Sandbox Reserved 1101: Difference between revisions

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*Interactions

**

Different types of interactions occur between specific residues during the NTD dimerization. Asp40, Lys65, Asp39 and Glu84 residues have been identified as being particularly important.

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Another intramolecular handshake interaction occurs also between Asp17 and Asp53 in subunit A. This interaction doesn’t exist in subunit B because of the orientation of subunit A with respect to subunit B, Asp17 and Asp53 are too far away in order to engage this interaction.

**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.

Residues T47B, M55B and K54B are more buried at the dimer interface creating specific contacts.

***Van der Waals

T47B engage in Van Der Waals contacts with I48A, A51A and L69A residues. Also, M55B is commited in Van Der Waals interactions with D40A and T43A.

***Hydrogen bonds and electrostatic interactions

K54B engage in a unique hydrogen bond to T43A and electrostatic interaction with D46A.

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.

*pH-dependent mechanism

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.

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.

~~<scene name='82/829354/Asp40-glu84/3'>intramolecular handshake interaction between Asp40 and Glu84</scene>~~

== Structural highlights ==

Sandbox Reserved 1101: Difference between revisions

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@@ Line 35: / Line 35: @@
 *Interactions
+**
 Different types of interactions occur between specific residues during the NTD dimerization. Asp40, Lys65, Asp39 and Glu84 residues have been identified as being particularly important.
@@ Line 40: / Line 42: @@
 Another intramolecular handshake interaction occurs also between Asp17 and Asp53 in subunit A. This interaction doesn’t exist in subunit B because of the orientation of subunit A with respect to subunit B, Asp17 and Asp53 are too far away in order to engage this interaction.
+**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.
+Residues T47B, M55B and K54B are more buried at the dimer interface creating specific contacts.
+***Van der Waals
+T47B engage in Van Der Waals contacts with I48A, A51A and L69A residues. Also, M55B is commited in Van Der Waals interactions with D40A and T43A.
+***Hydrogen bonds and electrostatic interactions
+K54B engage in a unique hydrogen bond to T43A and electrostatic interaction with D46A.
+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.
+*pH-dependent mechanism
+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.
+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.
 <Structure load='5IZ2' size='350' frame='true' align='right' caption='Insert caption here' scene='Insert optional scene name here' />
-<scene name='82/829354/Asp40-glu84/3'>intramolecular handshake interaction between Asp40 and Glu84</scene>
 == Structural highlights ==