Sandbox Reserved 1101: Difference between revisions

Line 55:

In one side, <scene name='82/829354/Asp40b_glu84b/1'>Asp40 and Glu84 of subunit B</scene> engage in the intramolecular handshake interaction. The asymmetric nature and the difference of topology of the subunits allow the formation of salt bridges. <scene name='82/829354/Lys_65-asp39_interaction/2'>Lys65 of subunit A and Asp39 of subunit B</scene> engage in a short-range intermolecular salt bridge of 2,6 Å. In the other side, <scene name='82/829354/Lys_65b-asp40a_interaction/1'>Asp40 of subunit A and Lys65 of subunit B</scene> engage in a short-range intermolecular salt bridge of 3,1 Å. Asp39 is not involved in this part of the dimer. The structure of ''N. clavipes'' dimer interface differs from those of other species due to the asymmetric nature of the interface and the involvement of Asp39. It has been reported that Asp39 is essential for the NTD dimerization in other species of spiders and seems to be also important in ''N.clavipes''. These interactions make subunits alignment better. Acidic residues are conserved around residues Asp39 and Asp40 and this allows the variability in the interactions that take place to Lys65. This variability provides a mechanism for plasticity in the dimer interface allowing the transition from loosely to stably associated dimer <ref>Atkison JH, Parnham S, Marcotte WR, Jr., and Olsen SK, 2016. Crystal Structure of the Nephila clavipes Major Ampullate Spidroin 1A N-terminal Domain Reveals Plasticity at the Dimer Interface, The Journal of Biological Chemistry, vol.291n no.36, p.19006-19017.</ref>

Another intramolecular handshake interaction occurs also between <scene name='82/829354/Asp17a-asp53a_interaction/1'>Asp17 and Asp53 in subunit A</scene>. 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.

Another intramolecular handshake interaction occurs also between <scene name='82/829354/Asp17a-asp53a_interaction/1'>Asp17 and Asp53 in subunit A</scene>. 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. <ref>Atkison JH, Parnham S, Marcotte WR, Jr., and Olsen SK, 2016. Crystal Structure of the Nephila clavipes Major Ampullate Spidroin 1A N-terminal Domain Reveals Plasticity at the Dimer Interface, The Journal of Biological Chemistry, vol.291n no.36, p.19006-19017.</ref>

======''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.

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>Atkison JH, Parnham S, Marcotte WR, Jr., and Olsen SK, 2016. Crystal Structure of the Nephila clavipes Major Ampullate Spidroin 1A N-terminal Domain Reveals Plasticity at the Dimer Interface, The Journal of Biological Chemistry, vol.291n no.36, p.19006-19017.</ref>

::*''Van der Waals''

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.

<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>Atkison JH, Parnham S, Marcotte WR, Jr., and Olsen SK, 2016. Crystal Structure of the Nephila clavipes Major Ampullate Spidroin 1A N-terminal Domain Reveals Plasticity at the Dimer Interface, The Journal of Biological Chemistry, vol.291n no.36, p.19006-19017.</ref>

::*''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>.

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>Atkison JH, Parnham S, Marcotte WR, Jr., and Olsen SK, 2016. Crystal Structure of the Nephila clavipes Major Ampullate Spidroin 1A N-terminal Domain Reveals Plasticity at the Dimer Interface, The Journal of Biological Chemistry, vol.291n no.36, p.19006-19017.</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.

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>Atkison JH, Parnham S, Marcotte WR, Jr., and Olsen SK, 2016. Crystal Structure of the Nephila clavipes Major Ampullate Spidroin 1A N-terminal Domain Reveals Plasticity at the Dimer Interface, The Journal of Biological Chemistry, vol.291n no.36, p.19006-19017.</ref>

::*''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.

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>Atkison JH, Parnham S, Marcotte WR, Jr., and Olsen SK, 2016. Crystal Structure of the Nephila clavipes Major Ampullate Spidroin 1A N-terminal Domain Reveals Plasticity at the Dimer Interface, The Journal of Biological Chemistry, vol.291n no.36, p.19006-19017.</ref>

====pH-dependent mechanism====

Line 77:

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.

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>Atkison JH, Parnham S, Marcotte WR, Jr., and Olsen SK, 2016. Crystal Structure of the Nephila clavipes Major Ampullate Spidroin 1A N-terminal Domain Reveals Plasticity at the Dimer Interface, The Journal of Biological Chemistry, vol.291n no.36, p.19006-19017.</ref>

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@@ Line 55: / Line 55: @@
 In one side, <scene name='82/829354/Asp40b_glu84b/1'>Asp40 and Glu84 of subunit B</scene> engage in the intramolecular handshake interaction. The asymmetric nature and the difference of topology of the subunits allow the formation of salt bridges. <scene name='82/829354/Lys_65-asp39_interaction/2'>Lys65 of subunit A and Asp39 of subunit B</scene> engage in a short-range intermolecular salt bridge of 2,6 Å. In the other side, <scene name='82/829354/Lys_65b-asp40a_interaction/1'>Asp40 of subunit A and Lys65 of subunit B</scene> engage in a short-range intermolecular salt bridge of 3,1 Å. Asp39 is not involved in this part of the dimer. The structure of ''N. clavipes'' dimer interface differs from those of other species due to the asymmetric nature of the interface and the involvement of Asp39. It has been reported that Asp39 is essential for the NTD dimerization in other species of spiders and seems to be also important in ''N.clavipes''. These interactions make subunits alignment better. Acidic residues are conserved around residues Asp39 and Asp40 and this allows the variability in the interactions that take place to Lys65. This variability provides a mechanism for plasticity in the dimer interface allowing the transition from loosely to stably associated dimer <ref>Atkison JH, Parnham S, Marcotte WR, Jr., and Olsen SK, 2016. Crystal Structure of the Nephila clavipes Major Ampullate Spidroin 1A N-terminal Domain Reveals Plasticity at the Dimer Interface, The Journal of Biological Chemistry, vol.291n no.36, p.19006-19017.</ref>
-Another intramolecular handshake interaction occurs also between <scene name='82/829354/Asp17a-asp53a_interaction/1'>Asp17 and Asp53 in subunit A</scene>. 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.
+Another intramolecular handshake interaction occurs also between <scene name='82/829354/Asp17a-asp53a_interaction/1'>Asp17 and Asp53 in subunit A</scene>. 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. <ref>Atkison JH, Parnham S, Marcotte WR, Jr., and Olsen SK, 2016. Crystal Structure of the Nephila clavipes Major Ampullate Spidroin 1A N-terminal Domain Reveals Plasticity at the Dimer Interface, The Journal of Biological Chemistry, vol.291n no.36, p.19006-19017.</ref>
 ======''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.
+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>Atkison JH, Parnham S, Marcotte WR, Jr., and Olsen SK, 2016. Crystal Structure of the Nephila clavipes Major Ampullate Spidroin 1A N-terminal Domain Reveals Plasticity at the Dimer Interface, The Journal of Biological Chemistry, vol.291n no.36, p.19006-19017.</ref>
 ::*''Van der Waals''
 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.
+<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>Atkison JH, Parnham S, Marcotte WR, Jr., and Olsen SK, 2016. Crystal Structure of the Nephila clavipes Major Ampullate Spidroin 1A N-terminal Domain Reveals Plasticity at the Dimer Interface, The Journal of Biological Chemistry, vol.291n no.36, p.19006-19017.</ref>
 ::*''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>.
+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>Atkison JH, Parnham S, Marcotte WR, Jr., and Olsen SK, 2016. Crystal Structure of the Nephila clavipes Major Ampullate Spidroin 1A N-terminal Domain Reveals Plasticity at the Dimer Interface, The Journal of Biological Chemistry, vol.291n no.36, p.19006-19017.</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.
+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>Atkison JH, Parnham S, Marcotte WR, Jr., and Olsen SK, 2016. Crystal Structure of the Nephila clavipes Major Ampullate Spidroin 1A N-terminal Domain Reveals Plasticity at the Dimer Interface, The Journal of Biological Chemistry, vol.291n no.36, p.19006-19017.</ref>
 ::*''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.
+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>Atkison JH, Parnham S, Marcotte WR, Jr., and Olsen SK, 2016. Crystal Structure of the Nephila clavipes Major Ampullate Spidroin 1A N-terminal Domain Reveals Plasticity at the Dimer Interface, The Journal of Biological Chemistry, vol.291n no.36, p.19006-19017.</ref>
 ====pH-dependent mechanism====
@@ Line 77: / Line 77: @@
 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.
+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>Atkison JH, Parnham S, Marcotte WR, Jr., and Olsen SK, 2016. Crystal Structure of the Nephila clavipes Major Ampullate Spidroin 1A N-terminal Domain Reveals Plasticity at the Dimer Interface, The Journal of Biological Chemistry, vol.291n no.36, p.19006-19017.</ref>