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J Biol Chem, Vol. 273, Issue 35, 22515-22518, August 28, 1998


Structure of an Fab Fragment against a C-terminal Peptide of hCG at 2.0 Å Resolution*

Constantina FotinouDagger , Jeremy BeauchampDagger , Paul EmsleyDagger , Annemarie deHaan§, Wim J. G. Schielen, Ebo Bos§, and Neil W. IsaacsDagger parallel

From the Dagger  Department of Chemistry, University of Glasgow, Glasgow G12 8QQ, United Kingdom, the § Department of Biotechnology and Biochemistry, N. V. Organon, Molenstraat 110, P. O. Box 20, 5340 BH, Oss, The Netherlands, and the  Chemistry Research Unit, Organon Teknika BV, P. O. Box 84, 5280 AB Boxtel, The Netherlands

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results & Discussion
References

3A2 is an antibody raised against human chorionic gonadotropin and recognizes a linear epitope on the C-terminal peptide of the human chorionic gonadotropin beta -subunit. Its three-dimensional structure has been determined to 2-Å resolution using molecular replacement and refined to a conventional R-factor of 18.2%. The protein exhibits the typical immunoglobulin fold, and the model contains 944 ordered water molecules and one sulfate ion. A comparison of the complementarity-determining regions of the Fab3A2 with those from the Protein Data Bank following the canonical structure method reveals a canonical main chain conformation. This antibody belongs to the canonical structure class (combination of canonical conformations of the complementarity determining loops) that shows a preference for haptens and not for peptides. However, the shape of the surface of the antigen binding loops resembles that of an anti-peptide antibody.

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results & Discussion
References

Human chorionic gonadotropin (hCG)1 is a glycoprotein hormone secreted in high levels during the first trimester of pregnancy (1, 2). It acts to sustain the action of progesterone, a hormone important to the early growth and nesting of the fertilized egg. Pregnancy testing kits work by detecting this hormone (3). Apart from its physiological action, hCG is found in pathological cases such as choriocarcinoma, hydatidiform mole and testicular cancer (4). Holo-hCG, its free subunits, or nicked parts of them are secreted during the course of these diseases and thus can be used as immunological markers of these conditions (3, 5).

The family of heterodimeric glycoprotein hormones comprises chorionic gonadotropin, luteinizing hormone (LH), follicle-stimulating hormone (FSH), and thyroid stimulating hormone (TSH). The hormones consist of two noncovalently linked glycosylated subunits, alpha  and beta  (2, 6). Within a given species, the shorter alpha -subunit is encoded by a single gene, is common to the four hormones, and is immunologically indistinguishable (2). The beta -subunits are distinctive for each hormone and confer specific biological and immunological activity. The beta -subunit of hCG has a unique 31-residue (residues 115-145) C-terminal peptide containing four O-linked glycosylation sites. Both subunits have two N-linked glycosylation sites.

The structure of HF-treated hCG has been determined (7, 8). However, the structure is incomplete because (i) the treatment with HF removes most of the carbohydrate and (ii) residues from both N and C termini of the subunits could not be positioned. In particular, residues 112-145 of the C-terminal region of the beta -subunit are missing from the current model.

Three-dimensional structural information of Fab fragments of anti-hCG antibodies and of Fab-hCG complexes will provide insight into interactions between hCG and its antibodies and possibly a more complete and accurate structure of hCG. This will be useful in designing immunometric assays for the diagnosis and monitoring of pregnancy and of hCG-producing tumors. We present here the first structure of an Fab fragment of a monoclonal antibody against an epitope on the C-terminal region of the beta -subunit of hCG.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results & Discussion
References

Sequencing of Monoclonal Antibody 3A2-- A hybridoma cell line for the production of the monoclonal 3A2 anti-hCG antibody was cultured in cell culture medium to an amount of 107 cells. mRNA was isolated from these cells, using the QuickPrep Micro mRNA purification kit (Amersham Pharmacia Biotech). Full-length first strand cDNA was generated from the isolated mRNA using the First-Strand cDNA synthesis kit (Amersham Pharmacia Biotech). The cDNA was amplified by PCR. Human primers were used for the variable region of the H- and L-chain. Mouse and human primers were used for the constant region of the H-chain. Human primers were used for the constant region of the L-chain. An agarose gel electrophoresis of the PCR product was carried out to isolate the DNA. The DNA was extracted out of the agarose gel, using the QIAEX II Agarose Gel Extraction Kit (QIAGEN). Ligation and transformation of the extracted DNA into Escherichia coli was performed with the aid of the Original TA Cloning Kit (Invitrogen). The transformed cells were spread on LB agar plates containing ampicillin and X-gal for screening. PCR was performed on white colonies and screened on agarose gel. Positive clones were cultured in LB medium containing 200 micrograms ampicillin/ml medium. DNA was isolated from the E. coli, and the DNA was fluorescein isothiocyanate labeled, using the AutoRead Sequencing Kit (Amersham Pharmacia Biotech). After dividing the labeled DNA over the four nucleotide mixes (A, C, G, and T, respectively), the DNA sequence of the H- and L-chain of the antibody was determined on the ALFexpress DNA Sequencer and Fragment Analysis System (Amersham Pharmacia Biotech). Translation from DNA sequence into protein sequence was performed with PC/GENE.

Data Collection and Processing-- The preparation, purification, and growth conditions of the Fab3A2 crystals and the method of data collection have been described previously (9). Data were integrated using DENZO (10) and scaled and merged using ROTAVATA/AGROVATA (11). Table I shows the data statistics.

                              
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Table I
Summary of the x-ray diffraction data for the Fab3A2

Crystal Structure Determination-- The crystal structure was determined by molecular replacement using AMoRe (12). The search model was the anti-peptide IgG1 Fab' B13I2 (13) (Protein Data Bank code 1igf, Ref. 14). Two separate searches for the variable and constant domain were performed using data in the resolution range of 10 to 3 Å. There were single clear solutions to both the rotation and translation functions in both cases. The packing of the molecules in the crystal was reasonable.

Structure Refinement-- A rigid body refinement was performed with X-PLOR (15) using the sequence of the search model and separating it into four protein chains, corresponding to the conventional VL, VH, CL, CH1 domains of the antibody. The initial Rcryst was 48.1% and Rfree 48.4%. Using "O" (16), a model of the Fab3A2 was generated by mutation of the Fab'B13I2 sequence where this differed from Fab3A2 (Table II).

                              
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Table II
Sequences of the variable domains of the Fab3A2
Sequences of the variable domains of the Fab3A2 with the molecular replacement search model, B13I2 (Protein Data Bank code 1igf). The vertical bar (|) indicates sequence identity and the dash (-) relative deletion. (a), numbering according to Kabat et al. convention (35). (b), sequence numbering. Loops classes correspond to the Fab3A2.

The residues of the CDR were omitted, and SIGMAA-DM 2 Fo - Fc and Fo - Fc maps (11, 17) were calculated. The fit of the model was good and correct density was found for almost all the omitted residues.

Positional refinement with X-PLOR lowered the Rcryst to 36% and the Rfree to 41%. New 2 Fo - Fc and Fo - Fc maps were calculated and appropriate corrections made. The ambiguous areas were resolved by positional refinement of the model omitting these residues and model building into the subsequent omit-maps. An unrestrained B-factor refinement using SFALL (11) was performed, which reduced the Rcryst and Rfree from 34.2 and 40.6% to 23.8 and 33.8%, respectively.

The data were of sufficient quality to allow the use of the program ARP (18) to find water positions. The Rcryst and the Rfree were decreased to 22.6% and 31.4%, respectively.

The refinement continued with the program REFMAC (19), resulting in final values of 18.2 (Rcryst) and 24.1% (Rfree).

The solvent accessible area of the CDR and the buried area between the light and heavy chains of the variable domain were calculated using the program SURFACE (11), with a 1.7-Å probe radius and standard van der Waals radii.

    RESULTS AND DISCUSSION
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Abstract
Introduction
Materials & Methods
Results & Discussion
References

Description of the Structure-- A drawing of the Calpha backbone of the Fab3A2 is shown in Fig. 1. The structure shows the typical immunoglobulin fold. The large number of the water molecules in the current model (944) is consistent with the data collection at a temperature of 100 K. Many of the water molecules are found to form 4- and 5-membered rings (Fig. 2). There is also bound one sulfate ion. The geometry of the model is good, with 92% of the residues in the allowed area of the Ramachandran plot (0.3% of the residues are in disallowed regions) and r.m.s deviations from ideality of bond distances is 0.011 Å. The average B-factor is 20.9 Å2 for the protein atoms and 37.6 Å2 for the solvent molecules. The residue L51 of the CDR2-L is in a strained conformation with angles phi = 65o and psi  = -33o. These are similar to the average angles that this residue adopts in 17 high resolution immunoglobulin structures analyzed by Al-Lazikani et al. (20). In common with other Fab structures (21-23), where disordered regions are observed for loops of the constant domain, the density for the residues L198-L205 is poor, and they cannot be located with confidence. The buried surface area between the light and heavy chain of the variable domain is 1822 Å2, which is larger than the average value (of about 1400 Å2) found in other Fabs (24).


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  		Fig. 1.   Ribbon drawing of the Fab3A2. The light chain is shown on the left side and the heavy chain on the right. L1, L2, L3, H1, H2, and H3 represent the hypervariable loops of the Fab3A2. The figure was produced using Setor (36).


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  		Fig. 2.   A section from a 2 Fo - Fc electron density map which shows hydrogen-bonded waters forming 4- and 5-membered rings. The map is contoured at 1 sigma .

Structure of the Antigen-binding Site-- A comparison of the observed conformations of the hypervariable loops of the Fab3A2 with those described by the canonical structure approach of Chothia et al. (25) shows that they are consistent with this classification. The CDR1-L belongs to the canonical group 3, as indicated by the presence of the key residues L2(Ile), L25(Ser), L33(Leu), and L71(Phe). The CDR2-L follows the conformation of group 1. The CDR3-L also belongs to group 1 (key residues L95(cis-Pro), L90(Gln)). The glutamine at L90 forms, as expected, hydrogen bonds to 7 of the 9 residues of the L3 loop. The key residues H26(Gly), H27(Phe), H29(Phe), H34(Met) and H94(Arg) give to the CDR1-H loop the canonical conformation 1. The CDR2-H loop adopts the class 4 conformation (key residues H54(Asn), H55(Tyr), and H71(Arg)). The CDR3-H has a length of 12 residues, which is a medium loop for mouse antibodies and among the most common occurring (26). At the moment there is not any canonical conformation for the CDR3-H. Recently, Shirai et al. (27) have found several rules that govern the CDR3-H conformation and the amino acid sequence. According to this classification the Fab3A2-CDR3-H conformation belongs to class A with a kinked base (Fig. 3) (27). Almost 50% of the Fab3A2-CDR3-H residues are tyrosines (there are 5 Tyr). It has been shown from the structures of antigen-antibody complexes that aromatic residues (especially Tyr) play a dominant role in antigen-antibody interactions (28, 29). Because the Fab3A2 H3 loop is rich in Tyr, we expect it to contribute significantly to the antigen binding.


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  		Fig. 3.   A Calpha backbone trace of the H3-loop showing its conformation and the hydrogen bonds that are formed between its residues. Note the hydrogen bond between the HNepsilon 1 of the H103Trp and the carbonyl oxygen of the H100dMet and the salt bridge between the H94Arg and H101Asp. Both, according to Shirai et al. (27), give to the H3 loop its kinked base conformation. Residue numbering is according to Kabat et al. convention (35).

The combination of the canonical structures of the CDR loops (1 4 3 1 1) for H1, H2, L1, L2, L3, respectively, is the same as that of antibodies, such as McPC603, that recognize small molecules (haptens) (30). Madrazo-Vargas et al. (30) have found that 89% of the antibodies which belong to this canonical class are specific for haptens and 11% for proteins. We found that the conformation of the Calpha backbone of the Fab3A2 is quite similar to that of the McPC6O3, Fig. 4 (the r.m.s. deviation is 0.76 Å for the 123 atoms of the light chain of the variable domain and 1.63 Å for the 220 atoms of the light chain of the Fab molecule after superposition of the molecules using lsq in "O" (16)).


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  		Fig. 4.   Comparison of the Fab fragment of the 3A2 antibody with that of the McPC603. The Calpha traces of the variable domains of both Fabs are shown. Superposition was done using code lsq in "O"(16), and the variable domain of the light chain (123 atoms) was used for the fitting. r.m.s. deviation is 0.76 Å .

The surface of the antigen binding site, however, is a groove (31) (Fig. 5A), with an accessible area of 2662 Å2, and it is less deep than grooves found in other anti-peptide antibodies. The surface of the CDR of the McPC603 is a cavity, typical for antibodies recognizing haptens (31). The difference in the shape of the surface of the CDR between the two Fabs, is explained by the different sequence of the H-loops (50% of the residues differ) and the L-loops (25% of the residues differ). Also, the H3-loop of the Fab3A2 is longer, its conformation is different and it is directed toward the center of the antigen binding area. This orientation of the H3-loop results in the relatively flat surface of the CDR.


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  		Fig. 5.   A, a top view of the surface of the antigen binding area of the Fab3A2 calculated using GRASP (37). The darker area shows the predicted, according to the method described in MacCallum et al. (33), antigen binding pocket. B, a Calpha backbone diagram of the variable domain of the Fab3A2 showing that the aromatic residues are located mainly in antigen binding site. The view is the same with panel A. Pictures were produced using GRASP (37).

It has been found that the majority of the residues which contribute to the antigen-antibody contacts are located centrally within the CDR area (32, 33). This area, for the Fab3A2, is dominated by aromatic residues (Fig. 5B) and is almost uncharged. The only charged residues are the ArgH99, ArgH52, and AspH50. This might suggest that the interactions with the epitope are expected to be mainly hydrophobic. However, the association between the Fab and antigen has been found by microcalorimetry titration to be enthalpically driven, with a decrease of entropy.2 According to Mariuzza et al. (34), this implies that the dominant forces on the association process are likely to be electrostatic. A possible explanation is that during the complex formation there is a rearrangement of the H3-loop and the side chains of two aspartic acids (AspH100, AspH101), the glutamic acid GluL55, the arginine ArgL54, and the lysine LysL24 in order to contribute to the antigen-binding. The real interactions will be revealed when the structure of the complex Fab3A2+hCG is determined.

Conclusions-- The knowledge of the Fab3A2 structure (especially when complexed with hCG) will aid in understanding their interactions, and it will contribute to a better understanding of the molecular recognition between antigens and antibodies. The 1.5-Å high resolution data that we have recently collected should reveal more about the structure and the dynamics of the antigen binding site of Fabs. When the structure of the complex with hCG is determined, the role of the waters in the antigen-Fab association may become more clear.

    ACKNOWLEDGEMENTS

We thank Steve Prince, John Maclean, and Adrian Lapthorn for help.

    FOOTNOTES

* This work was supported by the Human Capital and Mobility program of the European Community and the Medical Research Council.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The atomic coordinates and structure factors (codes 1sbs and rlsbssf) have been deposited in the Protein Data Bank, Brookhaven National Laboratory, Upton, NY.

parallel To whom correspondence should be addressed. Tel.: 0044 141 330-5954; Fax: 0044 141 330-4888; E-mail: neil{at}chem.gla.ac.uk.

The abbreviations used are: hCG, human chorionic gonadotropin; CDR, complementarity determining regions; HF, hydrofluoride; r.m.s., root mean square; PCR, polymerase chain reaction.

2 C. Fotinou, J. Beauchamp, P. Emsley, A. deHaan, W. J. G. Schielen, E. Bos, and N. W. Isaacs, unpublished results.

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Abstract
Introduction
Materials & Methods
Results & Discussion
References

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Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.
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