Allen B. Edmundson

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.

High resolution structures of the 4–4-20 Fab-fluorescein complex in two solvent systems: effects of solvent on structure and antigen-binding affinity

Three-dimensional structures were determined for three crystal forms of the antigen binding fragment (Fab) of anti-fluorescein antibody 4-4-20 in complex with fluorescein. These included 1) a triclinic (P1) form crystallized in 47% (v/v) 2-methyl-2,4-pentanediol (MPD); 2) a triclinic (P1) form crystallized in 16% (w/v) poly(ethylene glycol), molecular weight 3350 (PEG); and 3) a monoclinic (P21) form crystallized in 16% PEG. Solvent molecules were added to the three models and the structures were refined to their diffraction limits (1.75-A, 1.78-A, and 2.49-A resolution for the MPD, triclinic PEG, and monoclinic PEG forms, respectively). Comparisons of these structures were interesting because 4-4-20 exhibited a lower antigen-binding affinity in 47% MPD (Ka = 1.3 x 10(8) M-1) than in either 16% PEG (Ka = 2.9 x 10(9) M-1) or phosphate-buffered saline (Ka = 1.8 x 10(10) M-1). Even though the solution behavior of the antibody was significantly different in MPD and PEG, the crystal structures were remarkably similar. In all three structures, the fluorescein-combining site was an aromatic slot formed by tyrosines L32, H96, and H97 and tryptophans L96 and H33. In addition, several active site constituents formed an electrostatic network with the ligand. These included a salt link between arginine L34 and one of fluoresceins enolate oxygen atoms, a hydrogen bond between histidine L27d and the second enolic group, a hydrogen bond between tyrosine L32 and the phenylcarboxylate group, and two medium range (approximately 5 A) electrostatic interactions with lysine L50 and arginine H52. The only major difference between the triclinic MPD and PEG structures was the degree of hydration of the antigen-combining site. Three water molecules participated in the above electrostatic network in the MPD structure, while eight were involved in the PEG structure. Based on this observation, we believe that 4-4-20 exhibits a lower affinity in MPD due to the depletion of the hydration shell of the antigen-combining site.

Autoantibody-catalyzed Hydrolysis of Amyloid β Peptide

We describe IgM class human autoantibodies that hydrolyze amyloid β peptide 1–40 (Aβ40). A monoclonal IgM from a patient with Waldenströms macroglobulinemia hydrolyzed Aβ40 at the Lys-28—Gly-29 bond and Lys-16—Ala-17 bonds. The catalytic activity was inhibited stoichiometrically by an electrophilic serine protease inhibitor. Treatment with the catalytic IgM blocked the aggregation and toxicity of Aβ40 in neuronal cell cultures. IgMs purified from the sera of patients with Alzheimer disease (AD) hydrolyzed Aβ40 at rates superior to IgMs from age-matched humans without dementia. IgMs from non-elderly humans expressed the least catalytic activity. The reaction rate was sufficient to afford appreciable degradation at physiological Aβ and IgM concentrations found in peripheral circulation. Increased Aβ concentrations in the AD brain are thought to induce neurodegenerative effects. Peripheral administration of Aβ binding antibodies has been suggested as a potential treatment of AD. Our results suggest that catalytic IgM autoantibodies can help clear Aβ, and they open the possibility of using catalytic Abs for AD immunotherapy.

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.

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.

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)

Three-dimensional structure of a human Fab with high affinity for tetanus toxoid

BACKGROUND The wide range of antibody specificity and affinity results from the differing shapes and chemical compositions of their binding sites. These shapes range from discrete grooves in antibodies elicited by linear oligomers of nucleotides and carbohydrates to shallow depressions or flat surfaces for accommodation of proteins, peptides and large organic compounds. OBJECTIVES To determine the Fab structure of a high-affinity human antitoxin antibody. To explore structural features which enable the antibody to bind to intact tetanus toxoid, peptides derived from the sequence of the natural immunogen and antigenic mimics identified by combinatorial chemistry. To explain why this Fab shows a remarkable tendency to produce crystals consistently diffracting to d spacings of 1.7-1.8 A. To use this information to engineer a strong tendency to crystallize into the design of other Fabs. STUDY DESIGN The protein was crystallized in hanging or sitting drops by a microseeding technique in polyethylene glycol (PEG) 8000. Crystals were subjected to X-ray analysis and the three-dimensional structure of the Fab was determined by the molecular replacement method. Interactive computer graphics were employed to fit models to electron density maps, survey the structure in multiple views and discover the crystal packing motif of the protein. RESULTS Exceptionally large single crystals of this protein have been obtained, one measuring 5 x 3 x 2 mm (l x w x d). The latter was cut into six irregular pieces, each retaining the features of the original in diffracting to high resolution (1.8 A) with little decay in the X-ray beam. In an individual Fab, the active site is relatively flat and it seems likely that the protein antigen and derivative peptides are tightly held on the outer surface without significant penetration into the interior. There is no free space to accommodate even a dipeptide between VH and VL. One of the unique features of the B7-15A2 Fab is a large aliphatic ridge dominating the center of the active site. The CDR3 of the H chain contributes significantly to this ridge, as well as to adjoining regions projected to be important for the docking of the antigen. Both the ease of crystallization and the favorable diffraction properties are mainly attributable to the tight packing of the protein molecules in the crystal lattice. DISCUSSION The B7-15A2 active site provides a stable and well defined platform for high affinity docking of proteins, peptides and their mimotopes. The advantages for future developments are suggested by the analysis of the crystal properties. It should be possible to incorporate the features promoting crystallization, close packing and resistance to radiation damage into engineered human antibodies without altering the desired specificities and affinities of their active sites.

Incorporation of Long CDR3s into V Domains: Implications for the Structural Evolution of the Antibody-Combining Site

Available data suggest that ‘primitive’ antibody-combining sites often include longer than average HCDR3s. Long HCDR3 sequences have been reported in diverse vertebrates, including humans, cattle, camels and sharks. These long HCDR3 segments contain unusual sequence features such as stretches of Gly or Pro residues and multiple Cys residues. We examined how longer than average HCDR3s were accommodated in the V domains of human, murine and camel antibodies with known three-dimensional structures. The main conclusions were that (1) HCDR3s longer than 12 residues should protrude outward from the V domains; (2) descending HCDR3 polypeptides may utilize VL (including LCDR3) constituents as a platform, supporting the protruding segments; (3) intra- and inter-HCDR disulfides are frequently formed to rigidify the structure of HCDR3 or the combining site, and (4) V and C domains were possibly more similar in primordial antibodies than they are in their present day counterparts.

Crystal structure of a glycosylated Fab from an IgM cryoglobulin with properties of a natural proteolytic antibody.

The 2.6 A (1 A=0.1 nm) resolution structure has been determined for the glycosylated Fab (fragment antigen binding) of an IgM (Yvo) obtained from a subject with Waldenströms macroglobulinaemia. Dynamic light scattering was used to estimate the gel point and monitor the formation of an ordered hydroscopic gel of Yvo IgM upon cooling. If a cryoglobulin forms gels in peripheral tissues and organs, the associated swelling and damage to microvasculature can result in considerable morbidity and mortality. The three-dimensional structure of the branched N-linked oligosaccharide associated with the CH1 domain (first constant domain of heavy chain) is reported. The carbohydrate may act to shield part of the lateral surface of the CH1 domain and crowd the junction between the CH1 and CH2 domains, thereby limiting the segmental flexibility of the Fab arms in intact Yvo IgM, especially at low temperatures. Recently, Yvo IgM was shown to have the properties of a naturally occurring proteolytic antibody [Paul, Karle, Planque, Taguchi, Salas, Nishiyama, Handy, Hunter, Edmundson and Hanson (2004) J. Biol. Chem. 279, 39611-39619; Planque, Bangale, Song, Karle, Taguchi, Poindexter, Bick, Edmundson, Nishiyama and Paul (2004) J. Biol Chem. 279, 14024-14032]. The Yvo protein displayed the ability to cleave, by a nucleophilic mechanism, the amide bonds of a variety of serine protease substrates and the gp120 coat protein of HIV. An atypical serine, arginine and glutamate motif is located in the middle of the Yvo antigen-binding site and displays an overall geometry that mimics the classical serine, histidine and aspartate catalytic triad of serine proteases. Our present findings indicate that pre-existing or natural antibodies can utilize at least one novel strategy for the cleavage of peptide bonds.

Diverse binding site structures revealed in homology models of polyreactive immunoglobulins

We describe here computer-assisted homology models of the combiningsite structure of three polyreactive immunoglobulins. Template-based modelsof Fv (VL–VH) fragments were derived forthe surface IgM expressed by the malignant CD5 positive B cells from threepatients with chronic lymphocytic leukaemia (CLL). The conserved frameworkregions were constructed using crystal coordinates taken from highlyhomologous human variable domain structures (Pot and Hil). Complementaritydetermining regions (CDRs) were predicted by grafting loops, taken fromknown immunoglobulin structures, onto the Fv framework models. The CDRtemplates were chosen, where possible, to be of the same length and of highresidue identity or similarity. LCDR1, 2 and 3 as well as HCDR1 and 2 forthe Fv were constructed using this strategy. For HCDR3 prediction, adatabase containing the Cartesian coordinates of 30 of these loops wascompiled from unliganded antibody X-ray crystallographic structures and anHCDR3 of the same length as that of the B CLL Fv was selected as a template.In one case (Yar), the resulting HCDR3 model gave unfavourable interactionswhen incorporated into the Fv model. This HCDR3 was therefore modelled usingan alternative strategy of construction of the loop stems, using apreviously described HCDR3 conformation (Pot), followed by chain closurewith a β-turn. The template models were subjected to positionalrefinement using energy minimisation and molecular dynamics simulations(X-PLOR). An electrostatic surface description (GRASP) did not reveal acommon structural feature within the binding sites of the three polyreactiveFv. Thus, polyreactive immunoglobulins may recognise similar and multipleantigens through a diverse array of binding site structures.