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' | '''N-terminal domain of Major-ampullate Spidroin protein''' | ||
---- | ---- | ||
5IZ2 is the '''NTD | '''5IZ2''' is the N-terminal domain ('''NTD''') of a spider protein called Major ampullate [https://en.wikipedia.org/wiki/Spidroin_ Spidroin] 1A ('''MaSp1A'''), coming from the [https://en.wikipedia.org/wiki/Trichonephila_clavipes_ ''Nephila Clavipes''] species. This protein is a component of [https://en.wikipedia.org/wiki/Spider_silk#Types_of_silk_ '''dragline silk'''] produced in the major ampullate gland of spiders<ref name="Atkison"/>. The NTD domain of MaSp1A plays a major role in their combination during silk production <ref name="Atkison"/>. Indeed, thanks to the '''NTD''' | ||
The NTD domain of | [https://en.wikipedia.org/wiki/Dimer_(chemistry)_ '''dimerisation'''], two MaSps can be connected, leading to the formation of '''fibers''' with exceptional physical and biochemical qualities <ref name="José">José Roberto Aparecido dos Santos-Pinto, Helen Andrade Arcuri, Helga Priewalder, Heliana Clara Salles, Mario Sergio Palma and Gert Lubec, 2015. Structural Model for the Spider Silk Protein Spidroin‑1, Journal of Proteome research, 14, p.3859-3870.</ref>. It is of biotechnological interest to deeply understand the NTD dimerisation mechanism for the production of artificial spider silk, which can lead to '''innovative biomaterials'''. | ||
The study of the ''N. Clavipes'' NTD permits to compare its structure with other species thus to provide new insights into the mechanism of NTD dimerization. Moreover, silks produced from different spider breeds vary in physical properties such as toughness and elasticity. In this way, studying diverse species would allow to optimize '''artificial silk''' for different applications. | |||
==Generalities on fiber assembly of dragline silks== | |||
The process of the dragline fiber formation is the connection of '''soluble MaSp proteins''' into '''insoluble fibers'''. Indeed, MaSps are firstly secreted and stored in soluble form in the '''tail''' of the major ampullate gland which is located in the spider’s abdomen. On demand, they pass through the narrow '''duct''' where they experience mechanical and chemical forces that convert them into fibers. | |||
[[Image:Major ampullate gland of spiders.jpeg|600px|center|thumb| Schematic of Major ampullate gland of spiders.]] | |||
Actually, they deal with a '''pH dropping, an alteration of ion concentrations and''' [https://en.wikipedia.org/wiki/Redox_ '''oxidation'''] '''conditions''', which occur gradually along the duct. These changes promote the connection of MaSps extremities (i.e. '''homo-dimerisation of C- and N-terminal domains''') to form fibers. Finally, through flow rate and mechanical forces experienced in the duct, the fibers will '''agglomerate''' to create the dragline silk. | |||
== | [[Image:MaSps assembly.jpg|600px|center|thumb| Model of MaSps assembly into fibers according to the pH conditions.]] | ||
==Overall structure of Major-ampullate Spidroin protein== | |||
The dragline fiber is mainly composed of proteins termed Major ampullate Spidroin 1 and Major ampullate Spidroin 2 (MaSp1 and MaSp2). MaSp1 is found in both the core and periphery of the fiber, while MaSp2 is only assembled in the core <ref name="Cadle"/>. In ''Nephila clavipes'', there are two distinct MaSp1 genes ; MaSp1A and MaSp1B <ref>PMID:18828837</ref>. | |||
The MaSps are between 250 to 350 kDa <ref name="Cadle"/>. They are divided into three parts : [https://en.wikipedia.org/wiki/C-terminus_ C-terminal domain] '''(CTD)''', '''repeat domain (RD)''', and [https://en.wikipedia.org/wiki/N-terminus_ N-terminal domain] '''(NTD)'''. | |||
*'''Repeat domain (RD):''' | |||
The MaSp sequence corresponds to '''more than 90%''' of RD <ref>PMID:19221522</ref>. The RD is a long, flexible, highly repetitive central domain. It varies greatly between the types of silks, which makes it responsible for their '''different properties'''. | |||
MaSp1 contains poly-alanine (A)n motifs at the end of a repeat, as well as GA and GGX motifs where X is often A, Y, L, or Q <ref name="Cadle"/>. The poly-alanine motifs, usually present at the end of a repeat, form [https://en.wikipedia.org/wiki/Beta_sheet_ '''β-sheets'''] in the duct due to mechanical forces. The β-sheets will then line up in parallel, leading to the '''aggregation of the fibers'''. The GGX motifs form an amorphous matrix that connects the '''crystalline regions''' <ref>PMID:15556872</ref>. | |||
*'''C-terminal domain (CTD):''' | |||
The CTD is a non-repetitive sequence of about 150 amino acids <ref name="Cadle"/>. The sequence identity, secondary structure and overall physical properties of CTD is '''highly conserved''' across spider species <ref name="Cadle"/>. Its structure forms a bundle of five parallel α-helices. A single cysteine residue in the middle of its sequence is highly conserved and is responsible for the '''CTD homo-dimerisation'''. In other words, it allows the covalent connection between two CTDs through disulfide bond linkage. | |||
The CTD also plays a role in the change of '''MaSps solubility''' according to its localisation in the gland <ref name="Cadle"/>. Indeed, it presents a high number of charged and polar amino acids present in its sequence. In this way, when the pH is neutral in the ampullate, the hydrophobic residues are buried within the core and the hydrophilic residues are exposed. This permits to keep the MaSps soluble, preventing early fiber aggregation. On the contrary, when the CTDs are in the duct with lower pH, the acidic residues switch from a negative to a neutral charge. This leads to an increase of hydrophobic interactions that help with the formation of β-sheets and thus MaSps precipitation. | |||
*'''N-terminal domain (NTD):''' | |||
This domain is the most '''highly conserved''' domain. NTD '''dimerises''' in the duct upon '''conditions change''', which connects the MaSps to form '''fibers'''. | |||
== Monomer structure of the spidroin NTD domain == | |||
<Structure load='5iz2' size='350' frame='true' align='right' caption='NTD monomer' scene='Insert optional scene name here' /> | |||
One monomer of NTD (N-Terminal Domain) is composed of 5 parallel [https://en.wikipedia.org/wiki/Alpha_helix_ α-helix] (<scene name='82/829354/A/1'>H1 to H5</scene>)<ref name="Cadle"/>. | |||
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<ref name="Atkison"/>. | |||
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. 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<ref name="Atkison"/>. | |||
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 <scene name='82/829354/Chain_z/1'>chain Z</scene> composed of 3 amino acids (Ser, Tyr, Gly), but it role is not well established yet<ref name="Atkison"/>. | |||
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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 | 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 <ref name="Atkison"/>. These protonations are allowed by the lowering of the pH suffered by the NTD during its progression in the spinning duct <ref name="Cadle"/>. | ||
== Applications in Biotechnology == | == Applications in Biotechnology == | ||
The dragline silk represents the “'''toughest''' biopolymer on Earth” <ref>PMID:24119078</ref>. It also shows other beneficial properties including high '''tensile strength''', '''elasticity''' and '''biodegradability'''. That being, dragline fibers can have many uses in '''medical''' and '''industrial fields'''. | |||
Synthetic silk proteins are commonly produced by [https://en.wikipedia.org/wiki/Recombinant_DNA_ '''recombinant gene expression'''] and '''gene mimicry''' <ref>PMID:21999996</ref>. They can be spontaneously '''optimised''' by altering their form, size and composition. | |||
Indeed, DNA sections in silk protein sequence can be rearranged, added to or subtracted from to change the characteristics of the formed protein. | |||
For instance, silk proteins can be processed into many different forms such as fibers, sponges, films, capsules and gels <ref name="Cadle"/>. Their biodegradability can also be altered as required to increase or reduce their degradation time <ref name="Cadle"/>. | |||
In this way, the uses for spider silk can give rise to a wide range of '''novel materials'''. | |||
As far as the medical field is concerned, spider silk is naturally '''biocompatibility'''. This allows its use for applications like drug release materials, cell graft scaffolds, neuron regeneration and cartilage repair <ref name="Cadle"/>. Moreover, the spider silk can be recombinantly engineered to produce '''antimicrobial propertiers''', certainly useful in this sector <ref>PMID:21458065</ref>. | |||
== References == | == References == | ||
<references/> | <references/> | ||
