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K R Ely

Publications and source records attributed to K R Ely.

At least 37 records · Page 2Linked to original sources
PubMed1994

Recombinant human retinoic acid receptor beta. Binding of synthetic retinoids and transcriptional activation.

All-trans-retinoic acid mediates cell growth and differentiation by binding to and then activating nuclear retinoid receptor proteins that regulate gene transcription. Recombinant human retinoic acid receptor beta was cloned and expressed in Escherichia coli as a fusion protein rMBP-RAR beta with maltose-binding protein to facilitate purification. After isolation from bacterial lysates, rMBP-RAR beta was used for binding with selected retinoids. Scatchard analysis with [11,12-3H2]all-trans-retinoic acid gave a Kd of 0.34 nM. Competitive binding studies with a series of conformationally restricted aromatic retinoids indicated that the Ki values for binding to rMBP-RAR beta correlated with the logs of the EC50 values for gene transcriptional activation (p < or = 0.05) and with those for the relative activation compared to that of all-trans-retinoic acid (p < or = 0.01). Inspection of binding-activation correlation diagrams indicates candidate structures for improved retinoid agonists or antagonists.

Amino Acid Sequence↗
PubMed1994

Crystal structure of the tenth type III cell adhesion module of human fibronectin.

The crystal structure of the cell adhesion module of fibronectin (FNIII10) has been determined at 1.8 A resolution. A recombinant fragment corresponding to the tenth type III module of human fibronectin was crystallized in space group P2(1) with a = 30.7, b = 35.1 and c = 37.7 A and beta = 107 degrees. The structure was determined by molecular replacement and refined by least squares methods. The crystallographic R-factor for the final model of the 91 amino acid module plus 56 solvent atoms is 0.18 for 10 to 1.8 A data. The module consists of two layers of beta-sheet, one with three antiparallel strands and the other with four antiparallel strands. The beta-sheets enclose a hydrophobic core of 24 amino acid side-chains. The module contains the RGD cell recognition sequence in a flexible loop connecting two beta-strands. The tertiary structure of the FNIII10 module has been used to develop a structure-based sequence alignment of 17 type III modules in fibronectin based on the striking conservation of homologous hydrophobic residues. A similar pattern of homologous alternating hydrophobic residues is also evident in a comparison of type III modules in proteins unrelated to fibronectin such as cytokine receptors and muscle proteins.

Amino Acid Sequence↗
PubMed1993

Crystallization of the OP-G2 Fab fragment: a fibrinogen mimic with specificity for the platelet glycoprotein IIb/IIIa.

The OP-G2 monoclonal antibody binds to the platelet integrin, gpIIb/IIIa, in a mode that mimics fibrinogen binding. The specificity of this antibody is mediated by the third complementarity-determining region (CDR3) loop of the immunoglobulin heavy chain which contains a sequence (RYD) related to the RGD recognition sequence of fibrinogen. The OP-G2 Fab fragment has been crystallized by vapor diffusion from solutions containing polyethylene glycol and imidazole malate (pH 5.6). The crystals belong to space group P2(1)2(1)2 with a = 93.1, b = 83.8 and c = 53.7 A. One Fab molecule is present in the asymmetric unit. A complete data set to 2.0 A resolution has been collected.

Journal Article↗
PubMed1992

Control of translational repression by protein-protein interactions.

The coat protein of the RNA bacteriophage MS2 is a translational repressor and interacts with a specific RNA stem-loop to inhibit translation of the viral replicase gene. As part of an effort to dissect genetically its RNA binding function, mutations were identified in the coat protein sequence that suppress mutational defects in the translational operator. Each of the mutants displayed a super-repressor phenotype, repressing translation from the wild-type and a variety of mutant operators better than did the wild-type coat protein. At least one mutant probably binds RNA more tightly than wild-type. The other mutants, however, were defective for assembly of virus-like particles, and self-associated predominantly as dimers. It is proposed that this assembly defect accounts for their super-repressor characteristics, since failure to assemble into virus-like particles elevates the effective concentration of repressor dimers. This hypothesis is supported by the observation that deletion of thirteen amino acids known to be important for assembly of dimers into capsids also resulted in the same assembly defect and in super-repressor activity. A second class of assembly defects is also described. Deletion of two amino acids from the C-terminus of coat protein resulted in failure to form capsids, most of the coat protein having the apparent molecular weight expected of trimers. This mutant (dl-8) was completely defective for repressor activity, probably because of an inability to form dimers. These results point out the inter-dependence of the structural and regulatory functions of coat protein.

Amino Acid Sequence↗
PubMed1990

Three-dimensional structure of a hybrid light chain dimer: protein engineering of a binding cavity.

An attempt was made to engineer a binding site and check its structure by X-ray analysis. Two human light chains (Mcg and Weir), with "variable" domain sequences differing in 36 positions, were hybridized into a heterologous dimer and crystallized in ammonium sulfate by the same procedure used for the trigonal form of the Mcg dimer. The three-dimensional structure of the hybrid was determined at 3.5-A resolution by difference Fourier analysis, interactive model building with computer graphics and crystallographic refinement. In the heterologous dimer, the Weir protein behaved as the structural analog of the heavy chain in an antigen binding fragment, while the Mcg protein assumed the role of the light chain component. The hybrid and the Mcg dimer were closely similar in overall structure, an observation probably correlated with the deliberate cleavage of the intrachain disulfide bond in the variable domain of the Weir protein during the hybridization procedure. Examination of the crystal structure of the hybrid suggested that the cleavage resulted in the relaxation of restraints which might otherwise have interfered with the formation of an Mcg-like dimer. There were six substitutions among the residues lining the binding cavities of the hybrid and Mcg dimer. These substitutions significantly affected the sizes, shapes and binding properties of the two cavities.

Amino Acid Sequence↗
PubMed1989

Three-dimensional structure of a light chain dimer crystallized in water. Conformational flexibility of a molecule in two crystal forms.

The three-dimensional structure of an immunoglobulin light chain dimer (Mcg) crystallized in deionized water (orthorhombic form) was determined at 2.0 A resolution by phase extension and crystallographic refinement. This structure was refined side-by-side with that of the same molecule crystallized in ammonium sulfate (trigonal form). The dimer adopted markedly different structures in the two solvents. "Elbow bend" angles between pseudo 2-fold axes of rotation relating pairs of "variable" (V) and "constant" (C) domains were found to be 132 degrees in the orthorhombic form and 115 degrees in the trigonal form. Modes of association of the V domains and, to a lesser extent, the pairing interactions of the C domains were different in the two structures. Alterations in the V domain pairing were reflected in the shapes of the binding regions and in the orientations of the side-chains lining the walls of the binding sites. In the trigonal form, for instance, the V domain interface was compartmentalized into a main binding cavity and a deep pocket, whereas these spaces were continuous in the orthorhombic structure. Patterns of ordered water molecules were quite distinct in the two crystal types. In some cases, the solvent structures could be correlated with conformational changes in the proteins. For example, close contacts between V and C domains of monomer 1 of the trigonal form were not retained in orthorhombic crystals. Ordered water molecules filled the space created when the two domains moved apart.

Bence Jones Protein↗
PubMed1989

Synthetic site-directed ligands.

Complexes of nucleotides, peptides and aromatic hapten-like compounds with immunoglobulin fragments were studied by X-ray analysis. After tri- or hexanucleotides of deoxythymidylate were diffused into triclinic crystals of a Fab (BV04-01) with specificity for single-stranded DNA, extensive changes were detected throughout the structure of the protein. The Fab co-crystallized with a tri- or pentanucleotide in a different space group (monoclinic), an observation sometimes correlated with alterations in the structure of the 'native' protein. Structural analyses of the co-crystals are in progress for direct comparisons with the unliganded Fab. In crystals of a human (Mcg) Bence-Jones dimer, synthetic opioid peptides, chemotactic peptides or dinitrophenyl (DNP) derivatives could be diffused into a large conical binding cavity. The conformations of both the ligand and the protein were usually altered during the binding process. At the base of the cavity tyrosine residues could be displaced like trap-doors to permit entry of some opioid peptides and DNP compounds into a deep binding pocket. In co-crystals of the dimer and bis(DNP)lysine, two ligand molecules were bound in tandem, one in the main cavity and the second in the deep pocket. One ligand adopted an extended conformation, with the epsilon-DNP ring near the floor of the main cavity and the alpha-DNP group in solvent outside the binding site. There were no significant conformational changes in the protein. In contrast, the second ligand was very compact, with DNP rings immersed in the deep pocket, and the binding site was expanded to accommodate the oversized ligand. Peptides designed to be specific for the main cavity were incrementally constructed from minimal binding units by M. Geysen, G. Trippick, S. Rodda and their colleagues. A pentapeptide optimized for binding by this method was diffused into a crystal of the dimer and found by Fourier difference analysis to lodge exclusively in the main cavity as predicted. Binding regions in the BV04-01 Fab and the Mcg dimer were markedly different in size and shape. The Fab had a groove-type site, in which a layer of sidechains acted like a false floor over regions analogous to the cavity and deep pocket of the Bence-Jones dimer.

Antibodies, Monoclonal↗
PubMed1989

Cocrystallization of an immunoglobulin light chain dimer with bis(dinitrophenyl) lysine: tandem binding of two ligands, one with and one without accompanying conformational changes in the protein.

Previous studies showed that the Mcg dimer of immunoglobulin light chains bound bis(dinitrophenyl)lysine both in trigonal crystals and in solution. On prolonged storage in ammonium sulfate, mixtures of ligand and protein produced small trigonal cocrystals in low frequency. These crystals were nearly isomorphous with those of the unliganded dimer in which the subunits were covalently linked by an interchain disulfide bond. By difference Fourier analyses at 3.5 A resolution and subsequent crystallographic refinement, the cocrystals were found to contain molecules with two ligands aligned in tandem along the interface of the variable (V) domains of the protein. One ligand molecule adopted an almost fully extended conformation, with the epsilon-DNP ring situated near the floor, the alpha-carboxyl group directed toward the solvent at the entry, and the alpha-DNP ring outside the rim of the main cavity. As if architecturally designed, the ligand was located symmetrically between the two domains in an orientation that was compatible with both the unaltered structure of the cavity lining and with the known crystal packing interactions of neighboring protein molecules. The second ligand molecule in the cocrystal lodged in the deep pocket immediately under the floor of the main cavity. The ligand adopted a very compact conformation with the two DNP rings roughly antiparallel to each other. This molecule appeared to be semi-permanently sequestered in the pocket since it could not be dislodged by exhaustive perfusion with ammonium sulfate crystallizing media. Relative to its volume in the native dimer, the pocket was expanded to accommodate the oversized ligand. Within a single protein molecule, therefore, two types of binding of a flexible ligand were observed, one with and one without accompanying conformational changes in the protein. The number of cocrystals which could be produced was markedly increased if the interchain disulfide bond between the Mcg monomers was first reduced and alkylated.

Ammonium Sulfate↗
PubMed1987

The binding of opioid peptides to the Mcg light chain dimer: flexible keys and adjustable locks.

Enkephalins and beta-casomorphins (opioid peptides) were found to bind in a variety of conformations to a human light chain (Bence-Jones) dimer from a patient (Mcg) with amyloidosis. The peptides were diffused into crystals of the protein and their positions, relative occupancies and modes of binding were determined at 2.7 A resolution by difference Fourier analyses. Collectively, the opioid peptides occupied practically all of the available space in the concave, internal parts of the binding region, as well as flat or convex external surfaces around the rim of the binding cavity. Suitable ligands ranged in size from four to seven residues. As many as five residues could be accommodated inside the binding region, and there was space for at least four residues on the external surfaces. External binding was influenced by solvent effects and local packing interactions among adjacent protein molecules in the crystal lattice. In the enkephalin series the presence of amino-terminal tyrosine was necessary, but not sufficient for binding. [Met]-enkephalin, a pentapeptide, showed two different modes of binding in overlapping subsites. In one subsite, preferred over the second in a ratio of 1.3:1.0, the side chain of amino-terminal tyrosine penetrated through the floor of the main cavity to lodge in the deep binding pocket about 20 A from the entrance. The remainder of the peptide spanned the length of the main cavity in an extended conformation. In the second subsite the amino end was restricted to the main cavity and the peptide backbone turned abruptly upward at residue 3 to interact with external surfaces. An (Arg-6, Phe-7) heptapeptide extension of [Met]-enkephalin entered the deep pocket and assumed an extended conformation in the main cavity like the pentapeptide. Its last two residues flattened against the external surfaces. [Leu]-enkephalin and its analogues displayed a combination of internal and external binding like [Met]-enkephalin in its secondary subsite. Enkephalin analogues with D-amino acids in position 2 generally adopted conformations which were more convoluted than those in the L-isomers. Moreover, external interactions tended to be more prominent in the D-derivatives. The beta-casomorphin-7 heptapeptide penetrated into the deep pocket and traversed the main cavity in as extended a conformation as the presence of two proline residues would permit. On removal of the ligand there was an unexpected hysteresis effect involving permanent structural alterations in the walls of the binding region. beta-casomorphins-4 and -5 were bound in the main cavity with the carboxyl ends protruding from the entrance.(ABSTRACT TRUNCATED AT 400 WORDS)

Bence Jones Protein↗
PubMed1987

A mild method for the preparation of disulfide-linked hybrids of immunoglobulin light chains.

A method is described for the hybridization of immunoglobulin light chains (Bence-Jones proteins) from different patients. The interchain half-cystine residues in the light chains from one subject are converted into mixed disulfides with 2,2'-dithiodipyridine. In the Bence-Jones dimer from a second patient the interchain disulfide bond is reduced with dithiothreitol. A covalently linked hybrid molecule is produced by the reaction of the mixed disulfide with the reduced thiol. In favorable cases the mild treatment yields heterodimers which can be crystallized for X-ray diffraction studies. The procedure can also be employed for converting a monomer into a covalent dimer. The engineered dimer of one kappa chain (Jen) crystallizes in the same space group as an aggregate of monomers, but the unit cell is only one-third as large.

Bence Jones Protein↗
PubMed1986

Three-dimensional analyses of the binding of synthetic chemotactic and opioid peptides in the Mcg light chain dimer.

Synthetic peptides with chemotactic or opioid activity were bound to crystals of a light chain dimer and their three-dimensional structures and modes of binding were determined by X-ray analysis. The chemotactic series consisted of di- and tripeptides initiated with N-formylmethionine or N-formylnorleucine residues. Opioid peptides included the enkephalins and casomorphins ranging in length from four to seven residues. The binding region of the protein proved to be malleable in adjusting to the surface contours of the peptides. Aromatic contact residues, as well as polypeptide segments of hypervariable loops, moved to improve the complementarity with the ligands. The peptides were even more flexible and tended to conform fairly closely to the space and geometry available for occupancy in the binding sites. Binding interactions were not confined to the interior of the cavity. In both the chemotactic and opioid series, the carboxyl tails of the peptides encroached upon the outer surfaces of the rim and contributed to the binding energies for the protein-ligand complexes. The peptide bond in N-formylmethionyltryptophan was found to be in the energetically unfavourable cis configuration. There was also evidence for less severe distortions in peptide bond geometry when N-formyltripeptides were bound to the dimer.

Amyloidosis↗
PubMed1985

Pressure-induced conformational changes in a human Bence-Jones protein (Mcg).

The effect of high static pressures on the internal structure of the immunoglobulin light chain (Bence-Jones) dimer from the patient Mcg was assessed with measurements of intrinsic protein fluorescence polarization and intensity. Depolarization of intrinsic fluorescence was observed at relatively low pressures (less than 2 kbar), with a standard volume change of -93 mL/mol. The significant conformational changes indicated by these observations were not attributable to major protein unfolding, since pressures exceeding 2 kbar were required to alter intrinsic fluorescence emission maxima and yields. Fluorescence intensity and polarization measurements were used to investigate pressure effects on the binding of bis(8-anilino-naphthalene-1-sulfonate) (bis-ANS), rhodamine 123, and bis(N-methylacridinium nitrate) (lucigenin). Below 1.5 kbar the Mcg dimer exhibited a small decrease in affinity for bis-ANS (standard volume change approximately 5.9 mL/mol). At 3 kbar the binding activity increased by greater than 250-fold (volume change -144 mL/mol) and remained 10-fold higher than its starting value after decompression. With rhodamine 123 the binding activity showed an initial linear increase but plateaued at pressures greater than 1.5 kbar (standard volume change -23 mL/mol). These pressure effects were completely reversible. Binding activity with lucigenin increased slightly at low pressures (standard volume change -5.5 mL/mol), but the protein was partially denatured at pressures greater than 2 kbar. Taken in concert with the results of parallel binding studies in crystals of the Mcg dimer, these observations support the concept of a large malleable binding region with broad specificity for aromatic compounds.(ABSTRACT TRUNCATED AT 250 WORDS)

Acridines↗
PubMed1985

Binding of N-formylated chemotactic peptides in crystals of the Mcg light chain dimer: similarities with neutrophil receptors.

X-ray crystallographic techniques were used to study the modes of binding of N-formylated chemotactic peptides to the Mcg light chain (Bence-Jones) dimer. By difference Fourier analyses at 2.7-A resolution four N-formylated tripeptides were found to occupy similar sites in the main binding cavity of the dimer. In all cases the N-formyl group appeared to form a hydrogen bond with a phenolic hydroxyl group of a tyrosine residue (No. 38, monomer 1) at the base of the cavity. N-formylation was necessary, since di-, tri- and tetrapeptides with free alpha-amino groups failed to bind. Although methionine in ligand position 1 was optimal for binding, it could be replaced with norleucine. Position 2 was less critical, providing the side chain was bulky and hydrophobic (e.g. leucine, methionine or phenylalanine). An aromatic residue like phenylalanine was most favorable in position 3. These bound ligands were site-filling and wedge-shaped, with their side chains swept back toward the entrance of the cavity to conform to the space available for binding. In the binding site side chains and polypeptide segments of the hypervariable loops also moved in ways improving the complementarity between protein and ligand. The stereochemical requirements for binding were markedly similar to those found in interactions of neutrophil receptors with the same series of tripeptides. An N-formylated dipeptide, N-f-Met-Trp, was bound with equal occupancies in two overlapping subsites. In the deeper site the N-formyl group and methionine side chain were situated in positions comparable to those in the N-formyl tripeptides, but the peptide bond between methionine and tryptophan was in the cis configuration. In the outer site the corresponding peptide bond was in the energetically more favourable trans configuration.

Binding Sites↗
PubMed1985

Accessible intrachain disulfide bonds in hybrids of light chains.

The three-dimensional structure of the Mcg lambda-type Bence-Jones dimer crystallized in ammonium sulfate is known at 2.3-A resolution. A series of nine other human lambda-chains and two kappa-chains did not crystallize under the same conditions. After these proteins were hybridized with the Mcg light chain by the method of Peabody et al. [Biochemistry, 19, 2827 (1980)], however, crystals of six heterodimers were produced. Two of these (Mcg X Weir and Mcg X Hud) were suitable for X-ray analysis. The non-Mcg parental molecules in four of the crystallizable hybrids showed aberrant electrophoretic behavior after treatment with mild reducing agents. The results suggest that the intrachain disulfide bond in at least one domain (probably the variable domain) was susceptible to mild reductive cleavage in a significant proportion of light chains. Moreover, the loosening of the domain structure resulting from such disulfide cleavage in one parent appeared to promote the tendency of a hybrid molecule to crystallize.

Alkylation↗
PubMed1985

Unexpected similarities in the crystal structures of the Mcg light-chain dimer and its hybrid with the Weir protein.

The covalently linked hybrid of two human lambda-type light chains (Mcg and Weir) crystallizes as trigonal bipyramids in ammonium sulfate [Ely et al., Molec. Immun. 22, 85-92 (1985)]. While markedly different in appearance from the barrel-shaped crystals of the parental Mcg dimer, the bipyramids of the hybrid have the same space group: trigonal P3(1)21. Moreover, the unit cell dimensions are practically identical: a = 72.3 A in both proteins; c = 188.1 A in the hybrid and 185.9 A in the Mcg dimer. These observations imply that the crystal packing and the main features of the three-dimensional structures are closely similar in the Mcg X Weir hybrid and the Mcg dimer. The "constant" domains of the Mcg and Weir proteins belong to the same genetic subclass and were expected to interact in comparable ways in hybrids and parental dimers. However, the overall similarities in the "variable" domain pairs in the hybrid and Mcg dimer were completely unpredicted, since the amino acid sequences of the heterologous variable domains differ by 36 residues. By difference Fourier analysis the Weir light chain has been tentatively identified as monomer 1 (heavy-chain analogue) and the Mcg protein as monomer 2 (light-chain analogue) in the hybrid dimer. Substitutions in key positions in the hypervariable loops explain the differences in binding activity of the Mcg and Weir dimers. In the Mcg dimer bis(dinitrophenyl)lysine spans two relatively spacious subsites (A and B), with primary contacts involving tyrosines 34 and 38 of monomer 2. The Weir dimer, which does not bind dinitrophenyl ligands, has serine and phenylalanine in homologous positions. Moreover, the bilateral replacement of valine 48 and serine 91 in Mcg by leucine and methionine in the Weir dimer should effectively block access to subsite B. In the hybrid binding activity for bis(dinitrophenyl)lysine is restored because the Mcg light chain is present as the monomer 2 subunit.

Amino Acid Sequence↗
PubMed1984

A search for site-filling ligands in the Mcg Bence-Jones dimer: crystal binding studies of fluorescent compounds.

In trigonal crystals grown in 1.9 M ammonium sulfate buffered at pH 6.2, the Mcg light-chain (Bence-Jones) dimer has a highly aromatic binding cavity accessible to a wide range of hydrophobic and aromatic ligands. A search was made for site-filling ligands by diffusing compounds into the crystals and determining their locations, orientations and relative occupancies by difference Fourier analysis at 2.7-A resolution. 1-Anilinonaphthalene-8-sulfonate, a small ligand in comparison with the rest of the series, initially occupied a site in the main binding cavity. With time, however, this ligand changed its position to the deep binding pocket beyond the floor of the main cavity. The original binding site remained vacant, despite the presence of a large excess of ligand in the soaking solution. Ligands increasing in size from fluorescein to bis(N-methyl)acridine (lucigenin) to dimers of carboxytetramethylrhodamine were found to bind with stringent stereospecificity in the main cavity, but the mode of binding was different in each case. The dimer of the 6-isomer of carboxytetramethylrhodamine, in which the two carboxyl groups are in para positions on the phenyl moiety, proved to be an effective site-filling ligand. The differences in the binding properties of dimers of 5- and 6-carboxytetramethylrhodamine led to an explanation for isomeric discrimination in the binding site. There were extensive conformational changes in the binding cavity to accommodate the ligands, particularly 6-carboxytetramethylrhodamine. The second and third hypervariable loops proved very flexible, and moved in ways to expand the binding site. The side chains of key tyrosine and phenylalanine residues in the site were also highly mobile. Their orientations adjusted to optimize complementarity with the ligands. These conformational adjustments are consistent with the tenets of a limited neo-instructive theory of ligand binding.

Anilino Naphthalenesulfonates↗
PubMed1983

Three-dimensional structure of the Mcg IgG1 immunoglobulin.

The three-dimensional structure of an IgG1(lambda) immunoglobulin from a patient (Mcg) with amyloidosis was determined at 6.5-A resolution with X-ray diffraction techniques. The protein crystallized from water in the space group C2221, with a = 87.8, b = 111.3 and c = 186.3 A; the crystallographic asymmetric unit was a half-molecule consisting of one light and one heavy chain. The structure was solved by the multiple isomorphous replacement method with five heavy-atom derivatives. Electron density maps were interpreted with the aid of a protein modeling system used in conjunction with an Evans and Sutherland Picture System II graphics station. IgG1 molecules were tightly packed in the crystal lattice, with numerous intermolecular contacts. The two-fold axis relating identical halves of each molecule was found to be parallel to the y crystallographic axis. Electron density modules collectively representing one molecule were identified as three lobes representing the two antigen-binding (Fab) arms and the Fc region. An interchain disulfide bond connecting the two CL domains was located on the molecular diad and used as a landmark in the interpretation of the electron density map. A computer graphics method was developed to produce a solid image model of the IgG1 molecule in any prescribed orientation.

Computers↗