Domain Swapping and Different Oligomeric States for the Complex Between Calmodulin and the Calmodulin-Binding Domain of Calcineurin A

BACKGROUND: Calmodulin (CaM) is a ubiquitously expressed calcium sensor that engages in regulatory interactions with a large number of cellular proteins. Previously, a unique mode of CaM target recognition has been observed in the crystal structure of a complex between CaM and the CaM-binding domain of calcineurin A. METHODOLOGY/PRINCIPAL FINDINGS: We have solved a high-resolution crystal structure of a complex between CaM and the CaM-binding domain of calcineurin A in a novel crystal form, which shows a dimeric assembly of calmodulin, as observed before in the crystal state. We note that the conformation of CaM in this complex is very similar to that of unliganded CaM, and a detailed analysis revels that the CaM-binding motif in calcineurin A is of a novel '1-11' type. However, using small-angle X-ray scattering (SAXS), we show that the complex is fully monomeric in solution, and a structure of a canonically collapsed CaM-peptide complex can easily be fitted into the SAXS data. This result is also supported by size exclusion chromatography, where the addition of the ligand peptide decreases the apparent size of CaM. In addition, we studied the energetics of binding by isothermal titration calorimetry and found them to closely resemble those observed previously for ligand peptides from CaM-dependent kinases. CONCLUSIONS/SIGNIFICANCE: Our results implicate that CaM can also form a complex with the CaM-binding domain of calcineurin in a 1 ratio 1 stoichiometry, in addition to the previously observed 2 ratio 2 arrangement in the crystal state. At the structural level, going from 2 ratio 2 association to two 1 ratio 1 complexes will require domain swapping in CaM, accompanied by the characteristic bending of the central linker helix between the two lobes of CaM

Structure of the dimeric autoinhibited conformation of DAPK2, a pro-apoptotic protein kinase.

The death-associated protein kinase (DAPK) family has been characterized as a group of pro-apoptotic serine/threonine kinases that share specific structural features in their catalytic kinase domain. Two of the DAPK family members, DAPK1 and DAPK2, are calmodulin-dependent protein kinases that are regulated by oligomerization, calmodulin binding, and autophosphorylation. In this study, we have determined the crystal and solution structures of murine DAPK2 in the presence of the autoinhibitory domain, with and without bound nucleotides in the active site. The crystal structure shows dimers of DAPK2 in a conformation that is not permissible for protein substrate binding. Two different conformations were seen in the active site upon the introduction of nucleotide ligands. The monomeric and dimeric forms of DAPK2 were further analyzed for solution structure, and the results indicate that the dimers of DAPK2 are indeed formed through the association of two apposed catalytic domains, as seen in the crystal structure. The structures can be further used to build a model for DAPK2 autophosphorylation and to compare with closely related kinases, of which especially DAPK1 is an actively studied drug target. Our structures also provide a model for both homodimerization and heterodimerization of the catalytic domain between members of the DAPK family. The fingerprint of the DAPK family, the basic loop, plays a central role in the dimerization of the kinase domain

Interaction between the C-terminal region of human myelin basic protein and calmodulin: analysis of complex formation and solution structure-2

S titrated into buffer at +30°C, are indicated above, moved up by 0.1 μcal/s for clarity. B. Fitting for the specific binding site only. The solid line indicates the non-linear least squares fit for the integrated area under peaks 2–7. The fitting model is one binding site. C. Fitting of all data to a multi-site model. The solid line indicates the non-linear least squares fit for the integrated area under peaks 2–20. The used model is for 4 sequential binding sites. Only the parameters for first binding event were used for analysis. D. The observed enthalpy as a function of temperature. The heat capacity (= -0.13 kcal molK) is the slope calculated by using values at temperatures +25 and +30°C.<p><b>Copyright information:</b></p><p>Taken from "Interaction between the C-terminal region of human myelin basic protein and calmodulin: analysis of complex formation and solution structure"</p><p>http://www.biomedcentral.com/1472-6807/8/10</p><p>BMC Structural Biology 2008;8():10-10.</p><p>Published online 19 Feb 2008</p><p>PMCID:PMC2288786.</p><p></p

Interaction between the C-terminal region of human myelin basic protein and calmodulin: analysis of complex formation and solution structure-0

Mode with yellow colour. Note how the binding site is localised mainly to the hydrophobic pocket of the C-terminal (lower) lobe. B. Stereo view of the conformation of the CaM C-terminal lobe in free CaM (pink) and in the SAXS model of the complex (light blue). The peptide is shown in yellow, and superposition has been done using the residues of the central helix (pointing towards top right). The N-terminal lobe is omitted from the figure for clarity. An animation of the predicted movement is shown in Additional file .<p><b>Copyright information:</b></p><p>Taken from "Interaction between the C-terminal region of human myelin basic protein and calmodulin: analysis of complex formation and solution structure"</p><p>http://www.biomedcentral.com/1472-6807/8/10</p><p>BMC Structural Biology 2008;8():10-10.</p><p>Published online 19 Feb 2008</p><p>PMCID:PMC2288786.</p><p></p

Interaction between the C-terminal region of human myelin basic protein and calmodulin: analysis of complex formation and solution structure-4

Es contained 0.5 mM CaM, 120 mM NaCl, 2.5 mM CaCl, and 50 mM deuterated Tris-HCl (pH 7.5) in 90% HO and 10% DO. Changes in cross-peak positions were quantified by [(ΔN)+ (ΔH)], and some cross-peaks showing larger shifts are indicated on the figure. B. Chemical shift perturbations quantified by [(ΔN)+ (ΔH)]. Residues with large perturbations ([(ΔN)+ (ΔH)]> 10.0) are shown. Numbers of each bar show their residual numbers. Positions of four EF hands are also indicated under the graph<p><b>Copyright information:</b></p><p>Taken from "Interaction between the C-terminal region of human myelin basic protein and calmodulin: analysis of complex formation and solution structure"</p><p>http://www.biomedcentral.com/1472-6807/8/10</p><p>BMC Structural Biology 2008;8():10-10.</p><p>Published online 19 Feb 2008</p><p>PMCID:PMC2288786.</p><p></p

Interaction between the C-terminal region of human myelin basic protein and calmodulin: analysis of complex formation and solution structure-5

Ion volumes and molecular weights (in kDa) of the standard proteins used to calibrate the column. The positions of the collected fractions from the complex sample are indicated below the graph. The absorbances have been multiplied by 1000 for clarity. B. Electrophoretic analysis of the fractions obtained from the complex sample on a 16.5-% Tris-tricine peptide gel. The samples are as follows: 1, the CaM-peptide mixture injected into the column; 2, fraction 10; 3, fraction 11; 4, fraction 12; 5, CaM; 6, peptide. The positions of molecular weight markers (in kDa) are indicated on the left, and the positions of CaM and the peptide are indicated on right. Note how the peak fraction (lane 3) contains both CaM and the peptide. A weak low-molecular-weight shadow is also seen in the CaM sample (lane 5), but the peptide stains intensely dark with silver and can unequivocally be detected based on its colour. The faint band above CaM is most likely another conformation of CaM.<p><b>Copyright information:</b></p><p>Taken from "Interaction between the C-terminal region of human myelin basic protein and calmodulin: analysis of complex formation and solution structure"</p><p>http://www.biomedcentral.com/1472-6807/8/10</p><p>BMC Structural Biology 2008;8():10-10.</p><p>Published online 19 Feb 2008</p><p>PMCID:PMC2288786.</p><p></p

Interaction between the C-terminal region of human myelin basic protein and calmodulin: analysis of complex formation and solution structure-1

B. Thermal stability curves of CaM (black) and the complex (red), monitored at 222 nm. Mean residue ellipticity as a function of temperature was measured during heating from +20 to +98°C (thick lines). Refolding of the samples during cooling from +98 to +20°C is indicated by the thin lines.<p><b>Copyright information:</b></p><p>Taken from "Interaction between the C-terminal region of human myelin basic protein and calmodulin: analysis of complex formation and solution structure"</p><p>http://www.biomedcentral.com/1472-6807/8/10</p><p>BMC Structural Biology 2008;8():10-10.</p><p>Published online 19 Feb 2008</p><p>PMCID:PMC2288786.</p><p></p

Structural analysis of the complex between calmodulin and full-length myelin basic protein, an intrinsically disordered molecule.

Myelin basic protein (MBP) is present between the cytoplasmic leaflets of the compact myelin membrane in both the peripheral and central nervous systems, and characterized to be intrinsically disordered in solution. One of the best-characterized protein ligands for MBP is calmodulin (CaM), a highly acidic calcium sensor. We pulled down MBP from human brain white matter as the major calcium-dependent CaM-binding protein. We then used full-length brain MBP, and a peptide from rodent MBP, to structurally characterize the MBP-CaM complex in solution by small-angle X-ray scattering, NMR spectroscopy, synchrotron radiation circular dichroism spectroscopy, and size exclusion chromatography. We determined 3D structures for the full-length protein-protein complex at different stoichiometries and detect ligand-induced folding of MBP. We also obtained thermodynamic data for the two CaM-binding sites of MBP, indicating that CaM does not collapse upon binding to MBP, and show that CaM and MBP colocalize in myelin sheaths. In addition, we analyzed the post-translational modifications of rat brain MBP, identifying a novel MBP modification, glucosylation. Our results provide a detailed picture of the MBP-CaM interaction, including a 3D model of the complex between full-length proteins