A Human Igm Cold Agglutinin Three-Dimensional Structure of The

Three-Dimensional Structure of the Fab from a Human IgM Cold Agglutinin Ana Cauerhff, Bradford C. Braden, Julio Garcia Carvalho, Ricardo Aparicio, Igor Polikarpov, Juliana Leoni and This information is current as Fernando A. Goldbaum of September 24, 2021. J Immunol 2000; 165:6422-6428; ; doi: 10.4049/jimmunol.165.11.6422 http://www.jimmunol.org/content/165/11/6422 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2000 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Three-Dimensional Structure of the Fab from a Human IgM Cold Agglutinin1

Ana Cauerhff,* Bradford C. Braden,† Julio Garcia Carvalho,‡ Ricardo Aparicio,‡ Igor Polikarpov,‡ Juliana Leoni,* and Fernando A. Goldbaum2§

Cold agglutinins (CAs) are IgM autoantibodies characterized by their ability to agglutinate in vitro RBC at low temperatures. These autoantibodies cause hemolytic anemia in patients with CA disease. Many diverse Ags are recognized by CAs, most frequently those belonging to the I/i system. These are oligosaccharides composed of repeated units of N-acetyllactosamine, expressed on RBC. The three-dimensional structure of the Fab of KAU, a human monoclonal IgM CA with anti-I activity, was determined. The KAU combining site shows an extended cavity and a neighboring pocket. Residues from the hypervariable loops

VHCDR3, VLCDR1, and VLCDR3 form the cavity, whereas the small pocket is defined essentially by residues from the hyper- Downloaded from variable loops VHCDR1 and VHCDR2. This fact could explain the VH4-34 germline gene restriction among CA. The KAU combining site topography is consistent with one that binds a polysaccharide. The combining site overall dimensions are 15 Å wide and 24 Å long. Conservation of key binding site residues among anti-I/i CAs indicates that this is a common feature of this family of autoantibodies. We also describe the first high resolution structure of the human IgM CH1:CL domain. The structural analysis shows that the CH1-CL interface is mainly conserved during the isotype switch process from IgM to IgG1. The Journal of Immunology, 2000, 165: 6422–6428. http://www.jimmunol.org/ old agglutinins (CAs)3 are IgM autoantibodies character- isolated CA molecule. This phenomenon is called “idiotypic cross- ized by their ability to agglutinate in vitro RBC at low specificity” because these types of autoantibodies share an anti- C temperatures (4–22°C) (1, 2). These autoantibodies genic structure (or idiotope). The rat mAb 9G4 (11) served to cause hemolytic anemia in patients with CA disease (CAD). CAs identify the cross-reacting idiotope associated with the expression appear in the context of monoclonal gammopathies secondary to B of a particular variable domain of the heavy chain (VH) region. cell dyscrasias ranging from benign to malignant lymphoprolifera- Amino acid and nucleotide sequence analyses have confirmed this tion (3–5). They can also be detected in normal patients at low VH as derived from the VH4-34 (VH4-21) germline gene (12, 13). titers (6), though these titers increase with different infectious pro- The presence of a V chain derived from V 4-34 is necessary both

H H by guest on September 24, 2021 cesses (7–9). for the CA property and for the idiotope that is recognized by 9G4 Many diverse Ags are recognized by CAs, most frequently those mAb. Abs derived from the VH4-34 gene also recognize different belonging to the I/i system. These are oligosaccharides composed autoantigens as is the case of some rheumatoid factors (14), anti- of repeated units of N-acetyllactosamine, expressed in a linear DNA Abs (15), and the anti-D Abs (16). form (i) on fetal RBC or in a branched form (I) on adult RBC. Anti-I/i CAs show a great variability in VHCDR3, suggesting Early studies revealed that the vast majority of Abs with CA that this hypervariable region could not be directly involved in the activity reacted with a polyclonal antiserum (10) generated by an speciﬁc recognition or that the mode of binding is different among

CAs. Li et al. (17) postulated that the VHFR1 region is essential *Ca´tedra de Inmunologıá, Instituto de Estudios de la Inmunidad Humoral (IDEHU), in the specific recognition of the anti-I/i system, whereas the Facultad de Farmacia y Bioquı´mica UBA, Buenos Aires, Argentina; †Department of VHCDR3 and variable domain of the light chain (VL) dictate the Natural Sciences, Bowie State University, Bowie, MD 20715; ‡Laborato´rio Nacional de Luz Sıńcrotron, Campinas, Brazil; and §Instituto de Investigaciones Bioquı´micas fine specificity and strength of binding. Most of the anti-I CAs VL (Fundacioń Campomar, IIBBA-CONICET, FCEN-UBA), Buenos Aires, Argentina domains are derived from the V␬III germline gene, although some Received for publication March 2, 2000. Accepted for publication September 7, 2000. are encoded by V␬IorV␬II. In contrast, CAs with anti-i activity The costs of publication of this article were defrayed in part by the payment of page make no preferential usage of L chains. charges. This article must therefore be hereby marked advertisement in accordance The IgM KAU CA was obtained from the serum of a patient with 18 U.S.C. Section 1734 solely to indicate this fact. suffering CAD. Its amino acid sequence has been determined by 1 This work was supported by grants and fellowships from the Consejo Nacional de Investigaciones Cientı´ficas y Tećnicas (CONICET), Fundacioń Antorchas, and the Leoni and coworkers (18). As all CAs that recognize the I/i sys- University of Buenos Aires (to A.C., J.L., and F.A.G.). B.C.B. was supported by tem, the VH domain is derived from the VH4-34 germline gene, National Institutes of Health Grant 1R15AI44790-01 and National Aeronautics and showing a single point mutation in the VHCDR1 (Gly31Asp). The Space Administration Grant NCC5-232 (Model Institutes for Excellence). J.G.C., ␬ R.A., and I.P. were supported by Fundaçao de Amparo a`Pesquisa do Estado de Sao VL domain is derived from the kv305 germline gene (V IIIb). The Paulo (via Grant 1996/2285-5) and Conselho Nacional de Pesquisas (Brazil). Fab from IgM KAU was crystallized, and preliminary x-ray dif- 2 Address correspondence and reprint requests to Dr. Fernando Goldbaum, Instituto fraction data was reported (19). de Investigaciones Bioquı´micas, Fundacioń Campomar, Av. Patricias Argentinas 435, Here we present the three-dimensional structure of the Fab Buenos Aires 1405, Argentina. E-mail address: [email protected] KAU. Its combining site shows an extended cavity, as expected of 3 Abbreviations used in this paper: CA, cold agglutinin; CAD, CA disease; V , vari- H an anti-carbohydrate Ab. Conservation of key binding site residues able domain of the heavy chain; VL, variable domain of the light chain; CDR, complementarity-determining region; FR, framework region; CH1, first constant domain of among anti-I/i CAs indicates that this is a common feature of this the heavy chain; CL, constant domain of the light chain; C␮1, first constant domain of the IgM ␮ heavy chain; anti-Id, anti-idiotypic Ab; rmsd, root mean square deviation; family of autoantibodies. We also describe the first high-resolution PDB, Protein Data Bank. structure of the human IgM first constant domain of the heavy

chain:constant domain of the light chain (CH1:CL) domain and and 199–201 have no associated electron density and have been removed compare its features with those of other human and murine from the model. Likewise, CH1 residues 135–142 and 199–201 in the isotypes. second Fab have been removed due to lack of electron density. All residues in the VL,CL, and VH domains of both molecules are well resolved. Only two nonglycyl residues in each Fab, VL Ala52 and VH Asp31, are in dis- Materials and Methods allowed regions of a Ramachandran plot (data not shown). Similar occur- Molecular replacement and structure reﬁnement rences in tight turns have been observed in other Fabs (27–29). Calculation of a Fo-Fc difference Fourier map using the ﬁnal model

In a previous work (19) we described the preliminary x-ray diffraction of coordinates reveals electron densities at the CH1 glycosylation site. This a single crystal of Fab KAU to a resolution of 2.8 Å. Using synchrotron density is continuous with the side chain of CH1 Asn166, indicating co- light at the Laboratorio Nacional de Luz Sincrotron (LNLS, Campinas, valent bonding to the Fab. However, this density is unresolved, with breaks Brazil) we obtained a data set comprising 31,333 unique reflections (96.8% in the glycosidic linkages, and is only approximately the length of a tri- complete between 13.0 and 2.8 Å resolution, 99.0% complete in the last saccharide. Attempts to model and refine an oligosaccharide into this den- resolution shell between 2.9 and 2.8 Å). sity resulted in significant (Ͼ2.5 ␴) error peaks in a Fo-Fc Fourier. ϭ This crystal belongs to P3121 space group with cell dimension of a Superposition between the two Fabs using all C␣ atoms gave a rmsd of b ϭ 114, 23 Å, c ϭ 172, 78 Å; ␣ ϭ ␤ ϭ 90°, ␥ ϭ 120°. Estimation of 0.63 Å (0.48 Å using only C␣ atoms Ͻ2 rmsd differences). Superposition solvent content in this crystal, using the Matthews coefficient (20), indi- of the two Fvs gave values of 0.34 Å (0.30 Å); meanwhile, the superpo- ϭ 3 Ϫ1 cated that there are two Fabs in the asymmetric unit (Vm 3.38 Å Da sition of the CH1:CL modules resulted in a rmsd of 0.50 Å (0.27 Å). and solvent content ϭ 63%). The crystal structure of KAU Fab was ini- Solvent-excluded surface areas were computed with the probe radius of tially determined at 2.8 Å by the molecular replacement method using the 1.7 and 1.4 Å using the program GRASP (30). Superposition alignments program AMoRe (21). The search model consisted of the Fab of Ab 3d6 were made using the programs ALIGN (31) and Macromodel (32). The (22) (Protein Data Bank (PDB) code 1dfb). Several of the best rotation final coordinates have been deposited in the RCSB Protein Data Bank

function solutions were used for the translation search. The seven best (RCSB ID: PDB 1DN0). Downloaded from translation function solutions were subjected to rigid body refinement using reflections in the resolution range 13.8–3.5 Å. The two most significant Results solutions have correlation coefficients of 0.219 and 0.210, whereas all other Overall structure solutions were below 0.145. Combining these two solutions resulted in an R factor ϭ 0.497 and correlation coefficient ϭ 0.344. In all of these cal- The Fab KAU shows the usual Ig fold of Ab molecules (33). The culations, the search model elbow angle was not changed. two Fabs in the asymmetric unit are positioned in opposite direc- Several cycles of model building with the program O (23) followed by tions and have their interfaces at the C domains. The two Fabs can http://www.jimmunol.org/ refinement of atomic coordinates with Refmac (24) reduced the Rfree factor to 0.268, the R factor to 0.216, root mean square deviation (rmsd) bond be superimposed by a rotation of ϳ180°. lengths to 0.016 Å, and rmsd bond angles to 3.3°. Appropriate amino acid The final coordinates of the Fab A-B include 429 residues, 214 substitutions were made in the model structure using the KAU primary in the heavy chain and 215 in the light chain, whereas for the other structure (18). Omit maps were calculated after the final cycle of refine- Fab in the asymmetric unit, 212 amino acids of the heavy chain ment to check the integrity of the model. At this point, data to 2.28 Å from another trigonal crystal form became could be located. The quantitative agreement between the two Fabs ϭ ϭ ϭ available (space group P3121, cell dimensions a b 110.9 Å, c 170.8; gives a measure of the accuracy of the structural analysis (see ␣ ϭ ␤ ϭ ␥ ϭ ϭ 3 Ϫ1 90°, 120°; Vm 3.50 Å Da , and solvent content 65%). Materials and Methods). The elbow angle made by the two This data set was obtained from a single crystal grown as described (19). pseudodyads of the variable and constant domains is 170° for both Briefly, Fab KAU was concentrated to 5.5 mg/ml and crystallized using Fabs. This value falls within the range of those of other Fabs, from by guest on September 24, 2021 17% PEG 8000 and 0.1 M HEPES, pH 7.5, as mother liquor, and the temperature was kept at 4°C. This crystal was subsequently cryocooled 127° to 227° (34). cis-P proline residues occur at position 8, 96, using 12% PEG 400 as cryoprotectant, and a data set was collected at and 142 for the light chain and at position 155 for the heavy chain. Ϫ123°C using Synchrotron light at the Laboratorio Nacional de Luz Sin- crotron. At this step, molecular replacement using the 2.8 Å structure as a Variable region probe was performed with this new data set. Refinement was initiated with a rigid body procedure using the program CNS (25) followed by ten cycles The quaternary structure of this region is similar to the previously ␬ of manual model adjustment and addition of solvent water molecules using described human and mouse Abs. The Fab KAU VL (V IIIb) and the program TURBO (26) and simulated annealing refinement using the VH (VH4-34) domains form a compact module like other Fvs with program CNS, resulting in R factor ϭ 0.18 and R ϭ 0.22. This model 2 2 free an average buried surface area of 1587 Å (788 Å for VH and 799 consists of two Fab molecules with a total of 6594 protein atoms; 614 Å2 for V ). The V -V interaction is mediated by 220 atomic solvent molecules have been included in the model structure (see Table I). L L H contacts made mainly by VH residues Leu45, Trp47, Tyr106, and Quality of the model Trp110 and VL Pro44, Tyr92, Leu97, and Phe99. Residues Trp47H Results of the refinement of the KAU Fab crystal structure are summarized (VHFR2), Tyr92L, and Leu97L (VLCDR3) are also part of the combining site (see below). in Table I. In one of the Fabs present in the unit cell CH1 residues 135–140 The conformations of the hypervariable loops of the Fab KAU Table I. Refinement statistics summary of the Fab fragment of the anti- are all in defined canonical groups (35, 36). The complementarity- I KAU Ab determining regions (CDRs) H1, H2, L2, and L3 all belong to their respective class 1 canonical groups. KAU L1 could belong to ca- Program CNS nonical group 6, or a subset thereof, due to its length and similar Resolution range (Å) 17.0–2.28 sequence with the L1 loop of Fab 1f7 (PDB file 1fig). Fab 1f7 is Unique reflections (F Ͼ 0; F/␴ Ͼ 1) 45,494 at present the only structure assigned that exhibits the canonical a Final Rcryst 0.180 b length and conformation of L1 group 6 (37). However, the rmsd of Final Rfree 0.224 rmsd bond length (Å) 0.023 main chain atoms between KAU L1 and 1f7 L1 (1.70 Å) is quite rmsd bond angle (°) 2.4 high (Fig. 1B). Fab 409.5.3 (PDB file 1aif), like KAU and 1f7, also Mean temperature factor (Å2) contains 8 residues in L1. Likewise, the rmsd of the main chain Fab1 (main) 31.5 atoms with those of KAU (1.57 Å) is also too high for consider- Fab1 (side) 29.9 ation of canonical class. For the 1f7 structure, the difference in Fab2 (main) 29.9 Fab2 (side) 28.2 conformation of L1, relative to KAU L1, is dominated by two changes in the orientation of carbonyl groups (residues 26 and 29). a ϭ⌺ ʈ ͉ Ϫ ͉ ʈ ⌺ ͉ ͉ Rcryst hkl Fo Fc / hkl Fo b For the Fab 409.5.3 structure, the difference in conformation rel- Rfree is calculated in the same manner as Rcryst, but from 4599 reflections not used for refinement. ative to KAU appears to be by packing of residues 25 (Ala in 6424 STRUCTURE OF THE Fab OF A HUMAN IgM CA

FIGURE 1. A, Stereo view of the omit map of L1 KAU (residues 24–35) contoured at a significance level of 2.5 ␴. B, Stereo view of the superposition of L1 KAU (thick trace) and L1 1f7 (thin trace). Downloaded from http://www.jimmunol.org/ KAU, Val in 409.5.3) and 29 (Val in KAU, Ile in 409.5.3) with the The extended cavity floor is composed by residues Tyr92L, extra bulk of the Val-Ile interaction in 409.5.3, resulting in a higher Leu97L (VLCDR3), Pro98H (VHCDR3), and Glu50H (VHFR2). loop conformation at residue 29 and a subsequent change in the The cavity is surrounded by residues Gln58H (VHFR3), Trp47H positions of residues 30–31. Moreover, Tyr 71 in 409.5.3 makes a (VHFR2), Ser94L, Ser93L, Gly92L (VLCDR3), Ser30L, Ser31L, hydrogen bond to the amide of residue 31. This hydrogen bond is and Tyr33L (VLCDR1). missing in the KAU structure as a result of the Phe framework The cavity floor communicates with the pocket floor by means residue. As such, the conformational differences between KAU L1 of Pro98H (VHCDR3). The pocket floor is continued by Tyr33H and 409.5.3 L1 bear a striking similarity to the A and B subclasses and some main chain atoms of residues Asp31H (VHCDR1), by guest on September 24, 2021 of an L1 class 2 loop with the subclasses defined by interactions Asp101H, and Thr102H (VHCDR3). The pocket is completely sur- with residue 71. The resolution at which the 1f7 (3.0Å) and Fab rounded by polar and charged residues like Thr102H, Asp101H,

409.5.3 (2.9 Å) structures were solved, precluded their analysis of (VHCDR3), Asp31H (VHCDR1), His53H, Asn52H, Ser54H, and canonical structure by Al-Lazikani et al. (38). As such, the char- Ser56H (VHCDR2). acteristics of L1 canonical group 6 are still open to further developments. Constant region

The KAU combining site shows an extended cavity (39, 40) and The first CH domain of the human IgM (C␮1) displays the basic Ig a neighboring pocket (see Fig. 2A). Residues from the hypervari- fold as do other constant regions from different isotypes (41). The able loops VHCDR3, VLCDR1, and VLCDR3 form the cavity C␮1 and CL domains are covalently joined by a disulfide bond whereas the small pocket is defined essentially by residues from between the residues Cys134H and Cys215L. Both domains face the hypervariable loops VHCDR1 and VHCDR2. each other, related by a pseudodyad angle of 167.0°.

FIGURE 2. Analysis of the KAU combining site. A, Combining site surface colored by electrostatic po- tential (GRASP). Residues that constitute the extended groove are la- beled. B, Conservation of combining siteresiduesamong32sequencedanti- I/i CAs (as described in references and BLAST database). The ﬁgure is color coded based on the percentage of CAs containing the same residue at this particular position. Purple, 66–100%; red, 33–66%; yellow, 0–33%. The Journal of Immunology 6425

The interaction between C␮1 and CLk results in a buried surface at this interface. Eighteen of the twenty residues of C␮1 KAU that 2 2 2 ␬ ␭ of 2211 Å (1158 Å for C␮1 and 1053 Å for CL). The Van der contact CL make equivalent contacts on C␮1 RF-AN with CL . Waals contacts, hydrogen bonds, the disulﬁde bond, and the salt This analysis shows a remarkable conservation in this interface

bridge between C␮1 and CL are listed in Table II. The salt bridge regardless of the isotype of the partner light chain. occurs between Lys219H (N ) and Glu124L (O ) with a distance Z E2 The constant domains CH1 from KAU (human IgM), HIL (hu- of 2.5 Å and forming an angle of 104.5°. It is worth noting that man IgG1) (46), D44.1 (murine IgG1) (47) and Bv04–01 (murine Glu124L also participates in an equivalent salt bridge with IgG2b) (48) were aligned by a least-squares superposition to com- ␬ Lys221H of CH1 from mouse IgG2a isotype (41) (as shown in pare the folding conservation among these isotypes. The ␣-carbon Abs 17/9 and 33F12, PDB entries 1HIL, Ref. 42 and 1AXT, Ref. structure of the IgM KAU C 1 domain is very similar to all other 43, respectively) and mouse IgG ␬ (Ab NMC-4, PDB entry H 1 C 1 domains analyzed (Fig. 3). The rmsd obtained for the C␣ 1OAK) (44). H positions for the 73 spatially corresponding residues of KAU vs In total, C 1 and C establish 223 atomic contacts in Fab KAU ␮ L other isotypes are 0.83Å for human IgG1, 0.94Å for murine IgG1, A-B (247 contacts in Fab C-D). Light chain residues Phe119L, and 1.03Å for IgG2b. The C 1 domain superimposes well overall which makes 40 contacts, and Gln125L, which makes 22 contacts, ␮ with the exception of two segments, 163–168 and 194–204. account for 22% of contacts with C␮1. On C␮1, residues Phe129H, which makes 32 contacts, and Phe176H, which makes 32 contacts, In the region 163–168, the IgM CH1 domain deviates substan- tially from the other isotypes conformation (Fig. 3B), a fact that represent 27% of C␮1 contacts with CL. Residues Gln151, Arg174, Pro177, and Thr188 of the heavy chain make eight hydrogen bonds can be explained, at least in part, by the presence of a covalently

attached carbohydrate at residue Asn166 of the IgM. Human IgM Downloaded from with CL (see Table II). Electron density corresponding to a trisaccharide moiety (see conformation at residues 163–168 has a similar orientation to that

Materials and Methods) can be visualized in the C␮1 region, at- of murine IgG2b, where this loop is pointing toward the hinge tached to Asn166H by means of a N-glycan bond, but structural region, in clear contrast with the conformation observed in human

disorder prohibits placing the carbohydrate in the ﬁnal model due IgG1, murine IgG1, (Fig. 3B) IgG2a and IgG3 CH1 domains (49).

to breaks in electron density at the glycosidic linkages. The backbone conformation of the IgM CH1 domain also differs

signiﬁcantly from other isotypes structure in the segment 194– http://www.jimmunol.org/ Discussion 204. This loop is three amino acids longer in human IgM and Constant region displays a more extended conformation, even though residues This is the ﬁrst high-resolution structural analysis of the Fab from 199–201 were not modeled due to poor electron density. The cis-Pro155 in Fab KAU aligns with its counterpart cis in the a human IgM, allowing us to compare the module C␮1-CL with the corresponding human and murine domains from other isotypes. isotypes analyzed, as well as the intrachain S-S bond formed by

A previous Fab␮ structure was solved at a 3.2 Å resolution from Cys148-Cys208 in KAU. A very mobile segment formed by res- a human rheumatoid factor IgM (RF-AN, PDB 1ADQ) (45). In idues 135–140 in Fab KAU corresponds to a mobile segment in the rest of the isotypes at similar position.

that case, the structure of the constant segment is less well deﬁned by guest on September 24, 2021

due to disorder in this region. This mobility is less pronounced at The CH1 half-cystine forming the interchain disulﬁde bond with ␭ the C␮1/CL interface, allowing us to compare the contact residues the light chain is located at the N-terminal end of the domain in

Table II. Atomic contacts between the constant domains of the light and heavy chains of Fab KAUa

F P L V S C A L Q R G F P V L R T Q L K 129 130 131 132 133 134 145 149 151 174 175 176 177 179 180 181 188 190 192 219 Totals

F 117 2 9 11 I 118 2 1 3 F 119 16 10 9 5 40 P 120 1 1 S 122 5 3 8 E 124 8 5} 13 Q 125 19 3* 22 S 132 1 3* 4 V 134 5 5 L 136 88 N 138 6* 8 2 16 N 139 6* 6 Q 161 4 3 11 18 E 162 22 S 163 5 10** 2 17 V 164 44 T 165 3 5 1 9 S 175 4* 6 10 L 176 6 6 S 177 10 3* 13 T 179 44 T 181 22 C 215 1F 1 Totals 32 3 21 11 13 2 14 1 6 16 3 32 15 8 3 17 3 16 2 5 223

a The heavy chain residues are across the page and the light chain residues are down the page. The numbers represent van der Waals contacts otherwise indicated as follows: * represent a hydrogen bond, F represents a disulphide bond, and } represents a salt bridge. 6426 STRUCTURE OF THE Fab OF A HUMAN IgM CA

FIGURE 3. Superposition of the C␣ backbone of: human C␮1 (yellow), human C␥1 (blue), murine C␥1 (magenta), and murine C␥2b (red). A, Front stereo view of the CH1 face contacting CL. Cys- teine residues of human IgM (yellow sticks) and murine IgG1 (light blue sticks) are highlighted to show their dif- Downloaded from ferent location at the interface. B, Side view of the same domains, the conservation of the interface is clearly shown at the right side of the ﬁgure. http://www.jimmunol.org/ by guest on September 24, 2021

most of the isotypes. IgG1 is an exception, where the cystine do- 23–25). Accordingly, previous studies (52, 53) showed that bind- nated by the heavy chain is found at the carboxyl end of CH1 (50). ing of anti-idiotypic Ab (anti-Id) 9G4 to the FR1 region of VH4- As shown in the Fig. 3A, in human IgM the half-cystine is in the 34-encoded Abs blocked the hemagglutination activity of CAs loop between the strands 4-1 (A) and 4-2 (B) of the four-stranded with I/i activity. The KAU three-dimensional structure shows that ␤ sheet, whereas in murine IgG1 is located in the C-terminal loop the 9G4 cross-reactive idiotope (residues 23–25 of FR1) and the arising from the strand 3-3 (G), pertaining to the three-stranded ␤ combining site do not overlap. The inhibitory effect of 9G4 anti-Id sheet. Consequently, the half-cystine in both isotypes do not su- could be explained by an induced conformational change on FR1 perimpose, but their counterpart in the light chain (located also in upon binding of the anti-Id or by steric hindrance because of the a loop) can accommodate itself to interact to form the interchain bulky nature of the anti-Id. disulfide bond. The topography of the combining site (Fig. 2A) shows a pocket The structural alignment also shows that the C 1-C interface is H L and a cavity formed by hydrophobic and aromatic residues, sur- mainly conserved during the isotype switch process from IgM to rounded by polar and charged amino acids such as Asp31H, IgG isotypes. In effect, Fig. 3 shows the close correspondence of Asp101H, and Glu50H. Comparison with the binding site of the the ␣-carbon trace at the interface. This result is in agreement with soybean lectin bound to the Ag I of RBC (SBA, PDB files 2sba, the sequence alignment analysis of IgM against IgG1 isotypes made by Padlan and coworkers (51). 1sbd, 1sbe) (54, 55), shows that both have a similar topography and overall residue composition. The soybean agglutinin has a Variable region pocket formed by hydrophobic residues (Phe128, Ala87, Ile216, Leu214) and make hydrogen bonds with the I Ag by means of The usage of the VH4-34 gene is very common among autoantibodies, such us CAs, rheumatoid factors, anti-Rh, and anti-DNA surrounding residues, which typically participate in such interac- Abs. This restricted gene usage is striking in the case of anti-I/i tions with carbohydrate moieties (Asp88, Asp215, Asn130, CAs where all the analyzed Abs belong to this family. Therefore, His104). That pocket resembles the Fab KAU combining site topography, consequently, the way KAU interacts with the I Ag could the structural description of VH KAU, encoded by this gene, is important to understand the molecular basis of anti-I/i autoimmune be similar. recognition. The topography of the KAU combining site is consistent with The mAb 9G4 recognizes a public idiotope present in all CAs the binding activity and genetic origin of human CAs (56–59). encoded by VH4-34 (11). As was described before, the area react- Functional assays show that RBC agglutination produced by KAU ing with 9G4 is localized in framework region 1 (FR1; residues is abrogated by treatment of the cells with ␤-endogalactosidase The Journal of Immunology 6427 and inhibited by the linear (i) and branched (I) Ags (A. C., un- 13. Silberstein, L. E., L. C. Jefferies, J. Goldman, D. Friedman, J. S. Moore, published observation), suggesting that KAU recognizes a large P. C. Nowell, D. Roelcke, W. Pruzanski, J. Roudier, and G. J. Silverman. 1991. Variable region gene analysis of pathologic human autoantibodies to the related carbohydrate Ag. i and I red blood cell antigens. Blood 78:2372. To gain insight about the common structural features between 14. Kraj, P., D. F. Friedman, F. Stevenson, and L. E. Silberstein. 1995. Evidence for the overexpression of the VH4-34 (VH4.21) Ig gene segment in the normal adult anti-I/i autoantibodies, all known sequences of anti-I/i CAs (see human peripheral blood B cell repertoire. J. Immunol. 154:6406. Fig. 2B) were analyzed in terms of the Fab KAU CDRs and com- 15. Stevenson, F. K., C. Longhurst, C. J. Chapman, M. Ehrenstein, M. B. Spellerberg, T. J. Hamblin, C. T. Ravirajan, D. Latchman, and D. Isenberg. 1993. Utilization bining site. KAU H1 loop (residues 26–33 of the VH region) folds of the VH4-21 gene segment by anti-DNA antibodies from patients with systemic with the group 1 canonical structure. All the important residues for lupus erythematosus. J. Autoimmun. 6:809. this conformation (60) are conserved in all the analyzed anti-I/i 16. Borretzen, M., C. Chapman, F. K. Stevenson, J. B. Natvig, and K. M. Thompson. CAs, suggesting that the packing and conformation of KAU H1 1995. Structural analysis of VH4-21 encoded human IgM allo- and autoantibodies against red blood cells. Scand. J. Immunol. 42:90. loop is similar in all human anti-I/i. Most of sequenced anti-I/i CAs 17. Li, Y., M. B. Spellerberg, F. K. Stevenson, J. D. Capra, and K. N. Potter. 1996. have the same residues in the H2 loop (residues 52–56 of the VH The I binding specificity of human VH4-34 (VH4-21) encoded antibodies is de- region). As in KAU, they should have a canonical group 1 H2 termined by both VH framework region 1 and complementary determining region 3. J. Mol. Biol. 256:577. structure. 18. Leoni, J., J. F. Ghiso, F. Goni, and B. Frangione. 1991. The primary structure of It was also postulated that the VH4-34-encoded region is mainly the Fab fragment of protein KAU, a monoclonal immunoglobulin M cold agglutinin. J. Biol. Chem. 266:2836. responsible for Ag I/i specificity, while VHCDR3 and VL modulate 19. Cauerhff, A., I. Polikarpov, I. Mathov, L. Plotkin, C. Abatangelo, F. Goldbaum, the affinity (17). Fig. 2B shows the KAU combining site with its and J. Leoni. 1998. Crystallization and preliminary diffraction studies of a human residues colored according to the degree of conservation of those Fab␮ with anti-I activity. Prot. Pept. Letters 5:177.

20. Matthews, B. W. 1968. Solvent content of protein crystals. J. Mol. Biol. 33:491. Downloaded from amino acids among anti-I/i CAs. It is clear that most of VHCDR1 21. Navaza, J. 1994. AMoRe: an automated package for molecular replacement. Acta and all VHCDR2 residues are conserved among anti I/i CAs. These Crystallogr. A50:157. two loops form the pocket’s wall. FR2 residues Trp 47H, Glu 50H 22. He, X. M., F. Ruker, E. Casale, and D. Carter. 1992. Structure of a human and FR3 residue Asn 58H, that form the first segment of the ex- monoclonal antibody Fab fragment against gp41 of human immunodeficiency ternal perimeter, and Ser93L and Ser94L (both of V CDR3), that virus type 1. Proc. Natl. Acad. Sci. USA 89:7154. L 23. Jones, T. A., and M. Kjeldgaard. 1993. Refmac, a New Refinement Program: O extend this outer limit of the cavity, are remarkably conserved. Version 5.9, The Manual. Uppsala University, Uppsala, Sweden.

Therefore, all of these highly conserved residues constitute most of 24. Murshudov, G. N., A. A. Vagin, and E. J. Dodson. 1997. Acta Crystallogr. D53: http://www.jimmunol.org/ the external limit of the whole combining site. 240. 25. Brunger, A. T., P. D. Adams, G. M. Clore, W. L. Delano, P. Gros, The small pocket at the KAU combining site, formed essentially R. W. Grosse-Kunstleve, J. S. Jiang, J. Kuszewski, M. Nilges, N. S. Pannu, et al. 1998. Crystallography & NMR system: a new software system for macromolec- by VH4-34 encoded residues (VH CDR1, FR2, and CDR2) could explain the V 4-34 restriction among CAs. Differences in compo- ular structure determination. Acta Crystallogr. D54:905. H 26. Roussel, A., and A. G. Inisan. 1992. TURBO-FRODO. Technopole de Chateau- sition at VHCDR3, VLCDR3, and VLCDR1 would explain the di- Gombert, Europarc Bat. C. Marseille, France. versity in the fine specificity of this family of autoantibodies. Fur- 27. Arevalo, J. H., E. A. Stura, M. J. Taussig, and I. A. Wilson. 1993. Three-dimen- ther three-dimensional structural studies of other anti-I/i CAs, free sional structure of an anti-steroid Fab and progesterone complex. J. Mol. Biol. 231:103. and complexed with its carbohydrate Ags are needed to ascertain 28. Schulze-Gahmen, U., J. M. Rini, and I. A. Wilson. 1993. Detailed analysis of the the structural basis of this autoimmune recognition. free and bound conformations of an antibody: x-ray structures of Fab 17/9 and by guest on September 24, 2021 three different Fab-peptide complexes. J. Mol. Biol. 234:1098. 29. Braden, B. C., H. Souchon, J.-L. Eisele, G. A. Bentley, T. N. Bhat, J. Navaza, and Acknowledgments R. J. Poljak. 1994. Three-dimensional structures of the free and the antigen- We thank Dr. Roberto J. Poljak for the critical reading of the manuscript, complexed Fab from monoclonal anti-lysozyme antibody D44.1. J. Mol. Biol. and Dr. Ricardo A. Margni and Dr. Jose´M. Delfino for collaboration and 243:767. encouragement. 30. Nicholls, A., K. A. Sharp, and B. Honig. 1991. Protein folding and association- insights from the interfacial and thermodynamic properties of hydrocarbons. Pro- teins Struct. Funct. Genet. 11:281. References 31. Satow, Y., G. H. Cohen, E. A. Padlan, and D. R. Davies. 1986. Phosphocoline 1. Roelcke, D. 1989. Cold agglutination. Transfus. Med. Rev. III. 2:140. binding immunoglobulin Fab McPC603: an X-ray diffraction study at 2.7 Å. 2. Ciejka, J. Z., E. B. Cook, D. Lawler, J. Martin, R. D. Woodson, and F. Graziano. J. Mol. Biol. 190:593. 1999. Severe cold agglutinin disease and cryoglobulinemia secondary to a mono- 32. Mohamadi, F., N. G. Richards, W. C. Guida, R. Liskamp, M. Lipton, C. Caufield, clonal anti-Pr2 IgM lambda cryoagglutinin. Clin. Exp. Rheumatol. 17:227. G. Chang, T. Hendrickson, and C. Still. 1990. Macromodel: an integrated soft- 3. Ramos, R. R., B. R. Curtis, C. S. Eby, G. A. Ratkin, and H. Chaplin. 1994. 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