Structure Of Fab Research Articles

Structure of human IgM rheumatoid factor Fab bound to its autoantigen IgG Fc reveals a novel topology of antibody-antigen interaction.

Rheumatoid factors are the characteristic autoantibodies of rheumatoid arthritis, which bind to the Fc regions of IgG molecules. Here we report the crystal structure of the Fab fragment of a patient-derived IgM rheumatoid factor (RF-AN) complexed with human IgG4 Fc, at 3.2 A resolution. This is the first structure of an autoantibody-autoantigen complex. The epitope recognised in IgG Fc includes the C gamma 2/C gamma 3 cleft region, and overlaps the binding sites of bacterial Fc-binding proteins. The antibody residues involved in autorecognition are all located at the edge of the conventional combining site surface, leaving much of the latter available, potentially, for recognition of a different antigen. Since an important contact residue is somatic mutation, the structure implicates antigen-driven selection, following somatic mutation of germline genes, in the production of pathogenic rheumatoid factors.

The high-resolution structure of an anti-hCG Fab

The high-resolution structure of an anti-hCG Fab

X-ray Structure of the Uncomplexed Anti-tumor Antibody BR96 and Comparison with its Antigen-bound Form

The X-ray structure of the uncomplexed human chimeric Fab′ of the anti-tumor antibody BR96 has been determined at 2.6 Å resolution. The structure has been compared with Lewis Y antigen-complexed structures of BR96 which were determined previously. The comparison reveals segmental motions and/or conformational rearrangements of three CDR loops (L1, L3, and H2), whereas CDR H3 does not undergo changes upon complexation despite its significant main-chain contacts to the carbo hydrate antigen. In light of the uncomplexed chimeric Fab′ structure reported here, the previously observed high mobility of the CL:CH1 domains of the complexed chimeric BR96 Fab is rationalized as a “swinging” motion approximately about the axis of the elbow bend.

Crystallization and preliminary X-ray analysis of herpes simplex virus neutralizing antibody Fab fragment DL11.

An Fab fragment of a virus-neutralizing monoclonal antibody (DL11) that binds to herpes simplex virus glycoprotein D (HSV gD) has been purified, sequenced and crystallized. The biological activity of the purified Fab was verified by enzyme-linked immunosorbant assay, flow cytometry and by neutralization of HSV infectivity. The crystals have the space group P1 with cell dimensions a = 40.2, b = 49.2, c = 63.9 A, alpha = 76.1, beta = 77.4, gamma = 71.6 degrees. The unit-cell volume is consistent with it containing a single Fab molecule. The crystals grow to a maximum size of 0.7 x 0.3 x 0.3 mm and diffract X-rays to greater than 2.2 A resolution. The amino-acid sequences of the variable regions of the heavy and light chains of DL11 have been determined. These have been compared to those for other known Fab structures in the Protein Data Bank for selection of a starting model for crystallographic refinement by the molecular-replacement method.

ABGEN: a knowledge-based automated approach for antibody structure modeling.

Immunoglobulin (Ig) amino acid sequences are highly conserved and often have sequence homology ranging from 70 to 95%. Antigen binding fragments (Fab), variable region fragments (Fv), and single chain Fv (scFv) of more than 50 myeloma proteins and monoclonal antibodies (mAb) have been crystallized and display a high degree of structural similarity. Based on this observation, several homology modeling approaches have been developed for the prediction of Fab and Fv structures prior to their experimental determination. We have extracted features from existing Ig sequences, 44 known Fab and Fv structures to create an automated AntiBody structure GENeration (ABGEN) algorithm for obtaining structural models of antibody fragments. ABGEN utilizes a homology based scaffolding technique, and includes the use of invariant and strictly conserved residues, structural motifs of known Fab, canonical features of hypervariable loops, torsional constraints for residue replacements and key inter-residue interactions. The validity of the ABGEN algorithm has been tested using a five-fold cross validation with the existing Fab structures. Molecular mechanics and dynamics methods have been implemented with ABGEN models to accurately predict two Fab structures of anti-sweetener antibodies prior to crystallographic determinations.

Induced Pocket to Accommodate the Cell Attachment Arg-Gly-Asp Motif in a Neutralizing Antibody Against Foot-and-Mouth-Disease Virus

The three-dimensional structure of the Fab fragment of a neutralizing monoclonal antibody (SD6) elicited against foot-and-mouth disease virus (FMDV) has been determined at 2.5 Å resolution and refined to a crystallography agreement R-factor of 0.186. The structure has been compared with that of the same Fab molecule complexes with a 15 amino acid peptide (A15) representing a major antigenic site of FMDV, and determined at 2.8 Å resolution. The Fab quaternary structure, defined both by the elbow angle between modules and by the relative disposition of the light and heavy domains inside the modules, remains essentially unchanged. However, the comparison shows important conformational variations in the paratope, especially in the hypervariable loops of the heavy chain. The CDR-H3 loop has a peculiar amino acid sequence (RREDGGDEGF) with a high content of charged residues. Some of these Fab residues were fully reoriented upon complex formation. The reorientation resulted not only in an alteration of shape but also in an important redistribution of charges, providing multiple points of interaction with the A15 antigen and in particular with the cell attachment Arg-Gly-Asp motif in the peptide. Thus the recognition of A15 by SD6 represents an extreme example of the induced fit mechanism in antibody interactions. The electron density maps provide evidence that in the uncomplexed Fab structure some CDR residues show, with lower occupancy, the conformations found in the complex, suggesting that the rearrangements observed can have only minor energetic requirements.

Conformation of the hypervariable region L3 without the key proline residue.

The refined structure of the Fab fragment of the monoclonal antibody CRIS-I (IgG2a kappa) against the leukocyte differentiation antigen CD5, determined at 1.9 A resolution with an agreement R-factor of 18.3%, reveals a variant of the canonical conformations proposed for the light chain complementarity determining region L3 (CDR-L3). This is the first Fab structure available with a kappa light chain in which the CDR-L3 lacks the key proline residue in either position 94 or 95. The conformation found could be significant for about 10% of the murine IgG molecules with kappa light chains without proline in their CDR-L3 sequences.

PH Influences on the Crystal Structures and Mechanistic Properties of a Hydrolytic Antibody

AbstractAntibody CNJ206 catalyzes the hydrolysis of p‐nitrophenyl esters with significant rate enhancement and product specificity. We report here kinetic and affinity measurements for this antibody in the pH 4–9.5 range. We also report the X‐ray structures of CNJ206 Fab liganded with a transition‐state analog (TSA) and with one of the products of the hydrolysis, p‐nitrophenol, at pH 4. This product binds in the same hydrophobic pocket as the p‐nitrophenyl part of the TSA; its release at basic pH, where catalysis is usually measured, is facilitated by its ionized state and this avoids product inhibition. The conformation of the CNJ206 combining site is identical in this structure and in the previously determined structure of a CNJ206 Fab•TS A complex at pH 8. Taken together with the pH‐profile of the rate enhancement by CNJ206, the structural data support a mechanism for catalysis by CNJ206 involving stabilization of a tetrahedral oxyanion intermediate by the peptide NH groups of two consecutive residues in the H3 complementarity‐determining region.

The crystal structures of complexes formed between lysozyme and antibody fragments.

Type c lysozymes, and hen egg lysozyme in particular, have been extensively used to study the immune response because of their strong immunogenicity, the availability of many natural variants to study cross-reactivity, and the possibility to correlate these results with the known three-dimensional structure of lysozymes from several species. To date, the structure of six different murine monoclonal anti-lysozyme antibodies has been studied as a complex between the Fab fragment and antigen. In some cases, the structure of the uncomplexed Fab is also available, giving detail at the atomic level of the changes which take place during the formation of the antibody-antigen complex. The bacterially-expressed Fv molecule, the simplest fragment of an immunoglobulin retaining an intact antigen-binding site, has been studied for three of the monoclonal anti-lysozyme antibodies. Recombinant Fv fragments have opened up the possibility of using site-directed mutagenesis to study the effect of amino acid changes at the antibody-antigen interface. The six monoclonal antibodies appear to recognize epitopes which are localised on three different regions of the lysozyme surface.

Structure and humanization of a rat monoclonal Fab to human interleukin-5.

The X-ray crystal structure of a rat monoclonal Fab JES1-39D10, raised against recombinant human interleukin-5, has been determined with the use of molecular replacement techniques and refined at 2.7 A resolution by simulated annealing. The overall structure is similar to a murine Fab HyHEL-10 that is specific for hen egg white lysozyme. An interesting feature of the structure is the presence of leucine residues to support the H1 complementarity-determining region (CDR) loop. To our knowledge this is the first Fab crystal structure containing this unusual H1 loop support pattern. The activity of three humanized versions of 39D10 is explained by analysis of Fv interface residues and H1 support patterns of 39D10 and the human template HIL.

The use of antibody fragments for crystallization and structure determinations.

We thank Andrew Prongay for his earlier contributions to the crystallization of the HIV p24 Fab complex, Jan McClure (Bristol Myers-Squibb Pharmaceutical Corporation, Seattle, WA) for providing the anti-p24 monoclonals, Lorna Ehrlich and Carol Carter (SUNY, Stony Brook, NY) for the recombinant HIV p24, and Tianwei Lin for discussions on antibody sequencing. We are grateful for an MRC and an NIH grant for MGR and for a research scholar award to LCK from the American Foundation for AIDS Research.

Structural predictions of the binding site architecture for monoclonal antibody NC6.8 using computer-aided molecular modeling, ligand binding, and spectroscopy

Monoclonal antibody NC6.8 binds the superpotent sweetener ligand N-(p-cyanophenyl)-N'-(diphenylmethyl) guanidineacetic acid with high affinity (Kd = 53 nM). Using computer-aided molecular modeling and several experimental techniques, such as competitive ligand binding, absorbance spectroscopy, and fluorescence spectroscopy, we have predicted the structure of the variable domain fragment (Fv) and identified the key residues in the combining site of the antibody. We have identified nine specific amino acids as being involved in ligand recognition and complexation. Most notable are H:33W, which is responsible for ligand-induced tryptophan fluorescence quenching, H:56R, which forms a salt bridge with the carboxylate moiety of the ligand, and L:34H, which, deep in the binding site, interacts with the cyanophenyl portion of the ligand. Two residues located deep in the putative binding pocket, H:35E and H:50E, provide the negatively charged potential for interaction with the protonated aryl nitrogen and the positive guanidinium group. These modeling predictions were made before the solution of high-resolution structures of the native Fab (2.6 A) and the Fab-ligand complex (2.2 A). Comparisons between the theoretical model and experimental native and liganded Fab structures are made.

The x-ray structure of an anti-tumour antibody in complex with antigen.

The crystal structures of the murine BR96 Fab and its human chimera have been determined in complex with the nonoate methyl ester derivative of Lewis Y (nLey) at 2.8 A and 2.5 A resolution, respectively. BR96 binds the carbohydrate in a large pocket which is formed by residues of all CDR loops except L2. The binding of the carbohydrate is mediated predominantly by aromatic residues in BR96. Analysis of the structure suggests that BR96 is capable of recognizing a structure larger than the Le(y) tetrasaccharide, providing a possible explanation for its high tumour selectivity. The structure provides a rationale for mutagenesis experiments that have resulted in BR96 CDR loop mutants with increased affinity for nLey and/or tumour cells.

Structure of a monoclonal anti-ICAM-1 antibody R6.5 Fab fragment at 2.8 A resolution.

The specific binding of the monoclonal murine anti-intercellular adhesion molecule-1 (anti-ICAM-1) antibody, R6.5, inhibits the attachment of neutrophils to endothelium and prevents the attachment of major group human rhinovirus (HRV) to ICAM-1. This binding interferes with the host immune system and, as a result, the R6.5 antibody has been developed as a therapeutic anti-inflammatory and perhaps anti-HRV agent. The variable-region amino-acid sequence of R6.5 was determined from the anti-ICAM-1 cDNA. The crystallization conditions of the Fab fragment of R6.5 were established and the three-dimensional structure was determined by X-ray crystallography. The crystal space group is orthorhombic P2(1)2(1)2(1), a = 40.36, b = 137.76, c = 91.32 A, and the highest resolution of recorded reflections is 2.7 A. The molecular-replacement method using known Fab structures was employed to solve the R6.5 Fab structure. The final R-factor is 18.8% for a total of 3320 non-H protein atoms, 39 water molecules and 10 606 unique reflections. The protein exhibits the typical immunoglobulin fold. The surface contour of the antigen-combining site of the R6.5 antibody has a wide groove which resembles more the structure of an anti-polypeptide antibody than the structure of an anti-protein antibody.

Insights into DNA Polymerization Mechanisms from Structure and Function Analysis of HIV-1 Reverse Transcriptase

When the single-stranded RNA genome of HIV-1 is copied into double-stranded DNA, the viral enzyme reverse transcriptase (RT) catalyzes the addition of approximately 20,000 nucleotides; however, the precise mechanism of nucleotide addition is unknown. In this study, we attempt to integrate the genetic data and biochemical mechanism of DNA polymerization with the structure of HIV-1 RT complexed with a dsDNA template-primer. The first step of polymerization involves the physical association of a polymerase with its nucleic acid substrate. A comparison of the structures of HIV-1 RT in the presence and absence of DNA indicates that the tip of the p66 thumb moves approximately 30 A upon DNA binding. This conformational change permits numerous interactions between residues of alpha-helices H and I in the thumb subdomain and the DNA. Measurements of DNA binding affinity for nucleic acids with double-stranded DNAs that have an increasing number of bases in the template overhang and molecular modeling suggest that portions of beta 3 and beta 4 within the fingers subdomain bind single-stranded regions of the template. Measurements of nucleotide incorporation efficiency (kcat/Km) show that the binding and incorporation of the next complementary nucleotide are not dependent on the length of the template overhang. Molecular modeling of an incoming nucleotide triphosphate (dTTP), based in part on the position of mercury atoms in a RT/DNA/Hg-UTP/Fab structure, suggests that portions of secondary structural elements alpha C-beta 6, alpha E, beta 11b, and beta 9-beta 10 determine the topology of the dNTP-binding site. These results also suggest that nucleotide incorporation is accompanied by a protein conformational change that positions the dNTP for nucleophilic attack. Nucleophilic attack by the oxygen atom of the 3'-OH group of the primer strand could be metal-mediated, and Asp185 may be directly involved in stabilizing the transition state. The translocation step may be characterized by rotational as well as translational motions of HIV-1 RT relative to the DNA double helix. Some of the energy required for translocation could be provided by dNTP hydrolysis and could be coupled with conformational changes within the nucleic acid. A structural comparison of HIV-1 RT, Klenow fragment, and T7 RNA polymerase identified regions within T7 RNA polymerase which are not present in the other two polymerases that might help this polymerase to remain bound with nucleic acids and contribute to the ability of the T7 RNA polymerase to polymerize processively.

1.85 A structure of anti-fluorescein 4-4-20 Fab.

The crystal complex of fluorescein bound to the high-affinity anti-fluorescein 4-4-20 Fab (Ka = 10(10) M-1 at 2 degrees C) has been determined at 1.85 A. Isomorphous crystals of two isoelectric forms (pI = 7.5 and 7.9) of the anti-fluorescein 4-4-20 Fab, an IgG2A [Gibson et al. (1988) Proteins: Struct. Funct. Genet., 3, 155-160], have been grown. Both complexes crystallize with one molecule in the asymmetric unit in space group P1, with a = 42.75 A, b = 43.87 A, c = 58.17 A, alpha = 95.15 degrees, beta = 86.85 degrees and gamma = 98.01 degrees. The final structure has an R value of 0.188 at 1.85 A resolution. Interactions between bound fluorescein, the complementarity-determining regions (CDRs) of the Fab and the active-site mutants of the 4-4-20 single-chain Fv will be discussed. Differences were found between the structure reported here and the previously reported 2.7 A 4-4-20 Fab structure [Herron et al. (1989) Proteins: Struct. Funct. Genet., 5, 271-280]. Our structure determination was based on 26,328 unique reflections--four times the amount of data used in the previous report. Differences in the two structures could be explained by differences in interpreting the electron density maps at the various resolutions. The r.m.s. deviations between the variable and constant domains of the two structures were 0.77 and 1.54 A, respectively. Four regions of the light chain and four regions of the heavy chain had r.m.s. backbone deviations of > 4 A. The most significant of these was the conformation of the light chain CDR 1.

Computer-aided molecular modeling of the binding site architecture for eight monoclonal antibodies that bind a high potency guanidinium sweetener

Computer-aided molecular modeling of the antibody binding site of eight different monoclonal antibodies (mAb) that bind the intense sweetener ligand (N-(p-cyanophenyl)-N'-diphenylmethyl) guanidine acetic acid was completed using canonical loop structures and framework regions from known immuno globulins as “parent structures” for the molecular scaffoldings. The models of the fragment variable (Fv) region of the mAb were analyzed/or the presence and location of residues predicted to be involved in ligand binding. Several binding site tryptophan residues in these models were located in positions that support previous flurospectroscopic observations of the mAb-ligand complexation. Computer-aided renderings of the electrostatic potential at the van der Waals surface of the Fv region were compared and found to be consistent with the ligand binding specificity profiles for the different mAb. The Fv model of mAb NC6.8 was consistent with the binding site features determined in the Fab structure recently solved by X-ray diffraction techniques. These Fv models should provide an adequate basis for site-directed mutagenesis experiments in order to characterize interactive motifs in the mAb binding site.

Preparation, Characterization and Crystallization of an Antibody Fab Fragment that Recognizes RNA: Crystal Structures of Native Fab and Three Fab-mononucleotide Complexes

Fab fragments from Jel 103, an antibody which specifically binds to single-stranded poly(rl), were prepared by pepsin digestion, separated into eight isoforms and characterized by mass spectrometry. One of the purified isoforms yielded crystals suitable for structural studies by X-ray diffraction and its crystal structure was determined to 2·4 Å resolution. Soaking the crystals in solutions containing either of the mononucleotides inosine-5′-diphosphate, guanosine-5′-diphosphate or deoxyinosine-5′-monophosphate resulted in binding of the nucleotide in a single binding site. However, adenosine-5′-diphosphate does not bind to this antibody. The recognition of the base is achieved through hydrogen bonds to the C6 carbonyl oxygen and the imino NH group of the purine in a pattern similar to that of the base-base interactions in a double-stranded nucleic acid. Additional binding energy is provided by stacking of the base and the Tyr32L side-chain and by interaction of the α-phosphate with the antibody in an anionic binding site. Most of the side-chains interacting with the nucleotide come from the light chain. Surprisingly, this antibody shares the V L sequence with another nucleic acid-binding antibody, BV04-1. The latter binds to a single stranded DNA with a high preference for thymine bases. The structures of the unliganded and complexed Jel 103 Fab are compared to those of BV-04-1 Fab and while they show similarity in recognition of the base of the immunodominant nucleotide, their 5′ phosphates occupy different positions, suggesting different orientation of the nucleic acid bound to these two antibodies. Differences in the conformations of the L1 loops between the two Fabs have been noted.

Exploring the Mimicry of Polysaccharide Antigens by Anti-idiotypic Antibodies: The Crystallization, Molecular Replacement, and Refinement to 2·8 Å Resolution of an Idiotope-Anti-idiotope Fab Complex and of the Unliganded Anti-idiotope Fab

The monoclonal antibody YsT9.1 is specific for the lipopolysaccharide A antigen of Brucella abortus. A complex formed between the Fab of YsT9.1 (Ab1) and the Fab of its anti-idiotopic monoclonal antibody T91AJ5 (Ab2) has been crystallized, and data have been collected to 2·8 Å resolution. The space group is monoclinic P2 1, with one molecule per asymmetric unit. The structure was solved using a limited Patterson-correlation search over the asymmetric-unit of rotation space, and has been refined to an R-factor of 0·174. The complex is a head-to-head dimer, with the contact between the two Fabs almost completely restricted to their hypervariable loops. The interactions between the two Fabs in the complex are dominated by tyrosine residues, not only in the formation of hydrogen bonds, but in their participation in an aromatic ring network that spans the two Fv domains. The anti-idiotope was found to be unable to carry an internal image of the antigen and induce polysaccharide-specific "anti-anti-idiotopes" (Ab3) because the polysaccharide binding cleft on the Ab1 is too narrow and deep to allow comprehensive contact with the binding site of Ab2. The antibody T91AJ5 therefore is a class γ anti-idiotope. The contact surfaces of the two Fabs are highly complementary; however, they are distinctly different in character. Each Fab has two separate binding surfaces of approximately equal size, but while the two binding surfaces of Ab1 are partitioned between the light and heavy chains, the two binding surfaces of Ab2 are shared between the chains, with the heavy chain responsible for about 60% of the total binding area. The structure of the unliganded Fab of Ab2 has also been solved by molecular replacement, and refined to an R-factor of 0·152 at 2·8 Å resolution. This Fab crystallizes in the orthorhombic space group P2 12 12 1, with one molecule per asymmetric unit. The second hypervariable loop of the heavy chain of the Ab2 Fab is observed to undergo a significant and necessary conformational rearrangement in going from the unliganded to complexed form, and thus complex formation is an example of "induced fit" of antigen to antibody. The unliganded Ab1 Fab packs in the standard head-to-tail fashion observed for other Fabs. This mode of packing is precluded in the complex, by its head-to-head nature, and it is found to pack in tilted layers with most intermolecular contacts made between adjacent Ab2 Fab molecules. The unliganded Ab2 Fab packs in a manner that cannot be described as either head-to-tail or head-to-head.

Antibody-combining sites. Extending the natural limits.

The antibody repertoire is very large with at least 10(9) different antibody specificities, yet there are currently only 800 variable-region sequences known and < 23 Fab structures deposited with the Brookhaven Protein Data Bank. To engineer the antibody-combining site rationally, we need to define the rules that govern antibody structure. To understand the process of antibody-antigen recognition, we need not only to predict complementary determining regions accurately, but to simulate accurately the interaction of antibody with antigen. We have made progress in the modeling of antibody-combining sites and in the simulation of antibody complex formation. The combination of these approaches will allow us to extend the natural limits of antibody-combining sites in a more rational manner.