One monomer of NTD (N-Terminal Domain) is composed of 5 α-helix (H1 to H5).
In each subunit, the orientation of helices 2, 3 and 5 is different from the orientation of helices 1 and 4. Indeed, helices 1 and 4 form the rigid body of the NTD domain, while helices 2, 3 and 5 are involved in intermolecular contacts, so they play an important role in the dimerization process.
Moreover, at the opposite extremities of each subunits of the monomer there are clusters of acidic residus (Asp36, Asp39, Asp40, Glu79, Asp91) in one part, and clusters of basic residus (Lys54, Arg57, Lys60, Lys64, Lys65) in the other part. So, this create a dipole moment. In addition to this, the subunits A and B are organized antiparallel, which allows an access to charges poles.
The charged residues (the acidic and basic ones) are responsible for creating a dipole moment, which therefore implies a non-uniform charge arrangement within the subunits. This is important for the dimerization process, that is why they are highly conserved residues.
Compared with spidroin of other species of spider, the 2 subunits (A and B) of the dimerized NTD of the spidroin produced by N. Clavipes are slightly different, due to a different helices arrangement. So they do not completely overlap. This allows the creation of new intermolecular contact networks. There is also a chain Z composed of 3 amino acids (Ser, Tyr, Gly), but it role is not well established yet.
Dimerization of the spidroin by the NTD domain
Conformational change of the five-helix bundle
The dimerization of the spidroin by the NTD domain begins by a rearrangement of the five-helix bundle during the monomer to dimer transition. An acidification along the spinning duct results in a conformational change of the NTD. So, for the NTD dimerization, a lowering of pH from 7 to 6 is important. Then, a subunit selects a partner with a complementary binding interface. When the NTD forms a dimer, its positive and negative poles are opposed, creating an environment conducive to salt bridges formation. Moreover, dimerization is really triggered and stabilized by protonation of some residues. Studies have also shown that a lowering more important of the pH stabilize even more the dimer. The plasticity of the dimer interface could also be a factor of the conformational selection during transition from monomer to dimer or during the transition from loosely to stably dimer.
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.
In one side, engage in the intramolecular handshake interaction. The asymmetric nature and the difference of topology of the subunits allow the formation of salt bridges. engage in a short-range intermolecular salt bridge of 2,6Å. In the other side, engage in a short-range intermolecular salt bridge of 3,1Å. 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.
Another intramolecular handshake interaction occurs also between . 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.
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.
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. Also, . 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.
- Hydrogen bonds and electrostatic interactions
K54B engage in a unique hydrogen bond to and electrostatic interaction with .
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.
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Then, in subunits H5A and H5B, M126A and F127A also buried at the dimer interface, insert into hydrophobic pockets formed by S122B, L123B and M71B, S75B, E119B and I120B respectively.
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.
Applications in Biotechnology
