Chloride Intracellular Channel Protein 2: Difference between revisions
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== '''CLIC2''' == | == '''CLIC2''' == | ||
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== Introduction == | |||
CLIC proteins are a new class of soluble and membrane-bound proteins that have been grouped together on the basis of their sequence similarity. The proteins were named CLIC because the first members of this family to be characterized formed intracellular chloride channels (Heiss & Poustka, 1997). They display broad tissue and cellular distribution. They have been implicated in kidney function, cell division, and bone resorption. (Brett A. Cromer and al, 2007 1) They differ from the other classes of chloride ion channels in primary structure and in the transmembrane regions of the tertiary structure. Since the first member of CLIC, p64 (CLIC5), was discovered in bovine kidney, several members of the CLIC family have been found in other tissues from many species, including NCC27 (CLIC1), CLIC2, CLIC3, mtCLIC (CLIC4), and parchorin (CLIC6). (X. Meng and al, 2009) With the exception of p64 and parchorin, these proteins contain a conserved region of approximately 240 residues. (Brett A. Cromer and al, 2007 2) | CLIC proteins are a new class of soluble and membrane-bound proteins that have been grouped together on the basis of their sequence similarity. The proteins were named CLIC because the first members of this family to be characterized formed intracellular chloride channels (Heiss & Poustka, 1997). They display broad tissue and cellular distribution. They have been implicated in kidney function, cell division, and bone resorption. (Brett A. Cromer and al, 2007 1) They differ from the other classes of chloride ion channels in primary structure and in the transmembrane regions of the tertiary structure. Since the first member of CLIC, p64 (CLIC5), was discovered in bovine kidney, several members of the CLIC family have been found in other tissues from many species, including NCC27 (CLIC1), CLIC2, CLIC3, mtCLIC (CLIC4), and parchorin (CLIC6). (X. Meng and al, 2009) With the exception of p64 and parchorin, these proteins contain a conserved region of approximately 240 residues. (Brett A. Cromer and al, 2007 2) | ||
CLIC proteins can localize to distinct cellular membranes, including the nuclear membrane, lysosomal membranes, mitochondria, Golgi membranes, cell–cell junctions, and the plasma membrane. (Brett A. Cromer and al, 2007 1) | CLIC proteins can localize to distinct cellular membranes, including the nuclear membrane, lysosomal membranes, mitochondria, Golgi membranes, cell–cell junctions, and the plasma membrane. (Brett A. Cromer and al, 2007 1) | ||
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== Structure == | == Structure == | ||
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<applet load='2r5g' size='500' frame='true' align='right' caption='Insert caption here' /> | |||
Contrary to each members of the CLICs family, CLIC 2 is a monomer, no matter if it is oxydated or reduiced. It is composed of 247 amino acids, has a weight of 28.4kDa and an isoelectric point at 5.44(crystal structure). The CLIC2 molecule is box shaped (60×60×35 Å) and consists of a four strand core and two helices on one side. Comparing sequence similarities, the core is supposed to adopt the canonical fold of the glutathione S-transferase (GST) superfamily. This has been confirmed by the crystal structure determination of human CLIC1 at 1.4 Å resolution. Then, by analyzing CLIC genes sequences, this protein appears to have two potential transmembrane domains that would correspond to helices α1 and α6 in the GST-like structure of the soluble form. Thanks to immunological, electrophysical and proteolysis studies, we can say that membrane form of CLIC proteins cross the lipid bilayer an odd number of times. | Contrary to each members of the CLICs family, CLIC 2 is a monomer, no matter if it is oxydated or reduiced. It is composed of 247 amino acids, has a weight of 28.4kDa and an isoelectric point at 5.44(crystal structure). The CLIC2 molecule is box shaped (60×60×35 Å) and consists of a four strand core and two helices on one side. Comparing sequence similarities, the core is supposed to adopt the canonical fold of the glutathione S-transferase (GST) superfamily. This has been confirmed by the crystal structure determination of human CLIC1 at 1.4 Å resolution. Then, by analyzing CLIC genes sequences, this protein appears to have two potential transmembrane domains that would correspond to helices α1 and α6 in the GST-like structure of the soluble form. Thanks to immunological, electrophysical and proteolysis studies, we can say that membrane form of CLIC proteins cross the lipid bilayer an odd number of times. | ||
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<scene name='Sandbox123/Two_diprolines/1'>two diproline</scene>, Pro70-Pro71 and Pro96-Pro97 in this loop wich is called <scene name='Sandbox123/Joint_loop/2'>joint loop</scene>. Those diprolines lead to direction changing in the peptide chain. The largest deviations in the N-terminal domain occurs between the residues 55 and 69 that implies helix α2 and its surrounding sequences and also a loop (residues 80-84) that bind the β-strand 4 to helix α3. Crystallographic studies gave two forms of CLIC2, on each one we found out that this protein contain a right handed hook conformation. In fact, the long loop between helices 5 and 6 protrudes on the surface. | <scene name='Sandbox123/Two_diprolines/1'>two diproline</scene>, Pro70-Pro71 and Pro96-Pro97 in this loop wich is called <scene name='Sandbox123/Joint_loop/2'>joint loop</scene>. Those diprolines lead to direction changing in the peptide chain. The largest deviations in the N-terminal domain occurs between the residues 55 and 69 that implies helix α2 and its surrounding sequences and also a loop (residues 80-84) that bind the β-strand 4 to helix α3. Crystallographic studies gave two forms of CLIC2, on each one we found out that this protein contain a right handed hook conformation. In fact, the long loop between helices 5 and 6 protrudes on the surface. | ||
Finally, something that we have to highlight is the fact that this protein presents an intramolecular disulfide bridge. It is a well resolved disulfide bond between<scene name='Sandbox123/Disulfide_bond/ | Finally, something that we have to highlight is the fact that this protein presents an intramolecular disulfide bridge. It is a well resolved disulfide bond between<scene name='Sandbox123/Disulfide_bond/2'>Cys30 and Cys33</scene> on the N-terminal of helix 1 thanks to a CXXC motif. The Cys 30 is exposed to the cavity between the N- and the C-terminal domains, which means that this cystein is reachable by other molecules. On the opposite, the Cys 33 is located one turn after the Cys 30, thus is buried inside the N-terminal domain. Let us notice that the partial positive charge present on the N-terminal end of helix increases the nucleophility of the thiol group of the Cys 30, which is a characteristic of protein belonging to the glutaredoxin family. On another hand, we would like to point out the fact that this intramolecular disulfide bridge is the reason why CLIC2 is the only CLIC protein that exists only in the monomer form. In CLIC1, there is no intramolecular disulfide bridge in its monomer state but only in its dimer state where the bound is established between Cys 24 of the first CLIC1 and Cys 59 of the second one. Actually, this bridge is responsible for the dimerisation. In analogy, the corresponding residues in CLIC2 are Cys 30 and Ala 65; consequently, there is no possibility to establish a disulfide bridge. An experiment has been performed to check if CLIC2 cannot dimerise: mutant has been made in which Cys had been settled at the appropriate positions instead of Ala 65. Even in reducing conditions, no disulfide bridge can be created. That means that up to now, CLIC2 remains as a non dimerisable protein. | ||
Actually, the exhibited long loop is flexible and easily affected by crystal packing. When we analyze CLIC2’s electrostatic potential surface, we can observe that contrary to the cavity between the N- and C-terminal domains that is positively charged, the foot loop is negatively charged due to its six acid residues. Indeed, this loop has a crucial functional role; it can be considered as an anchor between CLIC2 and other protein. This foot loop can be inserted into a groove of a neighboring molecule thanks to electrostatic charges. The negative charges provided by the acid resides allow the foot loop to insert into positively charged cavity of another symmetry-related molecule. What’s more, there is an Asp in the 161 position that can form a salt bridge with a Lys from a neighbor molecule, thus the interaction between protein-protein will be increased. Then, the CXXC motif, close to the cavity can provide extensive hydrogen bonds which will stabilize again the interaction. We also can notice that hydrophobic interactions can be involved in the interaction with the highly conserved Ile 158 surrounded by Val 242, Tyr 239 and the alkyl chain of Lys 125 in the groove. This foot loop is an evidence of a structure-function link: the equivalent region in the structurally GST family corresponds to the active site. We can then suggest that CLIC2 will interact with other protein such as the ryanodine receptor for example through the same interaction way. | Actually, the exhibited long loop is flexible and easily affected by crystal packing. When we analyze CLIC2’s electrostatic potential surface, we can observe that contrary to the cavity between the N- and C-terminal domains that is positively charged, the foot loop is negatively charged due to its six acid residues. Indeed, this loop has a crucial functional role; it can be considered as an anchor between CLIC2 and other protein. This foot loop can be inserted into a groove of a neighboring molecule thanks to electrostatic charges. The negative charges provided by the acid resides allow the foot loop to insert into positively charged cavity of another symmetry-related molecule. What’s more, there is an Asp in the 161 position that can form a salt bridge with a Lys from a neighbor molecule, thus the interaction between protein-protein will be increased. Then, the CXXC motif, close to the cavity can provide extensive hydrogen bonds which will stabilize again the interaction. We also can notice that hydrophobic interactions can be involved in the interaction with the highly conserved Ile 158 surrounded by Val 242, Tyr 239 and the alkyl chain of Lys 125 in the groove. This foot loop is an evidence of a structure-function link: the equivalent region in the structurally GST family corresponds to the active site. We can then suggest that CLIC2 will interact with other protein such as the ryanodine receptor for example through the same interaction way. | ||