STRUCTURE OF THE RECOMBINANT PARAMECIUM-TETRAURELIA CALMODULIN AT 1.68 ANGSTROM RESOLUTION

The crystal structure of the recombinant calmodulin from Paramecium te traurelia (rPCaM, M(r)= 16 700, 148 residues) has been determined at 1 .68 Angstrom. resolution. X-ray intensity data were collected at 263 K using a Siemens-Nicolet area detector and Cu K alpha radiation from a rotating-anode source. A total of 35 936 observations were processed with XENGEN1.3 and scaled to yield 16255 unique reflections with R(sym m)(I) of 4.1%. The crystals are triclinic, with unit-cell dimensions a =29.89, b= 53.42, c = 25.35 Angstrom, alpha = 93.67, beta = 96.88, gam ma = 89.24 degrees, space group P1, with one molecule in the unit cell . The atomic coordinates of the wild-type Paramecium calmodulin (PCaM) studied in our laboratory provided the starting model. Refinement of the structure by X-PLOR and refitting it into omit maps yielded an R v alue of 0.194 for 15 965 reflections greater than 3 sigma(F) in the 6. 0-1.68 HL resolution range. The final model contained 1165 protein ato ms for all of the 148 residues, four Ca2+ ions, and 172 water molecule s. The dumbbell structure has seven alpha-helices including a long 7.8 turn central helix connecting the two terminal domains each containin g two EF-hand (helix-loop-helix motif) calcium-binding sites. The loop s within each pair of EF-hand motifs in the N- and C-terminal domains are brought into juxtaposition to form a pair of hydrogen-bonded antip arallel beta-sheets which are extended at either ends by water bridges . The four calcium-binding EF-hands are superposable with r.m.s. devia tions of 0.31-0.79 Angstrom. The best agreement is between site 1 and site 3 and the worst agreement is between site 1 and 4. The largest di fferences are in the ninth and tenth residues of the calcium-binding l oops probably because of their involvement in the mini beta-sheets. Th e calcium coordination distances vary between 2.04 and 2.69 Angstrom, average 2.34 Angstrom. The rPCaM and wild-type PCaM have an r.m.s. dev iation of 0.36 Angstrom for equivalent C-alpha atoms. The side chains of Lys13 and Lys115 are more extended in rPCaM compared to the wild ty pe where the posttranslational modified di- and tri-methylated lysine residues are more folded. The sequence of PCaM differs from those of m ammalian (MCaM) and Drosophila calmodulin (DCaM), but the overall stru ctures are very similar, with r.m.s. deviations of 0.44 and 1.68 Angst rom for equivalent C-alpha atoms, respectively. However, in rPCaM, the first four N-terminal residues stretch out and make intermolecular cr ystal contacts, in contrast to those in recombinant Drosophila calmodu lin (rDCaM), they stretch out in the opposite direction and towards th e second calcium-binding site (see note below), while in MCaM and wild -type PCaM, the N-terminal residues are not visible. The central helix in rPCaM has all its backbone hydrogen bonds intact with no unusually long separation between the carbonyl and amide groups as found in MCa M and rDCaM.