Abstract:As an essential structural protein required for tight compaction of the central nervous system myelin sheath, myelin basic protein (MBP) is one of the candidate autoantigens of the human inflammatory demyelinating disease multiple sclerosis, which is characterized by the active degradation of the myelin sheath. In this work, recombinant murine analogues of the natural C1 and C8 charge components (rmC1 and rmC8), two isoforms of the classic 18.5-kDa MBP, were used as model proteins to get insights into the stru… Show more
“…Binding of A2A-ct to vesicles was expected, as the vesicles contained negatively charged lipids, but the ability of CaM to release the whole C-terminus from the vesicles was an interesting finding. A similar competition between CaM and a lipid membrane surface was previously observed for the myelin basic protein (79). Most likely, CaM replaces the negative lipid head groups while binding to the arginine-rich epitope of the A2A-ct.…”
Understanding how ligands bind to G-protein-coupled receptors and how binding changes receptor structure to affect signaling is critical for developing a complete picture of the signal transduction process. The adenosine A2A receptor (A2AR) is a particularly interesting example, as it has an exceptionally long intracellular carboxyl terminus, which is predicted to be mainly disordered. Experimental data on the structure of the A2AR C-terminus is lacking, because published structures of A2AR do not include the C-terminus. Calmodulin has been reported to bind to the A2AR C-terminus, with a possible binding site on helix 8, next to the membrane. The biological meaning of the interaction as well as its calcium dependence, thermodynamic parameters, and organization of the proteins in the complex are unclear. Here, we characterized the structure of the A2AR C-terminus and the A2AR C-terminus-calmodulin complex using different biophysical methods, including native gel and analytical gel filtration, isothermal titration calorimetry, NMR spectroscopy, and small-angle X-ray scattering. We found that the C-terminus is disordered and flexible, and it binds with high affinity (Kd = 98 nM) to calmodulin without major conformational changes in the domain. Calmodulin binds to helix 8 of the A2AR in a calcium-dependent manner that can displace binding of A2AR to lipid vesicles. We also predicted and classified putative calmodulin-binding sites in a larger group of G-protein-coupled receptors.
“…Binding of A2A-ct to vesicles was expected, as the vesicles contained negatively charged lipids, but the ability of CaM to release the whole C-terminus from the vesicles was an interesting finding. A similar competition between CaM and a lipid membrane surface was previously observed for the myelin basic protein (79). Most likely, CaM replaces the negative lipid head groups while binding to the arginine-rich epitope of the A2A-ct.…”
Understanding how ligands bind to G-protein-coupled receptors and how binding changes receptor structure to affect signaling is critical for developing a complete picture of the signal transduction process. The adenosine A2A receptor (A2AR) is a particularly interesting example, as it has an exceptionally long intracellular carboxyl terminus, which is predicted to be mainly disordered. Experimental data on the structure of the A2AR C-terminus is lacking, because published structures of A2AR do not include the C-terminus. Calmodulin has been reported to bind to the A2AR C-terminus, with a possible binding site on helix 8, next to the membrane. The biological meaning of the interaction as well as its calcium dependence, thermodynamic parameters, and organization of the proteins in the complex are unclear. Here, we characterized the structure of the A2AR C-terminus and the A2AR C-terminus-calmodulin complex using different biophysical methods, including native gel and analytical gel filtration, isothermal titration calorimetry, NMR spectroscopy, and small-angle X-ray scattering. We found that the C-terminus is disordered and flexible, and it binds with high affinity (Kd = 98 nM) to calmodulin without major conformational changes in the domain. Calmodulin binds to helix 8 of the A2AR in a calcium-dependent manner that can displace binding of A2AR to lipid vesicles. We also predicted and classified putative calmodulin-binding sites in a larger group of G-protein-coupled receptors.
“…The result is an indication of possible membrane-induced aggregation of mutant P2 forms. The behaviour of the P2 mutants resembles somewhat the kinetics shown by another major myelin protein, MBP, which folds onto the membrane surface, forming an amorphous phase of protein, and dissociates very slowly 23,24 .Figure 4Membrane surface binding. ( A ) DMPC:DMPG (top) and DOPC:DOPG (bottom) sensorgrams with injection of 4 µM protein.…”
Charcot-Marie-Tooth (CMT) disease is one of the most common inherited neuropathies. Recently, three CMT1-associated point mutations (I43N, T51P, and I52T) were discovered in the abundant peripheral myelin protein P2. These mutations trigger abnormal myelin structure, leading to reduced nerve conduction velocity, muscle weakness, and distal limb atrophy. P2 is a myelin-specific protein expressed by Schwann cells that binds to fatty acids and membranes, contributing to peripheral myelin lipid homeostasis. We studied the molecular basis of the P2 patient mutations. None of the CMT1-associated mutations alter the overall folding of P2 in the crystal state. P2 disease variants show increased aggregation tendency and remarkably reduced stability, T51P being most severe. In addition, P2 disease mutations affect protein dynamics. Both fatty acid binding by P2 and the kinetics of its membrane interactions are affected by the mutations. Experiments and simulations suggest opening of the β barrel in T51P, possibly representing a general mechanism in fatty acid-binding proteins. Our findings demonstrate that altered biophysical properties and functional dynamics of P2 may cause myelin defects in CMT1 patients. At the molecular level, a few malformed hydrogen bonds lead to structural instability and misregulation of conformational changes related to ligand exchange and membrane binding.
“…Much of the early research was carried out using MBP purified from tissue [20][21][22][23][24][25]27], and more recently, recombinant MBP has also been intensively studied [29,30,48,49]. According to the current view, MBP is intrinsically disordered in solution, but folds into short helical segments upon membrane binding [87].…”
Section: Discussionmentioning
confidence: 99%
“…Full-length 18.5-kDa murine MBP and the MBP72-107 peptide underwent a disordered-to-ordered transition, when transferred from an aqueous to a membranelike environment in a trifluoroethanol (TFE) titration experiment [28,29]. We observed folding of recombinant 18.5-kDa MBP in various membrane-mimetic conditions, as well as in the presence of lipid vesicles [30]. Similarly, the MBP85-99 peptide acquired helical conformation in the presence of detergent micelles and lipid vesicles [31].…”
Background: Despite intense research, the causes of various neurological diseases remain enigmatic to date. A role for viral or bacterial infection and associated molecular mimicry has frequently been suggested in the etiology of neurological diseases, including demyelinating autoimmune disorders, such as multiple sclerosis. Pathogen mimics of myelin-derived autoimmune peptides have been described in the literature and shown to induce myelin autoimmune responses in animal models. Methods: We carried out a structural study on myelin-derived peptides, and mimics thereof from various pathogens, in aqueous and membrane-like environments, using conventional and synchrotron radiation circular dichroism spectroscopy. A total of 13 peptides from the literature were studied, and 290 circular dichroism spectra were analysed. In addition, peptide structure predictions and vesicle aggregation assays were performed.
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