Sandbox20: Difference between revisions
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Dimerisation is enabled by the association of α4 helices into an antiparellel coiled coil, and the conformation is subsequently stabilised by formation of interhelical salt bridges outside this region. An aromatic network forms on the inner surface to make a hydrophobic core, which confers additional stability. Residues that form part of this network are F11, F18, F45, F50 and Y22. The crystal structure of RTP in its unbound state was determined in 1995 (1BM9). A C110S mutant was then generated to prevent the aggregation of RTP through cysteine oxidation. With a very similar structure (1J0R) and almost no change in dimerisation and DNA-binding capacities, the mutant was the protein of choice for later studies. | Dimerisation is enabled by the association of α4 helices into an antiparellel coiled coil, and the conformation is subsequently stabilised by formation of interhelical salt bridges outside this region. An aromatic network forms on the inner surface to make a hydrophobic core, which confers additional stability. Residues that form part of this network are F11, F18, F45, F50 and Y22. The crystal structure of RTP in its unbound state was determined in 1995 (1BM9). A C110S mutant was then generated to prevent the aggregation of RTP through cysteine oxidation. With a very similar structure (1J0R) and almost no change in dimerisation and DNA-binding capacities, the mutant was the protein of choice for later studies. | ||
Herman's: | |||
The RTP monomer somewhat resembles winged-helix proteins in structure. These proteins are a subfamily of the helix-turn-helix proteins and have ‘wings’ that protrude from the loop between the final two β sheets of their αβααββ structure. The difference is that RTP has a β1 loop in place of the β1 sheet and has a fourth elongate α-helix, α4, at the C-terminus. | |||
For dimerisation, the α4 helices associate to form an antiparellel coiled coil. The conformation is then stabilised by interhelical salt bridges outside the coiled coil region. A aromatic network forms on the inner surface to make a hydrophobic core, which confers additional stability. Residues that form part of this network are F11, F18, F45, F50 and Y22. | |||
The crystal structure of RTP in its unbound state was determined in 1995 (1BM9). A C110S mutant was then generated to prevent the aggregation of RTP through cysteine oxidation. With a very similar structure (1J0R) and almost no change in dimerisation and DNA-binding capacities, the mutant was the protein of choice for later studies. | |||
http://www.ncbi.nlm.nih.gov/pubmed/10679470 | |||
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13-15 residues contribute to the attachment of an RTP molecule to the ''Ter'' DNA site. Most of this is contributed by the basic residues of the <scene name='Sandbox20/2efw/30'>α3 helix</scene>, which lies in the major groove of DNA. | 13-15 residues contribute to the attachment of an RTP molecule to the ''Ter'' DNA site. Most of this is contributed by the basic residues of the <scene name='Sandbox20/2efw/30'>α3 helix</scene>, which lies in the major groove of DNA. | ||
Herman's: | |||
When an RTP dimer binds to a TerA or TerB site, the basic residues of the a3 helix become positioned at the major groove and the beta ribbon rests at the minor groove. Non-specific ionic interactions between the N-terminus and the DNA backbone stabilise the complex. | |||
It is known, with some certainty, that the RTP dimer adopts an asymmetric arrangement upon binding of the TerB site. In the C110S mutant complexed with the native TerB sequence (2EFW), the two subunits interact differently with the DNA bases to produce wing-up and wing-down conformations. These can be distinguished by the angle the a2 helix makes with the a3 helix. It is likely that asymmetry is also introduced when the RTP dimer binds to the TerA site but the crystal structure of this complex has not been solved? | |||
Although the TerA site inherently has a lower binding affinity for RTP, as evident from the larger dissociation constant associated with the RTP-TerA complex, positive cooperativity from the binding of RTP to TerB facilitates the binding of RTP to TerA. It is proposed that RTP bends the DNA at the TerB site in a manner that favours RTP binding at TerA. The RTP dimer at TerB may also present a surface for stabilising interactions with the dimer at TerA. This is believed to happen through the b1 loop and b3 strand (need to standardise the names) | |||
The asymmetry of the dimer is shown by the names 'wing up' and 'wing down'. It is measured by the angle between the a2 and a3 heices, as shown <scene name='Sandbox20/2efw/14'>by clicking here</scene>. | The asymmetry of the dimer is shown by the names 'wing up' and 'wing down'. It is measured by the angle between the a2 and a3 heices, as shown <scene name='Sandbox20/2efw/14'>by clicking here</scene>. | ||
=== Replication Termination Activity=== | === Replication Termination Activity=== | ||
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The consequence of these different conformations is most prominent in the position of the B1 sheet. This is evident in the Tyr33 residue, <scene name='Sandbox20/2efw/20'>shown by clicking here</scene> | The consequence of these different conformations is most prominent in the position of the B1 sheet. This is evident in the Tyr33 residue, <scene name='Sandbox20/2efw/20'>shown by clicking here</scene> | ||
Space, which contact DNA only in the wing-down conformation. | Space, which contact DNA only in the wing-down conformation. | ||
Herman's: | |||
The asymmetric arrangement of the RTP dimer means that certain regions of the protein are accessible from one face only. In particular, Y33 in the wing-down monomer always comes in contact with the replication fork that is arrested. Of interest is that Y33 is found in a region that carries some similarity to DnaB, and this region is believed to interact with DnaB, likely in combination with the adjacent hydrophobic patch, in order to suppress its helicase activity.* At the moment, it is unclear why, but both the TerA and TerB sites must be occupied for full replication termination activity. (Reference??) | |||
*1bm9 paper | |||
==Tus== | ==Tus== | ||
