Structure Of Fab Research Articles

Crystal structure of the antigen-binding fragment of the murine anti-arsonate monoclonal antibody 36-71 at 2.9-A resolution.

The structure of the antigen-binding fragment (Fab) of an anti-phenylarsonate monoclonal antibody (36-71) bearing a major crossreacting idiotype of A/J mice has been solved and refined to an R factor of 19.3% at a resolution of 2.9 A. An initial electron density map was obtained with phase information from a total of six isomorphous heavy-atom derivatives (from two different compounds) and a molecular replacement solution using the HED10 Fab crystal structure as a model. The structure of the McPC603 Fab was used to provide an initial set of atomic coordinates. The electron density maps are clear and easily interpretable for the entire sequence except for sections from two of the heavy-chain complementarity-determining regions totaling 21 residues. These residues have been left out of the refinement and are not represented in our current model. The antigen-combining site was located by means of a difference Fourier synthesis with one of the heavy-atom derivatives, which contained arsanilic acid. It lies in a small pocket formed by residues from the hypervariable regions of both the heavy and the light chains. Interactions with the hapten from framework residues are also possible.

Three‐dimensional structure of a fluorescein–Fab complex crystallized in 2‐methyl‐2,4‐pentanediol

The crystal structure of a fluorescein-Fab (4-4-20) complex was determined at 2.7 A resolution by molecular replacement methods. The starting model was the refined 2.7 A structure of unliganded Fab from an autoantibody (BV04-01) with specificity for single-stranded DNA. In the 4-4-20 complex fluorescein fits tightly into a relatively deep slot formed by a network of tryptophan and tyrosine side chains. The planar xanthonyl ring of the hapten is accommodated at the bottom of the slot while the phenylcarboxyl group interfaces with solvent. Tyrosine 37 (light chain) and tryptophan 33 (heavy chain) flank the xanthonyl group and tryptophan 101 (light chain) provides the floor of the combining site. Tyrosine 103 (heavy chain) is situated near the phenyl ring of the hapten and tyrosine 102 (heavy chain) forms part of the boundary of the slot. Histidine 31 and arginine 39 of the light chain are located in positions adjacent to the two enolic groups at opposite ends of the xanthonyl ring, and thus account for neutralization of one of two negative charges in the haptenic dianion. Formation of an enol-arginine ion pair in a region of low dielectric constant may account for an incremental increase in affinity of 2-3 orders of magnitude in the 4-4-20 molecule relative to other members of an idiotypic family of monoclonal antifluorescyl antibodies. The phenyl carboxyl group of fluorescein appears to be hydrogen bonded to the phenolic hydroxyl group of tyrosine 37 of the light chain. A molecule of 2-methyl-2,4-pentanediol (MPD), trapped in the interface of the variable domains just below the fluorescein binding site, may be partly responsible for the decrease in affinity for the hapten in MPD.

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.

Canonical structures for the hypervariable regions of immunoglobulins

We have analysed the atomic structures of Fab and V L fragments of immunoglobulins to determine the relationship between their amino acid sequences and the three-dimensional structures of their antigen binding sites. We identify the relatively few residues that, through their packing, hydrogen bonding or the ability to assume unusual φ, ψ or ω conformations, are primarily responsible for the main-chain conformations of the hypervariable regions. These residues are found to occur at sites within the hypervariable regions and in the conserved β-sheet framework. Examination of the sequences of immunoglobulins of unknown structure shows that many have hypervariable regions that are similar in size to one of the known structures and contain identical residues at the sites responsible for the observed conformation. This implies that these hypervariable regions have conformations close to those in the known structures. For five of the hypervariable regions, the repertoire of conformations appears to be limited to a relatively small number of discrete structural classes. We call the commonly occurring main-chain conformations of the hypervariable regions “canonical structures”. The accuracy of the analysis is being tested and refined by the prediction of immunoglobulin structures prior to their experimental determination.

Three-Dimensional Structure of an Antigen-Antibody Complex at 2.8 Å Resolution

The 2.8 A resolution three-dimensional structure of a complex between an antigen (lysozyme) and the Fab fragment from a monoclonal antibody against lysozyme has been determined and refined by x-ray crystallographic techniques. No conformational changes can be observed in the tertiary structure of lysozyme compared with that determined in native crystalline forms. The quaternary structure of Fab is that of an extended conformation. The antibody combining site is a rather flat surface with protuberances and depressions formed by its amino acid side chains. The antigen-antibody interface is tightly packed, with 16 lysozyme and 17 antibody residues making close contacts. The antigen contacting residues belong to two stretches of the lysozyme polypeptide chain: residues 18 to 27 and 116 to 129. All the complementarity-determining regions and two residues outside hypervariable positions of the antibody make contact with the antigen. Most of these contacts (10 residues out of 17) are made by the heavy chain, and in particular by its third complementarity-determining region. Antigen variability and antibody specificity and affinity are discussed on the basis of the determined structure.

The galactan‐binding immunoglobulin Fab J539: An x‐ray diffraction study at 2.6‐Å resolution

The crystal structure of the Fab of the galactan-binding immunoglobulin J539 (a mouse IgA,kappa) has been determined at a resolution of approximately 2.6 A by X-ray diffraction. The starting model was that obtained from the real space search described previously (Navia, M.A., Segal, D.M., Padlan, E.A., Davies, D.R., Rao, D.N., Rudikoff, S. and Potter, M. "Crystal structure of galactan-binding mouse immunoglobulin J539 Fab at 4.5 A resolution." Proc. Nat. Acad. Sci. USA, 76:4071-4074, 1979). This Fab structure has now been refined by restrained least-squares procedures to an R-value of 19% for the 11,690 unique reflections between 8.0 A and 2.6 A. The rms deviation from ideal bond lengths is 0.025 A. The overall structure differs from McPC603 Fab, another mouse IgA,kappa antibody, in that the elbow bend, relating the variable and constant parts of the molecule, is 145 degrees vs. 133 degrees for McPC603. The region of the molecule expected to be the antigen binding site contains a large cavity with two clefts leading away from it. This has been fitted with a model of an oligo-galactan.

Preliminary crystallographic study of the Fab fragments of two monoclonal anti-2-phenyloxazolone antibodies.

We report on the preparation, crystallization, and preliminary x-ray crystallographic study of the Fab fragments of two monoclonal anti-2-phenyloxazolone antibodies obtained from the secondary response to this hapten. The Fab fragment from one of these (NQ10/12.5) has been crystallized from polyethylene glycol 8000 solutions in a form suitable for high-resolution x-ray crystallographic studies. These crystals are monoclinic, space group C2, with a = 129.2 A, b = 79.4 A, c = 57.7 A, beta = 96.2 degrees, and one Fab/asymmetric unit. Determination of the three-dimensional structure of Fab NQ10/12.5 should help clarify the role of somatic mutation in the maturation of an immune response. This antibody and an anti-lysozyme antibody also under study apparently use the same germ-line encoded VK and a similar VH gene, respectively, as the idiotypic anti-oxazolone antibodies characteristic of the primary response. A comparative study of the two structures should shed light on the role of the pairing of heavy and light chains in the antigen-binding function of antibodies.

On the specificity of antibody/antigen interactions: Phosphocholine binding to McPC603 and the correlation of three-dimensional structure and sequence data

Refined three-dimensional structures of McPC603 Fab and the complex with phosphocholine permit a detailed assessment of the residues crucial to determining the antibody specificity. Correlation with sequence data suggests that the structure of the binding site is highly conserved in immunoglobulins with phosphocholine-binding specificity. There is suggestive evidence that coupling of somatic mutations occurs to preserve antigen-binding specificity. The immune response is characterized by specificity and diversity. While each antibody appears to be specific for a single antigen, the immune response can generate up to 10(9) different specificities. In order to understand, at the molecular level, the nature of the interaction between antibody and antigen, it is necessary to have a high-resolution three-dimensional picture of the complex. Today it is possible to investigate antibody/antigen interactions directly by the crystallographic analysis of hybridoma products [5, 10]; in the past, structural studies were limited to myeloma proteins which, in some cases, could be shown to complex to certain haptens. Of the four Fab structures that have been determined by X-ray diffraction, only two have been demonstrated to bind hapten in the crystal. They are Fab NEW, which was shown to bind a vitamin K1 derivative [1] and McPC603, which binds to phosphocholine [6,9]. During the last few years, the McPC603 Fab structure has been refined at 2.7 A resolution and the complex of McPC603 Fab with phosphocholine has been refined independently at 3.1 A. In this communication, we make a comparative analysis of the sequences of a number of mouse phosphocholine-binding immunoglobulins based on the refined structure of the phosphocholine-binding site in McPC603.

Electron microscopy of rabbit FC crystals

Proteolytic digestion of the immunoglobulin IgG with papain cleaves the molecule into an antigen binding fragment, Fab, and a compliment binding fragment, Fc. Structures of intact immunoglobulin, Fab and Fc from various sources have been solved by X-ray crystallography. Rabbit Fc can be crystallized as thin platelets suitable for high resolution electron microscopy. The structure of rabbit Fc can be expected to be similar to the known structure of human Fc, making it an ideal specimen for comparing the X-ray and electron crystallographic techniques and for the application of the molecular replacement technique to electron crystallography. Thin protein crystals embedded in ice diffract to high resolution. A low resolution image of a frozen, hydrated crystal can be expected to have a better contrast than a glucose embedded crystal due to the larger density difference between protein and ice compared to protein and glucose. For these reasons we are using an ice embedding technique to prepare the rabbit Fc crystals for molecular structure analysis by electron microscopy.

Isotypes of IgG: Comparison of the primary structures of three pairs of isotypes which differ in their ability to activate complement

The comparison of three pairs of isotypic Cγ‡ primary structures is made with reference to the known crystallographic structure of Fab and Fc from human IgGl. Contiguous residue positions are assigned to sub-regions based on their occurrence in secondary structures, extended chains or the loops which connect them, in the crystallographic models. Simple inspection reveals that residue positions having identical amino acids in all six Cγ are found mainly in extended chain sub-regions forming parts of β-sheets. However, average structural dissimilarity (ASD) values in loops which mediate V H to Cγl and Cγ2-Cγ3 interactions are as low as those obtained for extended chain subregions, implying that these structures are conserved among IgGs. In contrast, loop structures which are candidates for Cγl-Cγ2 interactions show high values of ASD, indicating little structural homology is conserved. Longitudinal Fab-Fc interactions, if they occur at all, probably occur via remarkably different residue contacts in different IgGs. Our analysis yields no clear-cut evidence for a pathway by which isotypic IgGs diverged in biologic effector functions. Great variation in primary structure is seen in the ‘hinge’ region, and in some loop and extended chain subregions exposed to solvent in the Cγ2 Cγ2 domain. These variations may account for the ability or inability of the six IgGs compared to interact with the first component of complement (C1) since some structural features in those regions are shared among those Fcs which bind C1. We speculate that the C1. binding site is determined by the extended chain formed by residues Lys-290 to Glu-295 of human IgGl heavy chain.

Crystal structure of galactan-binding mouse immunoglobulin J539 Fab at 4.5-A resolution.

An electron-density map of the mouse galactan-binding immunoglobulin J539 (IgA2,kappa) Fab has been calculated to a resolution of 4.5 A by the method of heavy atom isomorphous replacement with four derivatives. The map has been interpreted with the aid of a computer program which systematically searched for the best fit between the electron-density map and the known coordinates of individual immunoglobulin domains. The quaternary structure of J539 Fab at this resolution appears similar to that of another mouse immunoglobulin, IgA2,kappa Fab, McPC603. The model coordinates for J539 Fab should allow us to proceed directly to a high-resolution structure determination without further heavy atom isomorphous replacement.

Magnetic resonance studies of the binding site interactions between phosphorylcholine and specific mouse myeloma immunoglobulin.

The interaction of phosphorycholine-binding mouse myeloma protein M603 and the isotopically substituted hapten phosphoryl[methyl-13C] choline has been investigated using 13C and 31P nuclear magnetic resonance (NMR) spectroscopy. Upon binding to antibody, upfield shifts of 0.7 and 1.5 ppm are observed for the hapten 13C and 31P resonances, respectively, and both spectra are in the "slow" exchange limit. Linewidth analysis indicates some immobilization of the phosphate group but essentially unrestricted methyl group rotation for the bound hapten. Hapten-antibody dissociation rate constants of 10 and 38 s-1 are calculated from 13C and 31P NMR spectra, respectively, suggesting the possibility of differential dissociation rates for the two opposing ends of the phosphorylcholine molecule. The NMR data are entirely consistent with the known x-ray structure of the M603 Fab'-phosporylcholine complex (Segal,D.M., Padlan, E.A., Cohen G.H., Rudikoff S., Potter,M., and Davies, D.R. (1974), Proc. Natl. Acad. Sci. U.S.A. 71, 4298).

Crystallographic structural studies of a human Fc-fragment. I. An electron-density map at 4 A resolution and a partial model.

The crystal structure of a human Fc fragment was analysed at 4 A resolution. A partial interpretation of the electron-density map in terms of domain structure was possible. The molecule has the shape of a mickey mouse. The spherical domain was interpreted visually and by domain Patterson function interpretation as the CH3 dimer. This dimer resembles closely the CH1-CL dimer found in Fab structures. The ellipsoidal "ears" of the molecule represent the CH2 domains. They are widely separated from each other, but closely connected to CH3. Their tertiary structure must be different from CH1, as Patterson domain interpretations were unsuccessful. A chain tracing in CH2 was not yet possible.

The three-dimensional structure of a phosphorylcholine-binding mouse immunoglobulin Fab and the nature of the antigen binding site.

The structure of the Fab of McPC 603, a mouse myeloma protein with phosphorylcholine binding activity, has been determined to 3.1-A resoltuion. The four domains are found to be structurally similar with a well-defined double-layer structure. A large cavity exists at one end of the fragment, the walls of which are formed exclusively of hypervariable residues. Phosphorylcholine binds in this cavity and forms specific interactions with several well-defined amino-acid side chains of the protein. The hapten is bound asymmetrically and interacts more with the heavy chain than with the light chain.

Studies on Fd-fragments of human immunoglobulins: II. The preparation and characterization of peptic (Fab′) 2-fragments and an Fd′-fragment of IgA-myeloma proteins

Pepsin digestion of unfolded human 7 S IgA1 myeloma proteins and of an unfolded IgA2 (1+) protein produced, under certain conditions, fragments which are similar with respect to gel filtration. The fragments contained light chains and part of the α-chains as judged by immunodiffusion experiments and urea starch gel electrophoresis. From approximate molecular weight determinations by thin layer gel filtration together with characterization of the fragments by ultracentrifuge experiments, it was concluded that (Fab′) 2-like fragments had been prepared. The nature of the fragments is discussed in relation to the observed antigenic heterogeneity of (Fab′) 2-like fragments from distinct myeloma proteins. Finally an Fd′ α-like fragment could be isolated from one of the studied myeloma proteins; the relevance of this result is discussed in the light of the structure of Fab from human immunoglobulins.

Crystal and molecular structure of a dimer composed of the variable portions of the Bence-Jones protein REI.

The structure of the variable part of a χ-type Bence-Jones protein RE1 has been determined at a resolution of 0.28 nm. It forms a dimer in the crystal related by a local diad, held together by hydrogen bonding interactions of residues Tyr-36, Gln-38, Ala-43, Pro-44, Tyr-87, Gln-89 and Phe-98, which are largely conserved in light chains. The structure consists of two hydrogen-bonded sheets covering a hydrophobic interior made up of mostly conserved amino-acid side chains. Approximately 50% of the residues form β-pleated sheets. Several of the pleated sheet strands are connected by hair-pin bends, which contain glycine residues conserved in most light chains. The arrangement of the hypervariable segments especially in comparison with the Fab structure suggests that the V dimers form a primitive antibody. The folding of the poly-peptide chain and the spatial relationship of the two monomers appear to be the same as in the λ-type Bence-Jones protein dimer and the Fab fragment.

Structure of Fab' New at 6 A resolution.

The structure of human immunoglobulin Fab′ fragment has been elucidated at a resolution of 6 A. There are two globular subunits, which presumably correspond to the variable and constant regions of the heavy and light polypeptide chains of immunoglobulins

Crystallographic data for the Fab fragment of a human myeloma immunoglobulin.

COMPARISONS of the physical properties and chemical structure of myeloma proteins with those of other immunoglobulins and antibodies have indicated that myeloma proteins are abnormal only in their relative homogeneity; they seem to be typical representatives of the immunoglobulin class to which they belong. In addition, antibody activity has been shown in several myeloma proteins and in macroglobulins associated with Waldenstrom's syndrome1–9. A knowledge of the tertiary structure of fragment Fab, which contains the combining site of an antibody10, should therefore be of particular interest.