1.96 A resolution. The structure was determined by a combination of molecular and single isomorphous replacement using data from two different monoclinic crystal lattices and collecting data from crystals soaked in 20% (w/v) methyl-pentanediol as cryoprotectant for shock-freezing at -150 degrees C. The multisubstrate adduct is bound in an extended crevice at the interface between the two functional domains of the enzyme. This inhibitor is positioned in the binding site by three sets of tight interactions with its phosphate, glutamate and pyrimidone ring moieties, while its interventing linker atoms are more flexible and adopt two distinct sets of conformations. The highly conserved Arg103, His108 and Gln170 residues that are key in ligand binding and catalysis (His108), have compensatory conformational variation that gives some clues as to their role in substrate specificity and in the formyl transfer. The molecular design of 1476U89 as a multisubstrate adduct inhibitor (Ki approximately 100 pM at pH 8.5), is confirmed as it closely mimics the shape, molecular interaction and combined binding constants of the natural 10-formyltetrahydrofolate (10-CHO-H4F; Km approximately 77.4 microM at pH 8.5) and glycinamide-ribonucleotide (GAR; Km approximately 8.1 microM at pH 8.5) substrates. The stereochemistry of this ligand complex suggests that His108 may act as an electrophile stabilizing the oxyanion of the tetrahedral intermediate that is formed as a result of the direct attack on the 10-CHO-H4F by the amino group of GAR. Structural comparison of the folate binding modes among GAR-Tfase, dihydrofolate reductase and thymidylate synthase reveals that folate derivates bound to GAR-Tfase differentially adopt the trans conformation for the dihedral angle between atoms C-6 and C-9 providing a handle for targeting specific folate-dependent enzymes. The structural information derived from two different discrete conformations of the ligand in the binding site also suggests several leads for the de novo design of inhibitors of GAR-Tfase that may develop into useful chemotherapeutic agents.

Acyltransferases↗
PubMed1995

Crystal structure of an H-2Kb-ovalbumin peptide complex reveals the interplay of primary and secondary anchor positions in the major histocompatibility complex binding groove.

Sequence analysis of peptides naturally presented by major histocompatibility complex (MHC) class I molecules has revealed allele-specific motifs in which the peptide length and the residues observed at certain positions are restricted. Nevertheless, peptides containing the standard motif often fail to bind with high affinity or form physiologically stable complexes. Here we present the crystal structure of a well-characterized antigenic peptide from ovalbumin [OVA-8, ovalbumin-(257-264), SIINFEKL] in complex with the murine MHC class I H-2Kb molecule at 2.5-A resolution. Hydrophobic peptide residues Ile-P2 and Phe-P5 are packed closely together into binding pockets B and C, suggesting that the interplay of peptide anchor (P5) and secondary anchor (P2) residues can couple the preferred sequences at these positions. Comparison with the crystal structures of H-2Kb in complex with peptides VSV-8 (RGYVYQGL) and SEV-9 (FAPGNYPAL), where a Tyr residue is used as the C pocket anchor, reveals that the conserved water molecule that binds into the B pocket and mediates hydrogen bonding from the buried anchor hydroxyl group could not be likewise positioned if the P2 side chain were of significant size. Based on this structural evidence, H-2Kb has at least two submotifs: one with Tyr at P5 (or P6 for nonamer peptides) and a small residue at P2 (i.e., Ala or Gly) and another with Phe at P5 and a medium-sized hydrophobic residue at P2 (i.e., Ile). Deciphering of these secondary submotifs from both crystallographic and immunological studies of MHC peptide binding should increase the accuracy of T-cell epitope prediction.

Amino Acid Sequence↗
PubMed1995

Crystallization and preliminary crystallographic data for class I deoxyribose-5-phosphate aldolase from Escherichia coli: an application of reverse screening.

X-ray quality crystals of class I-deoxyribose-5-phosphate aldolase from Escherichia coli have been obtained for the unliganded enzyme and in complex with its substrate, 2-deoxyribose-5-phosphate. The enzyme catalyzes the reversible cleavage of 2-deoxyribose-5-phosphate to acetaldehyde and D-glyceraldehyde-3-phosphate. The unliganded and complex crystals are prismatic long rods and belong to the orthorhombic space group P2(1)2(1)2(1) with cell dimensions a = 183.1 A, b = 61.4 A, c = 49.3 A and a = 179.2 A, b = 60.5, A, c = 49.1 A, respectively. Two molecules in the asymmetric unit are related by a noncrystallographic 2-fold axis. The crystals are stable in the X-ray beam and diffract to at least 2.6 A. A new method, reverse screening, designed to minimize protein utilization during the screening process was used to determine supersaturation and crystallization conditions.

Aldehyde-Lyases↗
PubMed1995

Protein-peptide interactions.

Proteins can interact with short peptide sequences in a variety of ways that can be sequence dependent or independent. The bound peptides are frequently in an extended conformation but may also adopt beta-turns or alpha-helices as motifs for recognition. The peptides can be completely buried in cavities, bound in grooves or pockets, or form beta-strand type interactions at the protein surface. These various recognition motifs are illustrated by peptide interactions with antibodies, calmodulin, OppA periplasmic binding protein, PapD chaperone, MHC class I and class II molecules, and Src homology (SH) domains 2 and 3.

Amino Acid Sequence↗
PubMed1994

Crystallization and preliminary crystallographic data for Rab guanine nucleotide dissociation inhibitor (RabGDI) from bovine brain.

X-ray quality crystals of Rab guanine nucleotide dissociation inhibitor (RabGDI) from bovine brain expressed in Escherichia coli have been obtained from 1.73 M ammonium sulfate. The crystals are prismatic long rods and belong to the monoclinic space group P21 with approximate cell dimensions a = 91.9 A, b = 43.5 A, c = 63.2 A, beta = 104.5 degrees and one molecule per asymmetric unit. The crystals are stable in the X-ray beam and diffract to at least 2.3 A. Reverse screening, streak seeding and macroseeding methods were used to obtain and improve the crystals.

Animals↗
PubMed1994

Structural analysis of antibody specificity. Detailed comparison of five Fab'-steroid complexes.

Structures of the Fab' fragment of the anti-progesterone antibody DB3 in complex with five cross-reactive steroids (aetiocholanolone, 5 beta-androstane-3,17-dione, 5 alpha-pregnane-20-one-3 beta-ol-hemisuccinate, progesterone-11 alpha-ol-hemisuccinate and progesterone) have been determined by X-ray crystallography to a maximum resolution of 2.7 A. These different steroids compete with progesterone binding with affinities in the nanomolar range despite substantial differences in their three-dimensional structures. Comparison of the unliganded DB3 Fab' and these five steroid-Fab' complexes reveals that all the steroid ligands bind to an "open" conformation of the Fab' as defined by the orientation of the indole side-chain of TrpH100, whereas in the unliganded or "closed" form the binding site is occluded by TrpH100. Small but significant conformational changes take place in the antibody to maximize the physical and chemical complementarity with each ligand. The various cross-reactive ligands are accommodated in the binding site in two distinct orientations. We term these binding modes syn and anti, as they are defined by the orientation of the steroid beta face relative to TrpH50. In all cases, the steroid D ring is inserted into a hydrophobic cavity formed mainly by TrpH50, TyrH97, TrpH100 and PheH100b; a hydrogen bond interaction with AsnH35 to the keto group at position C17 or C20 orients the steroid in the pocket. The AsnH35 hydrogen bond and the interaction with TrpH50 account for the restricted heavy chain response to immunization with progesterone-like steroids derivatized at the 11 alpha position. Cross-reactivity of the antibody with different steroids is explained by alternative binding pockets for the A ring, which generates different ligand orientations in the binding site. This study suggests which factors are most likely to contribute to the observed antibody specificity, such as linker position and the paucity of functional groups on the immunogenic hapten.

Amino Acid Sequence↗
PubMed1994

Crystal structure of a peptide complex of anti-influenza peptide antibody Fab 26/9. Comparison of two different antibodies bound to the same peptide antigen.

The three-dimensional structure of the complex of a second anti-peptide antibody (Fab 26/9) that recognizes the same six-residue epitope of an immunogenic peptide from influenza virus hemagglutinin (HA1; 75-110) as Fab 17/9 with the peptide has been determined at 2.8 A resolution. The amino acid sequence of the variable region of the 26/9 antibody differs in 24 positions from that of 17/9, the first antibody in this series for which several ligand-bound and free structures have been determined and refined. Comparison of the 26/9-peptide with the 17/9-peptide complex structures shows that the two Fabs are very similar (r.m.s.d. 0.5 to 0.8 A) and that the peptide antigen (101-107) has virtually the same conformation (r.m.s.d. 0.3 to 0.8 A) when bound to both antibodies. A sequence difference in the 26/9 binding pocket (L94; His in 26/9, Asn in 17/9) results in an interaction with a bound water molecule that is not seen in the 17/9 structures. Epitope mapping shows that the relative specificity of 26/9 and 17/9 antibodies for individual positions of the peptide antigen are slightly different. Amino acid substitutions in the peptide, particularly at position SerP107, are tolerated to different extents by 17/9 and 26/9. Structural and sequence analysis suggests that amino acid differences near the peptide-binding site are responsible for altering slightly the specificity of 26/9 for three peptide residues and illustrates how amino acid substitutions can modify antibody-antigen interactions and thereby modulate antibody specificity.

Amino Acid Sequence↗
PubMed1994

Crystal structure of the principal neutralization site of HIV-1.

The crystal structure of a complex between a 24-amino acid peptide from the third variable (V3) loop of human immunodeficiency virus-type 1 (HIV-1) gp 120 and the Fab fragment of a broadly neutralizing antibody (59.1) was determined to 3 angstrom resolution. The tip of the V3 loop containing the Gly-Pro-Gly-Arg-Ala-Phe sequence adopts a double-turn conformation, which may be the basis of its conservation in many HIV-1 isolates. A complete map of the HIV-1 principal neutralizing determinant was constructed by stitching together structures of V3 loop peptides bound to 59.1 and to an isolate-specific (MN) neutralizing antibody (50.1). Structural conservation of the overlapping epitopes suggests that this biologically relevant conformation could be of use in the design of synthetic vaccines and drugs to inhibit HIV-1 entry and virus-related cellular fusion.

Amino Acid Sequence↗
PubMed1994

Routes to catalysis: structure of a catalytic antibody and comparison with its natural counterpart.

The three-dimensional structure of a catalytic antibody (1F7) with chorismate mutase activity has been determined to 3.0 A resolution as a complex with a transition state analog. The structural data suggest that the antibody stabilizes the same conformationally restricted pericyclic transition state as occurs in the uncatalyzed reaction. Overall shape and charge complementarity between the combining site and the transition state analog dictate preferential binding of the correct substrate enantiomer in a conformation appropriate for reaction. Comparison with the structure of a chorismate mutase enzyme indicates an overall similarity between the catalytic mechanism employed by the two proteins. Differences in the number of specific interactions available for restricting the rotational degrees of freedom in the transition state, and the lack of multiple electrostatic interactions that might stabilize charge separation in this highly polarized metastable species, are likely to account for the observed 10(4) times lower activity of the antibody relative to that of the natural enzymes that catalyze this reaction. The structure of the 1F7 Fab'-hapten complex provides confirmation that the properties of an antibody catalyst faithfully reflect the design of the transition state analog.

Antibodies, Catalytic↗