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L M Amzel

Publications and source records attributed to L M Amzel.

At least 19 recordsLinked to original sources
PubMed1992

Three-dimensional structure of an angiotensin II-Fab complex at 3 A: hormone recognition by an anti-idiotypic antibody.

The elucidation of bioactive conformations of small peptide hormones remains an elusive goal to structural chemists because of the inherent flexibility of these molecules. Angiotensin II (AII), the major effector of the renin-angiotensin system, is an octapeptide hormone for which no clear structural models exist. Peptide hormones such as AII share the property that they bind to their receptors with high affinities, in spite of the fact that they must overcome an extremely large conformational entropy barrier to bind in one conformation. A "surrogate system" that consists of a high-affinity monoclonal antibody (MAb) and AII has been used to study a bound conformation of AII. The crystallographic structure of the complex reveals a structure of AII that is compatible with predicted bioactive conformations of AII derived from structure-activity studies and theoretical calculations. In the complex, the deeply bound hormone is folded into a compact structure in which two turns bring the amino and carboxyl termini close together. The antibody of this complex (MAb 131) has the unusual property that it was not generated against AII, but rather against an anti-idiotypic antibody reactive with a MAb to AII, which renders this antibody an anti-anti-idiotypic antibody. The high affinity for AII of the original MAb to AII was passed on to MAb 131 through a structural determinant on the anti-idiotypic antibody. Strikingly, the conformation of AII in this complex is highly similar to complementarity determining region loops of antibodies, possibly indicating that a true molecular mimic of bound AII was present on the anti-idiotypic antibody against which MAb 131 was elicited.

Amino Acid Sequence↗
PubMed1992

Recognition of angiotensin II: antibodies at different levels of an idiotypic network are superimposable.

Genetic and sequence information are reported for an angiotensin II-reactive antibody (Ab1, MAb 110) and an anti--anti-idiotypic antibody (Ab3, MAb 131) that have identical antigen binding properties and that are related by an anti-idiotypic antibody (Ab2-beta) that satisfies accepted biochemical criteria for an internal image-bearing antibody. The sequences of the variable regions of the Ab3 and of the Ab1 are nearly identical, even though the Ab1 is an antibody to a peptide and the Ab3 is an antibody to a globular protein. Significantly, amino acid residues that make critical contacts with antigen in the crystal structure of the Ab3-antigen complex are highly conserved in Ab1, suggesting that the epitopes of the Ab2-beta recognized by the Ab3 do indeed resemble the bound structure of the antigen.

Amino Acid Sequence↗
PubMed1992

ATP synthase: structure-function relationships.

Recent work has focused on obtaining a better understanding of the three-dimensional structural relationships between the alpha and beta subunits of the F1 moiety and the location of nucleotide binding domains within these subunits. Four types of approach are currently being pursued: X-ray crystallographic, chemical, molecular biological and biochemical. Here we briefly review some of the major conclusions of these studies, and point out some of the problems that must be resolved before an adequate model that relates structure to function in the ATP synthase molecule can be formulated.

Amino Acid Sequence↗
PubMed1992

Quaternary structure of ATP synthases: symmetry and asymmetry in the F1 moiety.

It has been proposed that during ATP synthesis/hydrolysis F1 ATPases experience a complex pattern of nucleotide binding and release during the catalytic cycle (binding change mechanism). This type of mechanism has implications that can be correlated with the structure of the enzyme. F1-ATPases (stoichiometry alpha 3 beta 3 gamma delta epsilon) are essentially a symmetrical trimer of pairs of the major subunits (alpha and beta); the minor subunits (gamma, delta and epsilon) are in single copies and interact with the trimer in an asymmetrical fashion. The asymmetry introduced by the minor subunits has important structural and functional consequences: (1) it introduces differences between the potentially equivalent binding and catalytic sites in the major subunits, (2) it restricts the ways in which a binding change mechanism can occur, and (3) it governs the way in which the F1 interacts with the (asymmetrical) F0 sector.

Models, Molecular↗
PubMed1991

Mitochondrial ATP synthase. Quaternary structure of the F1 moiety at 3.6 A determined by x-ray diffraction analysis.

The F1 moiety of the mitochondrial ATP synthase is composed of five different subunits with stoichiometry alpha 3 beta 3 gamma delta epsilon and exhibits the capacity to synthesize ATP from ADP and Pi. We have previously crystallized rat liver F1 and described its structure at 9-A resolution (Amzel, L. M., McKinney, M., Narayanan, P., and Pedersen, P. L. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 5852-5856). Here we present an x-ray map of this complex enzyme at 3.6 A, which provides a much more informative description of its quaternary structure. The overall dimensions of the F1 molecule are 120 A x 120 A x 74 A. The enzyme exhibits 3-fold symmetry relating the three copies of each of the two major subunits, alpha and beta. As the alpha subunits (but not the beta subunits) contain cysteine residues, it has been possible to identify the alpha subunits by heavy atom labeling with mersalyl and to relate their positions in the F1 molecule to the beta subunits. Significantly, the alpha and beta subunits each exist as trimeric arrays which are organized in two slightly offset, interdigitated layers along the 3-fold axis. In one trimeric layer the alpha subunits are located close to the axis with homologous subunits interacting with each other; in the other trimeric layer the beta subunits are far from the axis, and they interact only with alpha subunits and not with one another. At one end of the structure, part of the interface between each alpha and beta subunit encloses a space or "pocket" that is accessible to the solvent; at the other end the interfaces between the subunits are more open and exposed. The present work represents the highest resolution map reported to date for the F1 moiety of an ATP synthase complex.

Animals↗
PubMed1991

Structure of human beta 1 beta 1 alcohol dehydrogenase: catalytic effects of non-active-site substitutions.

The three-dimensional structure of human beta 1 beta 1 alcohol dehydrogenase (ADH; EC 1.1.1.1) complexed with NAD+ has been determined by x-ray crystallography to 3.0-A resolution. The amino acids directly involved in coenzyme binding are conserved between horse EE and human beta 1 beta 1 alcohol dehydrogenase in all but one case [serine (horse) vs. threonine (human) at position 48]. As a result, the coenzyme molecule is bound in a similar manner in the two enzymes. However, the strength of the interactions in the vicinity of the pyrophosphate bridge of NAD+ appears to be enhanced in the human enzyme. Side-chain movements of Arg-47 and Asp-50 and a shift in the position of the helix comprising residues 202-212 may explain both the decreased Vmax and the decreased rate of NADH dissociation observed in the human enzyme vs. the horse enzyme. It appears that these catalytic differences are not due to substitutions of any amino acids directly involved in coenzyme binding but are the result of structural rearrangements resulting from multiple sequence differences between the two enzymes.

Alcohol Dehydrogenase↗
PubMed1990

Crystallization and preliminary x-ray analysis of soybean lipoxygenase-1, a non-heme iron-containing dioxygenase.

Crystals of lipoxygenase-1 from soybeans have been grown by the method of vapor diffusion in the presence of sodium formate, ammonium acetate, and lithium chloride at pH 7.0. This enzyme contains a non-heme iron and is closely related to a human lipoxygenase found in leukocytes that participates in the biosynthesis of leukotrienes and lipoxins. The crystals are monoclinic space group C2 with cell dimensions of a = 183.8 A, b = 123.2 A, c = 94.3 A and beta = 102.9 degrees. They diffract beyond 2.7 A, are stable for several days in the x-ray beam, and appear to be suitable for x-ray diffraction studies.

Chromatography, High Pressure Liquid↗
PubMed1989

Crystallization and preliminary X-ray diffraction data of an anti-angiotensin II Fab and of the peptide-Fab complex.

mAb-131 is a monoclonal antibody that binds with high affinity (K alpha = 7.4 x 10(9) M-1) to the 8-residue peptide hormone angiotensin II, the major effector of the renin/angiotensin system. mAb-131 is a member of a well characterized idiotypic antibody network since it was raised as an anti-anti-idiotype of an antibody raised against angiotensin II. mAb-131 Fabs prepared with papain contain four major charge isoforms that can be separated by pH gradient elution from an anion-exchange column. Diffraction quality isomorphous crystals of two of the isoforms and of the Fab.peptide complexes have been grown. The crystals diffract to 3.5 A resolution, are tetragonal, space group P4(1) (or P4(3] with cell dimensions a = b = 78.6 A, c = 125.2 A, and have two Fab molecules per asymmetric unit. By using a different buffer, a second crystal form has been grown which diffracts to 3.3 A. It also belongs to space group P4(1) (or P4(3] but has cell dimensions of a = b = 109.6 A and c = 125.2 A. Knowledge of the three-dimensional structure of this Fab and of the peptide.Fab complex will give insight into two problems: 1) the recognition of small peptide hormones (which exist as random coils in solution) with high affinity by proteins, and 2) the nature of conservation of antibody combining sites in idiotypic networks.

Angiotensin II↗
PubMed1989

Crystallization and preliminary X-ray diffraction studies of an anti-4-hydroxy-3-nitrophenylacetic acid monoclonal antibody Fab fragment complexed with immunizing and heteroclitic haptens.

The Fab fragment of the anti-4-hydroxy-3-nitrophenylacetic acid monoclonal antibody, 88C6/12 has been crystallized in the presence of the eliciting hapten, 4-hydroxy-3-nitrophenacetyl-epsilon-aminocaproic acid (NP-aminocap) and the heteroclitic iodinated analog, 4-hydroxy-3-iodo-5-nitrophenylacetyl-epsilon-aminocaproic acid (NIP-aminocap). Crystals obtained by precipitation with 32% (w/v) polyethylene glycol 3400 in the presence of 40 to 400 microM of either NP-aminocap or NIP-aminocap, belong to the orthorhombic space group P2(1)2(1)2(1) with cell dimensions a = 81.2 A, b = 86.9 A, c = 131.1 A. The cell volume suggests the presence of two molecules of the complex per asymmetric unit. Analysis of the Patterson function indicates that these two molecules are related by a local 2-fold axis parallel to the crystallographic b axis located at x = 0.218 and z = 0.25.

Antibodies, Monoclonal↗
PubMed1989

Recognition in cell adhesion. A comparative study of the conformations of RGD-containing peptides by Monte Carlo and NMR methods.

Many of the proteins that mediate cell adhesion processes processes-fibronectin, fibrinogen, vitronectin, von Willebrand factor, osteopontin, laminin and various collagens--contain the amino acid sequence Arg-Gly-Asp. Short peptides that include this sequence have been shown to inhibit the interactions of cell adhesion proteins with their receptors and to have dramatic effects on developmental processes involving cellular recognition. In order to determine which conformations are accessible to Arg-Gly-Asp containing peptides, we analyzed tri-, tetra- and pentapeptides using molecular mechanics and Monte Carlo methods, and studied the solution conformations of the pentapeptide Gly-Arg-Gly-Asp-Ser using nuclear magnetic resonance techniques. The Monte Carlo method was used to: (a) identify the low energy conformations of the peptides and (b) evaluate their thermodynamic properties. In the case of the pentapeptide Gly-Arg-Gly-Asp-Gly, the four stable conformations include three with reverse turns and one open structure. The conformations found in this analysis are compatible with the nuclear magnetic resonance (nuclear Overhauser effect) data.

Amino Acid Sequence↗
PubMed1988

ATP synthases--structure of the F1-moiety and its relationship to function and mechanism.

A great deal of progress has been made in understanding both the structure and the mechanism of F1-ATPase. The primary structure is now fully known for at least five species. Sequence comparison between chloroplast, photobacteria, aerobic bacteria, and mitochondrial representatives allow us to infer more general functional relationships and evolutionary trends. Although the F1 moiety is the most studied segment of the H+-ATPase complex, there is not a full understanding of the mechanism and regulation of its hydrolytic activity. The beta subunit is now known to contain one and probably two nucleotide binding domains, one of which is believed to be a catalytic site. Recently, two similar models have been proposed to attempt to describe the "active" part of the beta subunits. These models are mainly an attempt to use the structure of adenylate kinase to represent a more general working model for nucleotide binding phosphotransferases. Labelling experiments seem to indicate that several critical residues outside the region described by the "adenylate kinase" part of this model are also actively involved in the ATPase activity. New models will have to be introduced to include these regions. Finally, it seems that a consensus has been reached with regard to a broad acceptance of the asymmetric structure of the F1-moiety. In addition, recent experimental evidence points toward the presence of nonequivalent subunits to describe the functional activity of the F1-ATPase. A summary diagram of the conformational and binding states of the enzyme including the nonequivalent beta subunit is presented. Additional research is essential to establish the role of the minor subunits--and of the asymmetry they introduce in F1--on the physiological function of the enzyme.

Amino Acid Sequence↗
PubMed1988

A molecular-mechanics study of the conformation of the interchain disulfide of human immunoglobulin G4 (IgG4).

The conformation of the interchain disulfide bond between the light and the heavy chains of human immunoglobulin G4 (IgG4) was modeled based on the known structure of a human IgG1 Fab. Exploration of a large number of conformations followed by energy minimization using molecular mechanics methods rendered a plausible model for the disulfide. In this model the disulfide adopts the long, trans-gauche-trans configuration found in immunoglobulin intrachain bridges and not the conformations most commonly observed in other proteins (left-handed spiral or right-handed hook). Heavy chain residues H217 and H218 pack tightly against the disulfide and put restrictions on which sequences can exist in the proposed conformation. Sequence analysis at these positions shows that: (a) all human IgG4 and IgG3 are compatible with the model; (b) IgG2 and human immunoglobulins of other classes cannot adopt the conformation proposed for IgG4.

Disulfides↗
PubMed1988

Cell volume regulation by the thin descending limb of Henle's loop.

Thin descending limb cells from Henle's loop (from the inner strip of the outer medulla of long loops) were studied with optical and video techniques to identify the mechanisms of ion transport and cell volume regulation. Increasing the K+ concentration in the basolateral solution from 5 to 90 mM caused the cells to swell. This K+-induced swelling was inhibited by exposure of the basolateral membrane to 9 mM Ba2+ and was abolished by removing Cl- from the perfusion solutions. Decreasing the perfusion osmolality caused an increase in cell volume followed by a return to the preexposure volume. The latter regulatory decrease in hypoosmolality was slowed by basolateral Ba2+ and the removal of HCO-3 from the solutions. Further slowing occurred when both HCO-3 and Cl- were removed. Exposure of cells to ouabain abolished volume regulation. These data suggest that the basolateral cell membrane of the thin descending limb has a Cl- -dependent K+ permeability, which is important in cell volume regulation. The cells also possess Cl- and HCO-3 transport pathways that participate in volume regulation. Finally, volume regulation is dependent upon the operation of the Na/K pump.

Animals↗
PubMed1986

Preliminary crystallographic X-ray data for an NAD(P)H:quinone reductase from mouse liver.

The hydrophilic form of NAD(P)H:(quinone acceptor) oxidoreductase (EC 1.6.99.2) from mouse liver cytosol has been crystallized in the presence of FAD. X-ray diffraction analysis showed these crystals to be tetragonal and to belong to the I422 space group. The unit cell measured a = b = 246.5 A and c = 70.4 A. Since the subunit molecular weight is 30,000, the asymmetric unit probably contains two pairs of dimers.

Animals↗