D19

F. Designed CBD-site II

Figure 4. The local structures of the classic EF-hand calcium binding site 1 of calmodulin (A) and the relocated Site 1 (B). The local structures of the calcium binding site lof calbindinD9k with a pseudo-EF-hand in the natural protein (C) and the relocated Site 1 (D). The calcium binding site 2 of calbindinD9k with a classic-EF-hand in the natural protein (E) and the relocated Site 2 (F).

Figure 4. The local structures of the classic EF-hand calcium binding site 1 of calmodulin (A) and the relocated Site 1 (B). The local structures of the calcium binding site lof calbindinD9k with a pseudo-EF-hand in the natural protein (C) and the relocated Site 1 (D). The calcium binding site 2 of calbindinD9k with a classic-EF-hand in the natural protein (E) and the relocated Site 2 (F).

All four natural calcium-binding sites in calmodulin are successfully identified. Their parameters for the bond length of Ca-O and angle O-Ca-O are listed in Tables 1 and 2. All the redesigned natural calcium-binding sites use the expected ligand residue and corresponding oxygen atoms. As shown in Fig. 4, the geometric parameters, such as bond lengths of Ca-O andO-Ca-O angles of the designed calcium binding sites, are very similar to that in natural calcium binding sites. Except Site 4 of calmodulin, the deviations of bond lengths of Ca-O and angles O-Ca-O are within 0.35 A and 18 O, respectively.

Natural calcium binding sites have the lowest U(p) numbers.

The U(p) numbers for all the reconstructed calcium binding sites in calmodulin are plotted in Fig. 5. The highest number is 150.55 and the lowest one is 5.02 with a mean of 58.21. The U(p) number indicates the deviation of a site from the target site. For example, Site 3091 has the highest U(p) value of 150.55, its bond lengths vary from 1.9 to 3.7 A and its O-Ca-O angles have 50 o deviation from the target values. Interestingly, all natural calcium-binding sites have small U(p) numbers less than 25, suggesting small deviations from the target ideal pentagonal bipyramidal. These results are consistent with the observation from X-ray studies that the geometry of Sites 1-4 in calmodulin resemble closely the ideal bipyramidal geometry (Babu et al., 1988).

All the constructed sites with U(p) numbers close to that of natural sites share several similarities. First, they all contain the correct bidentate Glu residues from positions 31, 67, 104 and 140 in the natural calcium binding sites of calmodulin. Second, mainchain oxygen atoms from T26, T62, Y99, and Q135 in natural calcium sites (1 to 4) are always used. Third, residue numbers used for the remaining four ligand positions are the same as those in the natural calcium binding sites with either Asp replaced by Asn or Asn replaced by Asp. These calcium-binding sites are introduced mainly because both Asn and Asp are used for four positions and both residues have very similar sidechain rotamer configurations.

Table 3. The pseudo-energy value of relocated sites and natural sites.

Protein

Site

Natural

Relocated

CaM

0 0

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