Dimerization by colicin E3 in the absence of immunity protein.

133

104

Small-angle X-ray and neutron scattering data were used to study the solution structure of calmodulin complexed with a synthetic peptide corresponding to residues 577-603 of rabbit skeletal muscle myosin light chain kinase. The X-ray data indicate that, in the presence of Ca2+, the calmodulin-peptide complex has a structure that is considerably more compact than uncomplexed calmodulin. The radius of gyration, Rg, for the complex is approximately 20% smaller than that of uncomplexed Ca2+.calmodulin (16 vs 21 A), and the maximum dimension, dmax, for the complex is also about 20% smaller (49 vs 67 A). The peptide-induced conformational rearrangement of calmodulin is [Ca2+] dependent. The length distribution function for the complex is more symmetric than that for uncomplexed Ca2+.calmodulin, indicating that more of the mass is distributed toward the center of mass for the complex, compared with the dumbell-shaped Ca2+.calmodulin. The solvent contrast dependence of Rg for neutron scattering indicates that the peptide is located more toward the center of the complex, while the calmodulin is located more peripherally, and that the centers of mass of the calmodulin and the peptide are not coincident. The scattering data support the hypothesis that the interconnecting helix region observed in the crystal structure for calmodulin is quite flexible in solution, allowing the two lobes of calmodulin to form close contacts on binding the peptide. This flexibility of the central helix may play a critical role in activating target enzymes such as myosin light chain kinase.

Section: Resultsmentioning

confidence: 99%

Changes in the structure of calmodulin induced by a peptide based on the calmodulin-binding domain of myosin light chain kinase

Heidorn

Seeger²,

Rokop³

et al. 1989

133

104

“…70, when extrapolated to zero protein concentration, was found to be the same for calmodulin in the presence of calcium or EGTA. The molecular weight corresponding to this forward scatter value was determined as previously described (Levinson et al, 1983) using hen egg-white lysozyme as a standard (molecular weight 13 900; v = 0.71). The molecular weight of calmodulin was found to be 16 500 ± 200, which agrees closely with the value of 16 680 derived from the amino acid sequence (Watterson et al, 1980).…”

Section: Resultsmentioning

confidence: 99%

“…Typical data collection times ranged from 2 to 12 h. Data Analysis. Data were analyzed as previously described (Levinson et al, 1983), using both the Guinier approximation (Guinier, 1939) and the indirect transformation method of Moore (Moore, 1980) to incorporate slit desmearing. The X-ray wavelength of 1.54 Á was used.…”

Section: Methodsmentioning

confidence: 99%

Calcium-induced increase in the radius of gyration and maximum dimension of calmodulin measured by small-angle x-ray scattering

Seaton¹,

Head²,

Engelman³

et al. 1985

138

117

We have used solution small-angle X-ray scattering to characterize bovine brain calmodulin in the presence and absence of calcium. In the presence of calcium, calmodulin exists in solution as an elongated molecule with a radius of gyration of 21.5 A and a maximum vector length of approximately 62 A. These values are consistent with the dimensions recently determined for the crystal form of rat testis calmodulin. In the absence of calcium, the calmodulin molecule is shorter, the radius of gyration decreases to 20.6 A, and the maximum vector length decreases to approximately 58 A. This change in dimensions is consistent with an overall contraction of the protein through movement of the two lobes closer to each other upon removal of calcium from calmodulin.

“…Reconstitution of the complex by addition of Imm to Imm-free colE3 restored the effective cytotoxic concentration to the level of native colE3Imm (data not shown). The small decrease in cytotoxicity of Imm-free colE3 may be explained by reversible dimerization of the Imm-free colicin (26). 21) that the 58 kDa colicin E3 molecule can efficiently (concentration, 10 nM) and specifically occlude the large well-characterized ion channels of OmpF incorporated into planar bilayers (15,27,28).…”

Section: Release Of Immunity Protein In Vitromentioning

confidence: 99%

The Colicin E3 Outer Membrane Translocon: Immunity Protein Release Allows Interaction of the Cytotoxic Domain with OmpF Porin

Zakharov

Zhalnina²,

Sharma³

2006

The crystal structure previously obtained for the complex of BtuB and the receptor binding domain of colicin E3 forms a basis for further analysis of the mechanism of colicin import through the bacterial outer membrane. Together with genetic analysis and studies on colicin occlusion of OmpF channels, this implied a colicin translocon consisting of BtuB and OmpF that would transfer the C-terminal cytotoxic domain (C96) of colicin E3 through the Escherichia coli outer membrane. This model does not, however, explain how the colicin attains the unfolded conformation necessary for transfer. Such a conformation change would require removal of the immunity (Imm) protein, which is bound tightly in a complex with the folded colicin E3. In the present study, it was possible to obtain reversible removal of Imm in vitro in a single column chromatography step without colicin denaturation. This resulted in a mostly unordered secondary structure of the cytotoxic domain and a large decrease in stability, which was also found in the receptor binding domain. These structure changes were documented by near- and far-UV circular dichroism and intrinsic tryptophan fluorescence. Reconstitution of Imm in a complex with C96 or colicin E3 restored the native structure. C96 depleted of Imm, in contrast to the native complex with Imm, efficiently occluded OmpF channels, implying that the presence of tightly bound Imm prevents its unfolding and utilization of the OmpF porin for subsequent import of the cytotoxic domain.