Sandbox Reserved 1101: Difference between revisions
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======''Secondary interactions'' ====== | ======''Secondary interactions'' ====== | ||
These asymmetric contacts play a well-defined role in dimer formation in many species of spiders but in ''N. clavipes'' several other novel interactions occur. For example, in comparison with the ''Euprosthenops australis'' NTD, ''N. clavipes'' NTD engage more than 38,5% of novel interactions. These ones result from the distinct topology of the three helices (H2, H3 and H5) compared to other species. Indeed, the specific angles at which the H2, H3 and H5 helices cross their counterparts in the asymmetric interface allow the correct positioning of residues and the establishment of these interactions. <ref | These asymmetric contacts play a well-defined role in dimer formation in many species of spiders but in ''N. clavipes'' several other novel interactions occur. For example, in comparison with the ''Euprosthenops australis'' NTD, ''N. clavipes'' NTD engage more than 38,5% of novel interactions. These ones result from the distinct topology of the three helices (H2, H3 and H5) compared to other species. Indeed, the specific angles at which the H2, H3 and H5 helices cross their counterparts in the asymmetric interface allow the correct positioning of residues and the establishment of these interactions. <ref name="Atkison"/> | ||
::*''Van der Waals'' | ::*''Van der Waals'' | ||
Residues T47B, M55B and K54B are more buried at the dimer interface creating specific contacts. | Residues T47B, M55B and K54B are more buried at the dimer interface creating specific contacts. | ||
<scene name='82/829354/T47b-i48a-a51a-l69a/1'>T47B engage in Van Der Waals contacts with I48A, A51A and L69A residues</scene>. Also, <scene name='82/829354/M55b-d40a-t43a/2'>M55B is commited in Van Der Waals interactions with D40A and T43A</scene>. In subunits H2A and H2B, T47A and A51B engage in a Van Der Waals interaction of 4,1 Å, and that contribute to the plasticity of the dimer interface. <ref | <scene name='82/829354/T47b-i48a-a51a-l69a/1'>T47B engage in Van Der Waals contacts with I48A, A51A and L69A residues</scene>. Also, <scene name='82/829354/M55b-d40a-t43a/2'>M55B is commited in Van Der Waals interactions with D40A and T43A</scene>. In subunits H2A and H2B, T47A and A51B engage in a Van Der Waals interaction of 4,1 Å, and that contribute to the plasticity of the dimer interface.<ref name="Atkison"/> | ||
::*''Hydrogen bonds and electrostatic interactions'' | ::*''Hydrogen bonds and electrostatic interactions'' | ||
K54B engage in a unique hydrogen bond to <scene name='82/829354/K54b-t43a/1'>T43A</scene> and electrostatic interaction with <scene name='82/829354/K54b-d46a/1'>D46A</scene>. <ref | K54B engage in a unique hydrogen bond to <scene name='82/829354/K54b-t43a/1'>T43A</scene> and electrostatic interaction with <scene name='82/829354/K54b-d46a/1'>D46A</scene>. <ref name="Atkison"/> | ||
On the other side on the dimer interface, there are also other specific contacts but distinct due to the different topology. But residues T47A, K54A and M55A are less buried than their counterparts in subunit A in particularly K54A which doesn’t engage any interaction. <ref | On the other side on the dimer interface, there are also other specific contacts but distinct due to the different topology. But residues T47A, K54A and M55A are less buried than their counterparts in subunit A in particularly K54A which doesn’t engage any interaction. <ref name="Atkison"/> | ||
::*''Hydrophobic pockets'' | ::*''Hydrophobic pockets'' | ||
Then, in subunits H5A and H5B, <scene name='82/829354/M126/1'>M126A</scene> and <scene name='82/829354/F127/1'>F127A</scene> also buried at the dimer interface, insert into hydrophobic pockets formed by S122B, L123B and M71B, S75B, E119B and I120B respectively. <ref | Then, in subunits H5A and H5B, <scene name='82/829354/M126/1'>M126A</scene> and <scene name='82/829354/F127/1'>F127A</scene> also buried at the dimer interface, insert into hydrophobic pockets formed by S122B, L123B and M71B, S75B, E119B and I120B respectively. <ref name="Atkison"/> | ||
====pH-dependent mechanism==== | ====pH-dependent mechanism==== | ||
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In order to observe the pH-dependent NTD dimerization mechanism, a tryptophan fluorescence assay was used. The ''N. clavipes'' NTD contains a single tryptophan (Trp10) near the N-terminus. During the transition from the NTD monomer to the NTD dimer, a conformational change occurs for Trp10 that increases its solvent exposure. As a consequence, a quenching of its fluorescence emission is observed. The transition from the NTD monomer to the NTD dimer occurs at pH 6,1. At pH above 6,1, NTD is in the form of monomer and the formation of dimer occurs after pH 6,1. | In order to observe the pH-dependent NTD dimerization mechanism, a tryptophan fluorescence assay was used. The ''N. clavipes'' NTD contains a single tryptophan (Trp10) near the N-terminus. During the transition from the NTD monomer to the NTD dimer, a conformational change occurs for Trp10 that increases its solvent exposure. As a consequence, a quenching of its fluorescence emission is observed. The transition from the NTD monomer to the NTD dimer occurs at pH 6,1. At pH above 6,1, NTD is in the form of monomer and the formation of dimer occurs after pH 6,1. | ||
Mutations in residues Asp40, Lys65 involved in salt bridges result in decrease in dimer stability. This assay shows that short-range asymmetric salt bridges between Asp39, Asp40 and Lys65 are essential to the NTD dimerization. | Mutations in residues Asp40, Lys65 involved in salt bridges result in decrease in dimer stability. This assay shows that short-range asymmetric salt bridges between Asp39, Asp40 and Lys65 are essential to the NTD dimerization. | ||
Next, a mutation of residue Glu84 completely destabilize the dimer formation, that shows the importance of the handshake interaction and also the protonation of Glu84, which must be preceded by protonation of Glu79 and Glu119. Similarly, the protonation of Asp17 and Asp53 plays also a key role in the mechanism of NTD dimerization. These protonations are allowed by the lowering of the pH suffered by the NTD during its progression in the spinning duct.<ref | Next, a mutation of residue Glu84 completely destabilize the dimer formation, that shows the importance of the handshake interaction and also the protonation of Glu84, which must be preceded by protonation of Glu79 and Glu119. Similarly, the protonation of Asp17 and Asp53 plays also a key role in the mechanism of NTD dimerization. These protonations are allowed by the lowering of the pH suffered by the NTD during its progression in the spinning duct.<ref name="Atkison"/> | ||
