**5. Binding of copper ions in the constant region domain**

#### **5.1. Preparation of mutants and their uptake of copper ions**

potential of each molecular form is at a comparable level after the preparation of the molecule (CL) without Cu2+, as shown in **Figure 17a**. In this case, transfer of the potential well A to B (or C) is easy because the walls of the potential energies of the wells are low (**Figure 17b**). However, the energy potential is drastically changed when copper ions are added. The multimolecular forms of the constant region domain, which are sitting in each potential well, drop in one deep potential energy level, as shown in **Figure 17c**, resulting in the formation of a mono-molecular form from the multi-molecular forms. Once the molecule dropped into the deep potential well, the form would be no longer able to transfer to other forms. As a consequence, the monomolecular form of the constant region domain molecule became stable. This situation can be achieved by the presence of copper ion in a ratio of more than 0.5 eq. of Cu2+

**Figure 17.** Consideration about conversion of unstable forms to a stable form of CL. (a) State A (corresponding to peak 1 in **Figure 10**), State B (corresponding to peaks 2, 3, and 4 in **Figure 10**), and State C (corresponding to peaks 5 and 6 in **Figure 10**) may stay in a chemical equilibrium. (b) Assumed situation in potential energy for the case without Cu2+: each potential energy level for the case without Cu2+ may be comparable in wells of A, B, and C. The walls among the potential energy wells are not high. (c) Assumed situation of potential energy for the case with Cu2+: when Cu2+ is incorporated, a deep potential level can be generated, and all molecules showing a different heterogeneity may drop into the well and

to the constant region domain molecule.

248 Antibody Engineering

exist as a stable form.

In order to clarify the copper-binding site, two mutants were prepared from the C51 light chain, because the light chain has no histidine residues in the variable region compared to the sequence of the constant region domain comprising 2 His residues (**Figure 8**). Both histidine and cysteine residues are considered as the most plausible candidates for the binding site. Therefore, the residues of His195, His204, and Cys220 present in the constant region domain of the C51 light chain were mutated to Ala. As the consequence, two mutants were made. One is Cys220Ala (C220A: mono-mutant) and another is His195Ala, His204Ala, and Cys220Ala (H195A/H204A/C220A: triple-mutant; **Figure 18a**). The locations of the mutated residues are shown in **Figure 18b**.

**Figure 18.** C51 mutants and locations of His and Cys residues. There are no histidine residues in the variable region of the C51 light chain. (a) Location of Cys220, His195 and His204 in wild type. The mutated positions, C220A and H195A/ H204A/C220A, are also indicated with green colored character. (b) Three-dimensional structure of the C51 light chain. Light blue is sheet structure and red is helix structure.

These mutants were similarly expressed and purified as stated in the previous experiments. Fifty μM of Cu2+ (1.25 eq.) was added to both the cell suspension and the Ni-NTA eluent, where all light chains gave a single peak in the cation exchange chromatography. The copper uptake by the wild type and each mutant was chemically analyzed and the results are presented in **Table 2**. The wild type possessed 0.75 atoms of Cu2+ per one C51 light chain of full length. That of the C220A and H195A/H204A/C220A mutant was 0.54 and 0.25, respectively.

As stated in the above section, the value in the case of the constant region domain was 0.55. Taking together this finding and the full-length cases into account, it is considered that the variable region domain uptakes 0.20 atom (0.75–0.55 = 0.20). Therefore, the mono-mutant C220A is supposed to bind with 0.34 atom-Cu (0.54–0.20 = 0.34) and the triple-mutant H195A/H204A/C220A 0.05 atom-Cu (0.25–0.20 = 0.05). These facts are strongly implying that histidine residues at positions 195th and 204th as well as cysteine at position 220th are responsible for the copper-binding site.

#### **5.2. Possibility of a zinc finger in the constant region domain**

It is not well known that there is a zinc finger-like motif in the constant region of the antibody light chain. Interestingly, Radulescu pointed out that the motif is a type of Cys-X<sup>3</sup> -His [35]. The sequence Cys-X<sup>3</sup> -His-X15-Cys-X<sup>3</sup> -His is a complete motif of a zinc finger. The aa sequence of the constant region domain used in this article is presented in **Figure 8**. The sequence from positions 190th–224th of the constant region domain is CEVTHQGLSSPVTKSFNRGECLEHH. The sequence of LEHH was adducted as a His-tag was introduced. The underlined amino acids agree with those of the zinc finger motif, Cys-X<sup>3</sup> -His-X15-Cys-X<sup>3</sup> -His, in which His224 is a part of the His-tag. This motif can uptake a metal ion such as Zn2+, which is a divalent metal ion. As Cu2+ is also a divalent metal ion, it can bound to the motif. Based on the results of the chemical analysis of copper ions in mutants, those histidine and cysteine residues must be responsible to uptake the copper ion. It is plausible that a copper ion is able to bind to the zinc finger motif instead of a zinc ion. As seen in the investigation of divalent metal ions on the structural diversity, zinc ions showed some effect. This maybe ascribed to the presence

of a zinc finger motif in the constant domain in the light chain (note that the zinc finger motif is conserved in both human and mouse antibody light chain (kappa type)). In addition, the similar motif composed of the same amino acids exists in the CH1 domain of the heavy chain. As well known, a zinc finger can function as a transcription factor influencing gene regulation and protein expression. Few studies on the relationship between antibody and zinc finger have been made so far. From the viewpoint that one protein can have dual or multi-functions in case, the chemical and biochemical functions of each domain of an antibody should be

to 224th (His224 is a part of a His-tag) is existing in the constant region domain of the light chain (CL). As Cu2+ is also a divalent metal ion, it can be bound with the motif instead of a zinc ion, unless the ion is present in the solution. By the addition of copper ions, the CL is easily becoming the dimer form. Considering the chemical analysis of copper ions, one


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251


Taking those facts mentioned above and discussed, the binding site of copper is assumed as illustrated in **Figure 19**. Cu2+ must be coordinated with histidine and cysteine residues of dimeric CL molecules. In this situation, the copper ion mediates the CL molecule placing the

AFM analysis was performed using the #4 light chain as shown in **Figure 20a**–**g**. **Figure 20a, c,** and **e** demonstrates the wild type of the #4 light chain. The results obtained with the mutant (C220A) of the #4 light chain are shown in **Figure 20f** and **g**. The red circles in the figures rep-

investigated in detail.

lowest potential energy level.

resent the clear image of the AFM.

**6. Molecular forms**

**6.1. AFM analysis**

**5.3. Assumed binding site of a copper ion**

**Figure 19.** Assumed binding site for copper ion. A zinc finger motif, Cys-X<sup>3</sup>

copper atom binds with two CL molecules (CL1 and CL2) via His and Cys residues.


\*Result: The ratio of full length of C51 vs Cu and the constant region domain of the light chain (CL) vs Cu was 0.75 and 0.55, respectively. This result suggests that the contribution of the variable domain of the light chain (VL) should be 0.20 atom. (0.75-0.55)=0.20)

\*\*: calculation

\*\*\*: assumption

#### **Table 2.** Copper uptake by each light chain and constant region domain (CL).

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**Figure 19.** Assumed binding site for copper ion. A zinc finger motif, Cys-X<sup>3</sup> -His-X15-Cys-X<sup>3</sup> -His, from positions 190th to 224th (His224 is a part of a His-tag) is existing in the constant region domain of the light chain (CL). As Cu2+ is also a divalent metal ion, it can be bound with the motif instead of a zinc ion, unless the ion is present in the solution. By the addition of copper ions, the CL is easily becoming the dimer form. Considering the chemical analysis of copper ions, one copper atom binds with two CL molecules (CL1 and CL2) via His and Cys residues.

of a zinc finger motif in the constant domain in the light chain (note that the zinc finger motif is conserved in both human and mouse antibody light chain (kappa type)). In addition, the similar motif composed of the same amino acids exists in the CH1 domain of the heavy chain.

As well known, a zinc finger can function as a transcription factor influencing gene regulation and protein expression. Few studies on the relationship between antibody and zinc finger have been made so far. From the viewpoint that one protein can have dual or multi-functions in case, the chemical and biochemical functions of each domain of an antibody should be investigated in detail.

#### **5.3. Assumed binding site of a copper ion**

Taking those facts mentioned above and discussed, the binding site of copper is assumed as illustrated in **Figure 19**. Cu2+ must be coordinated with histidine and cysteine residues of dimeric CL molecules. In this situation, the copper ion mediates the CL molecule placing the lowest potential energy level.
