Anti-DNA antibodies (Abs)1 are of biomedical interest because of their association with autoimmune diseases such as systematic lupus erythematosus and multiple sclerosis.2, 3 Naturally occurring anti-DNA Abs generally do not exhibit sequence specificities.2, 3 Instead, they can be classified as specific for single-stranded (ss) DNA,1, 4, 5 double-stranded (ds) DNA,6, 7 or both ssDNA and dsDNA5, 8 with a preference for certain DNA sequences such as poly(dT) or poly(dG-dC) sequences.2, 3
In the previous study, we isolated an anti-DNA monoclonal Ab 3D8 from the spleen cells of the MRL-lpr/lpr mouse, which spontaneously develops an autoimmune syndrome resembling human systemic lupus erythematosus.9 Very recently, we have shown that each single domain of heavy chain (VH) and light chain (VL) as well as recombinant single-chain variable fragment (scFv) degraded both ss-DNA and ds-DNA without sequence specificities in a Mg2+-dependent manner. Structural and subsequent mutational studies of 3D8 scFv showed that His35 of VH and His94 of VL were critically involved in the catalysis of 3D8 scFv. However, the DNA-hydrolyzing activity of single-domain 3D8 VL was not affected by H94A mutation of VL. Further there were no additional His residues in the VL domain. These observations indicate that VL uses different catalytic mechanism including distinct active site environment from those of 3D8 scFv.10
In this study, we found that single-domain 3D8 VL also hydrolyzes DNA substrates, when a Mg2+ ion was replaced with either Co2+ or Cd2+ ions. To provide the molecular information on putative catalytic site of 3D8 VL, we elucidated high-resolution crystal structure of VL in complex with a small molecule bis-(2-hydroxyethyl)imino-tris(hydroxymethyl)methane (Bis-Tris, BTB) and complex crystal structures with two metal ions Co2+ and Cd2+.
The 3D8 VL derived from a mAb 3D8 IgG (IgG2a/κ) (IgG) that had been originated from autoimmune-prone MRL-lpr/lpr mouse9 was subcloned using XmaI/NcoI sites into a bacterial expression vector pIg20, which has a bacterial alkaline phosphatase leader sequence at the N-terminus for targeting of the protein expression to the periplasm under the control of T7 promoter and a thrombin cleavage site followed by Staphylococcal protein A (SPA) tag at the C-terminus.9 The construct was confirmed by DNA sequencing and transformed into E. coli BL21(DE3)pLysE (Novagen).
Bacterial expression and purification of recombinant 3D8 VL
The transformed cells were grown at 37°C to an OD600 of ∼0.8 in 1-L Luria-Bertani medium containing 100 μg/mL ampicillin and 20 μg/mL chloramphenicol, and then induced at 30°C for 16 h by adding 0.5 mM IPTG. The induced cells were harvested and the 3D8 VL-SPA protein was purified from the supernatant with an IgG-Sepharose column (Pharmacia Biotech). The SPA tag was removed from the fused proteins by thrombin cleavages and then the cleaved VL proteins were further purified using IgG-Sepharose column as minutely described earlier.9, 11 Protein concentration was determined using extinction coefficient of 1.674 for 3D8 VL in units of mg/(mL cm) at 280 nm, which was calculated from its amino acid sequence.
DNA hydrolyzing assay by agarose gel electrophoresis
pUC19 was purified by a plasmid miniprep kit (Intron, Korea) and used as a substrate. The DNA-hydrolyzing experiments were initiated by mixing 3D8 VL protein (5 μM) with the substrate (2.2 nM) in a reaction buffer consisting of TBS (50 mM Tris-HCl, pH 7.5, and 50 mM NaCl) containing 2 mM MgCl2. If necessary, 2 mM MgCl2 was replaced with 2 mM CoCl2, 2 mM CdCl2, or 50 mM EDTA in the buffer. After reactions were performed at 37°C for 1 or 12 h and terminated by mixing with a gel loading dye containing 1% SDS,8 samples were analyzed on 0.8% agarose gels by electrophoresis. The agarose gels were stained with ethidium bromide.
Crystallization, data collection, and structure determination
For crystallization, the purified 3D8 VL protein was concentrated into a buffer of 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), pH 6.5, and 50 mM NaCl. The initial crystallizing conditions of 3D8 VL were obtained from Sparse Matrix Screening.12 Crystals suitable for X-ray diffraction experiments were obtained at 18°C within 7 days from the precipitant conditions of 30–40% polyethyleneglycol 3350 and 0.1M BTB, pH 6.5. For data collection, crystals were immediately placed in a −173°C nitrogen-gas stream. Diffraction data of 3D8 VL were collected to 1.60 Å at the 4A beamline of Pohang Accelerator Laboratory (PAL, Korea). The data were then indexed and scaled using the HKL2000.13 Crystals belonged to the hexagonal space group P3121, with unit-cell parameters of a = 61.75 Å, b = 61.75 Å, c = 95.75 Å, α = β = 90°, and γ = 120°. For metal complexes, VL crystals were soaked into the precipitant solutions containing 2 mM CoCl2 or 2 mM CdCl2 for more than 10 h at 18°C. The phasing problem for 3D8 VL-BTB complex was solved with molecular replacement program Phaser-1.3,14 using the VL sub-domain of our previous 3D8 scFv structure (PDB ID 2GKI) as a search model. Further model building was manually performed using program O15 and the refinement was done at 1.60 Å with CNS.16 The metal complex structures were refined at 2.0 and 2.4 Å for the Co2+ and Cd2+ complex structures, respectively. The data statistics are summarized in Table I.
Table I. Data Collection and Refinement Statistics
Values in parentheses are for the highest-resolution shell.
Rsym = Σhkl Σj|Ij−<I>|/ΣhklΣjIj, where <I> is the mean intensity of reflection hkl.
Rfactor = Σhkl||Fobs|−|Fcalc||/Σhkl|Fobs|; where Fobs and Fcalc are the observed and calculated structure factor amplitude, respectively, for reflections hkl included in the refinement.
Rfree is the same as Rfactor but calculated over a randomly selected fraction (10%) of reflection data not included in the refinement.
The refined model was deposited in the Protein Data Bank (PDB ID 3BD3 for VL-BTB complex, 3BD4 for VL-BTB-Cd complex, and 3BD5 for VL-BTB-Co complex).
RESULTS AND DISCUSSION
Crystal structure of VL-BTB complex
Crystal structure of 3D8 VL was elucidated by molecular replacement and two molecules are located in the asymmetric unit (molA and molB), which have different intermolecular interactions in the crystalline state. Among the cloned 123 residues, 112 amino acids were built into the experimental electron density. The missing residues are located in the both terminal regions of the construct, and the Tyr93–Tyr96 residues of the CDR3 region in molB have rather weak electron density map. Ala51 in both molecules and Ser77 in molA have strong electron density map, but they belong to the disallowed and generously allowed regions, respectively, upon analysis of the Ramachandran plot by PROCHECK.17
3D8 VL structure is composed of two β-sheets of a five- and a four-stranded β-sheet, which form a frequently observed β-sandwich fold [Fig. 1(A)]. The single-domain VL structure is well superposed with the VL sub-domain structure of 3D8 scFv except the hypervariable loop CDR3 between Ser91 and Tyr96 [Fig. 1(B)]. This loop contains the sole His residue of the sub-domain VL (sub-VL) in 3D8 scFv, which was necessary for the catalytic activity of 3D8 scFv. Further, single-domain VLs expose new surfaces that were buried in the domain interface of 3D8 scFv structure [Fig. 1(C)]. The exposed new grooves contain polar residues of Glu55, Lys89, and Ser91, and aromatic residues of Tyr32, Tyr36, Tyr49, Trp50, and Tyr96, as well as hydrophobic residues of Leu46 and Phe98.
At the final refining step, an extra electron density was observed in the exposed new groove of one of the two molecules (molA) in the asymmetric unit. Careful analysis of the electron density and the chemicals used during purification and crystallization interpreted it as a BTB molecule derived from the precipitant solution [Fig. 1(D)]. The bound BTB molecule interacts with VL protein atoms using its four out of five hydroxyl groups, directly and indirectly through water molecules. The OH of Ser91 and the NE1 of Trp50 are simultaneously hydrogen-bonded to one hydroxyl group (O6) of BTB. Another hydroxyl group (O3) of BTB interacts with the NZ of Lys89 and the OH of Tyr36. The third hydroxyl group (O4) is directly hydrogen-bonded to the side chain of Ser91 and indirectly through a water molecule to the peptidyl carbonyl oxygen atom of Lys89.
Together with protein atoms, the bound BTB molecule traps two water molecules [Wat1 and Wat2 in Fig. 1(D)], which make hydrogen bonds between them and mediate interactions between the bound BTB molecule and the VL protein. Wat1 interacts with OH of Ser91, carbonyl oxygen of Lys89, and the hydroxyl group (O4) of BTB. Wat2 makes polar interactions with NE1 of Trp50 and one OH (O6) of the bound BTB molecule [Fig. 1(D)].
The other VL molecule molB, whose groove is exposed in solvent region, is, however, occupied by two water molecules (Wat3 and Wat4) without BTB. Like Wat1 and Wat2 trapped by BTB and protein atoms, these two water molecules make hydrogen bonds between them and to proteins atoms. Wat3 interacts with OH of Ser91 and with the carbonyl oxygen atoms of Lys89 and Tyr32, whereas Wat4 molecule interacts with peptidyl nitrogen atom of Trp50.
Crystal structures of VL-BTB-metal complex
In the previous study, we have shown that divalent Mg2+ ion, not Ca2+ ion, is required for DNA-hydrolyzing activity of 3D8 VL as well as 3D8 scFv.10 To get further insight on the catalytic site of 3D8 VL, the DNA-degrading activity of VL was checked in the presence of heavy atoms such as divalent Co2+ and Cd2+ ions. When Mg2+ ion was replaced with Co2+ ion, 3D8 VL efficiently hydrolyzed the used pUC19 plasmid DNA with an almost same kinetics as that in the presence of Mg2+ ion. However, VL exhibited a little bit lowered enzymatic activity in the presence of Cd2+ ion, about 80% compared with that of Mg2+ ion [Fig. 2(A)]. The metal-chelating EDTA totally abolished the hydrolyzing activity of 3D8 VL [Fig. 2(A)], which is same as earlier.11
To find out the metal binding site of 3D8 VL using crystallography, VL crystals were soaked into the precipitant solutions for 10 h, which contain 2 mM each metal ion of CoCl2 or CdCl2. The complex structures of VL-BTB-Co and VL-BTB-Cd were obtained at 2.0 and 2.4 Å resolution, respectively (Table I). Like the VL-BTB complex, the CDR3 regions in molBs of both complex structures are partially disordered. The strong electron density of two crystals commonly indicated that both Cd2+ and Co2+ ions replace one of the two water molecules trapped in the exposed new grooves in both monomers [Fig. 2(B,C)]. In molA of VL-BTB-metal complex structures, Wat2 is replaced by Co2+ or Cd2+ but the interaction between a hydroxyl group (O6) of BTB and NE1 of Trp50 observed in VL-BTB structure is lost. The bound metal ions are coordinated by two hydroxyl atoms (O4 and O6) of BTB and Wat1 [Fig. 2(B)].
In VL-BTB-Cd complex, a large, strong electron density was observed in the groove of molB, which was interpreted as a Cd2+ and three water molecules [Fig. 2(C)]. However, the interaction between water molecules and protein atoms is totally different from that observed in complex with BTB in molA. Cd2+ ion is surrounded by three water molecules, which interact with side chain atoms of Ser91and Tyr36 and with main chain atoms of peptide carbonyl oxygen of Leu33 and peptide nitrogen of Trp50, respectively.
Interestingly enough, metal replacement of water molecule in the groove of molA relocates the bound BTB molecule slightly far away from the protein molecule [Fig. 2(D)]. Therefore, the interaction between one hydroxyl group (O6) of the bound BTB and NE1 of Trp50 is missing in both the metal complex structures.
Putative active site of 3D8 VL
BTB molecule has five hydroxyl groups. Using hydroxyl groups, BTB takes up catalytic cavity of a protein or coordinates a metal ion. For example, the BTB in a cell wall inhibitor of β-fucosidase from Nicotina tabacum coordinates a cadmium ion that was necessary for its activity.18 On the other hand, BTB is bound in a large pocket of the small domain of type III CoA transferase from E. coli in carnitine metabolism, which was proposed to constitute the binding site for substrate moieties participating in the CaiB transfer reaction.19
Each VH and VL sub-domain of 3D8 scFv structure has similar folding of β-sandwich and has grooves at the domain interface, which were completely hidden from the solvent-accessible 3D8 scFv surface. As shown in Figure 1(C), however, these grooves are exposed toward solvent region in forming a single-domain VL structure. This exposed new groove in VL structure provides a cavity to trap one BTB molecule along with two water molecules [Fig. 1(D)]. Further, the cavity was a binding site for both Co2+ and Cd2+ in the metal complex structures, regardless of the presence of BTB [Fig. 2(B,C)]. The divalent metal ions such as Mg2+, Co2+, and Cd2+ are essential for the catalytic activity of single-domain 3D8 VL [Fig. 2(A)], suggesting the metal binding pocket observed in the complex structures is most likely the catalytic site of single-domain VL for the DNA-hydrolyzing activity [Fig. 2(A)].10
The purified 3D8 VL Ab migrated as a monomer,10 which excludes any possibility to self-assemble to form a quaternary structure resembling the scFv structure. Therefore, the DNA-degrading activity of 3D8 VL should be contributed by a monomeric property of VL. Based on the structural and the biochemical features of 3D8 scFv and 3D8 VL and the generation of new enzymatic activity upon forming a monomeric 3D8 VL, we strongly suggest that active site of 3D8 VL might include this exposed new groove including Ser91 and Trp50 as catalytically important residues. Additional structural studies of DNA complexes and subsequent mutational studies are needed to understand the enzymatic mechanism of this novel anti-DNA degrading enzyme 3D8 VL.
We thank Dr. K. J. Kim, Pohang Light Source, for helping with data collection.