**6. Molecular forms**

#### **6.1. AFM analysis**

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.

light chain. Interestingly, Radulescu pointed out that the motif is a type of Cys-X<sup>3</sup>

It is not well known that there is a zinc finger-like motif in the constant region of the antibody

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

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

Cu / full length

Clone name of C51 Cu / CL

0.54\*

\*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.

0.25\* 0.05\*\*

0.75\*



0.55\* 0.20\*\*

Contribution\* By VL

0.20\*\*\*

0.20\*\*\*

0.34\*\*



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


acids agree with those of the zinc finger motif, Cys-X<sup>3</sup>

Wild Type

C220A

(0.75-0.55)=0.20) \*\*: calculation \*\*\*: assumption

H195A/H204A/C220A

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

The sequence Cys-X<sup>3</sup>

250 Antibody Engineering

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 represent the clear image of the AFM.

In the case of the #4 wild type, two kinds of form were observed. One is the dimer circled with #1 red color (**Figure 20a**). This seems to be a *cis* form, whose molecular conformation corresponds to that of **Figure 20b**. A variable region faces to another variable region. On the other hand, the dimer circled with #2 red color (**Figure 20c**) seems *trans* form, whose molecular conformation corresponds to that of **Figure 20d**. **Figure 20e** was another spot, where *cis* and *trans* forms were observed. In contrast, a very simple form was observed in the case of #4 mutant C220A, as shown in **Figure 20f** and **g**. In any spots, only monomeric forms were observed.

**6.2. X-ray diffraction analysis**

We are trying to determine the detailed steric conformation of a catalytic light chain. At present, the structure of the main chain of the #4 mutant C220A was clarified as a preliminary experiment. **Figure 21a** shows a single crystal of the #4 mutant C220A formed in the experiment. By using the single crystal, X-ray diffraction analysis was performed. The result is presented in **Figure 21b**, where a 3.1 Å resolution was attained. Interestingly, there are eight

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**Figure 21.** X-ray diffraction analysis for #4 C220A. (a) crystallization: a single crystal (like a small pillar) of the #4 mutant C220A is seen in a red dotted circle. (b) Conformation of the main chain: As the preliminary experiment, the conformation of the main chain for the #4 mutant 220A was determined. Each #4 mutant molecule is indicated with each color such as red, yellow, green, blue, etc. Eight molecules of the mutant were packed in one lattice, indicating that the

mutant molecules may interact with each other by hydrophobic interaction.

molecules of the #4 mutant in one lattice mediated by hydrophobic interaction.

If copper ion is incorporated into the zinc finger motif residing in the constant region domain, the dimeric form observed in the #4 wild type is easily formed. On the other hand, the mutant C220A exists as the monomeric form, which indicates that the cysteine locating at position 220th is a crucial amino acid to bind to a copper ion.

**Figure 20.** AFM analysis for #4 light chains. These AFM images were taken under the same condition as in **Figure 7**. (a) #4 wild type (*cis form*). (b) Molecular modeling for *cis form.* (c) #4 wild type (*trans form*). (d) Molecular modeling for *trans form.* (e) Mixture of *cis and trans forms.* In the case of the #4 wild type, two kinds of forms were observed. One is the dimer circled with #1 red color (a). This seems to be a *cis* form, whose molecular conformation corresponds to that of (b). A variable region faces to another variable region. The dimer circled with #2 red color (c) seems to be a *trans* form, whose molecular conformation corresponds to that of (d). **Figure 20e** was another view, where *cis* and *trans* forms were observed. (f) #4 mutant (C220A). (g) #4 mutant (C220A) (another spot). For #4 mutant (C220A), one simple form was observed. In any views, only monomeric forms were observed.

#### **6.2. X-ray diffraction analysis**

**Figure 20.** AFM analysis for #4 light chains. These AFM images were taken under the same condition as in **Figure 7**. (a) #4 wild type (*cis form*). (b) Molecular modeling for *cis form.* (c) #4 wild type (*trans form*). (d) Molecular modeling for *trans form.* (e) Mixture of *cis and trans forms.* In the case of the #4 wild type, two kinds of forms were observed. One is the dimer circled with #1 red color (a). This seems to be a *cis* form, whose molecular conformation corresponds to that of (b). A variable region faces to another variable region. The dimer circled with #2 red color (c) seems to be a *trans* form, whose molecular conformation corresponds to that of (d). **Figure 20e** was another view, where *cis* and *trans* forms were observed. (f) #4 mutant (C220A). (g) #4 mutant (C220A) (another spot). For #4 mutant (C220A), one simple form was

In the case of the #4 wild type, two kinds of form were observed. One is the dimer circled with #1 red color (**Figure 20a**). This seems to be a *cis* form, whose molecular conformation corresponds to that of **Figure 20b**. A variable region faces to another variable region. On the other hand, the dimer circled with #2 red color (**Figure 20c**) seems *trans* form, whose molecular conformation corresponds to that of **Figure 20d**. **Figure 20e** was another spot, where *cis* and *trans* forms were observed. In contrast, a very simple form was observed in the case of #4 mutant C220A, as shown in **Figure 20f** and **g**. In any spots, only monomeric forms were observed.

If copper ion is incorporated into the zinc finger motif residing in the constant region domain, the dimeric form observed in the #4 wild type is easily formed. On the other hand, the mutant C220A exists as the monomeric form, which indicates that the cysteine locating at position

observed. In any views, only monomeric forms were observed.

220th is a crucial amino acid to bind to a copper ion.

252 Antibody Engineering

We are trying to determine the detailed steric conformation of a catalytic light chain. At present, the structure of the main chain of the #4 mutant C220A was clarified as a preliminary experiment. **Figure 21a** shows a single crystal of the #4 mutant C220A formed in the experiment. By using the single crystal, X-ray diffraction analysis was performed. The result is presented in **Figure 21b**, where a 3.1 Å resolution was attained. Interestingly, there are eight molecules of the #4 mutant in one lattice mediated by hydrophobic interaction.

**Figure 21.** X-ray diffraction analysis for #4 C220A. (a) crystallization: a single crystal (like a small pillar) of the #4 mutant C220A is seen in a red dotted circle. (b) Conformation of the main chain: As the preliminary experiment, the conformation of the main chain for the #4 mutant 220A was determined. Each #4 mutant molecule is indicated with each color such as red, yellow, green, blue, etc. Eight molecules of the mutant were packed in one lattice, indicating that the mutant molecules may interact with each other by hydrophobic interaction.

In the case of the AFM analysis, the #4 mutant C220A was a monomer. This is ascribed that the mica as the supporting material firmly interacts with the #4 mutant molecule. In solution, the #4 mutant may interact with each other with a strong Van der Waals force.

4 Tottori College of Nursing, Kurayoshi-shi, Tottori, Japan

Nanotechnologies (ISIT), Fukuoka, Japan

Journal of Medicine. 2005;**353**:1734-1736

Japan

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5 Graduate School of System Life Science, Kyushu University, Nishi-ku, Fukuoka, Japan

7 Nanotechnology Laboratory, Institute of Systems, Information Technologies and

6 Department of Applied Chemistry, Faculty of Engineering, Oita University, Oita-shi, Oita,

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