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Mutational analysis of the Notch2 negative regulatory region identifies key structural elements for mechanical stability.

Stephenson NL, Avis JM - FEBS Open Bio (2015)

Bottom Line: Here, mutations are made within the heterodimerization (HD) domain of the NRR that are known to cause constitutive activation of Notch1 whilst having no effect on the chemical stability of Notch2.Comparison of the mechanical stability and simulated forced unfolding of recombinant Notch2 NRR proteins demonstrates a reduced stability following mutation and identifies two critical structural elements of the NRR in its response to force - the linker region between Lin12-Notch repeats LNRA and LNRB and the α3 helix within the HD domain - both of which mask the S2 cleavage site prior to Notch activation.In two mutated proteins, the LNRC:HD domain interaction is also reduced in stability.

View Article: PubMed Central - PubMed

Affiliation: Faculty of Life Sciences, Manchester Institute of Biotechnology, University of Manchester, 131 Princess Street, Manchester M1 7DN, United Kingdom.

ABSTRACT
The Notch signalling pathway is fundamental to cell differentiation in developing and self-renewing tissues. Notch is activated upon ligand-induced conformational change of the Notch negative regulatory region (NRR), unmasking a key proteolytic site (S2) and facilitating downstream events. The favoured model requires endocytosis of a tightly bound ligand to transmit force to the NRR region, sufficient to cause a structural change that exposes the S2 site. We have previously shown, using atomic force microscopy and molecular dynamics simulations, that application of force to the N-terminus of the Notch2 NRR facilitates metalloprotease cleavage at an early stage in the unfolding process. Here, mutations are made within the heterodimerization (HD) domain of the NRR that are known to cause constitutive activation of Notch1 whilst having no effect on the chemical stability of Notch2. Comparison of the mechanical stability and simulated forced unfolding of recombinant Notch2 NRR proteins demonstrates a reduced stability following mutation and identifies two critical structural elements of the NRR in its response to force - the linker region between Lin12-Notch repeats LNRA and LNRB and the α3 helix within the HD domain - both of which mask the S2 cleavage site prior to Notch activation. In two mutated proteins, the LNRC:HD domain interaction is also reduced in stability. The observed changes to mechanical stability following these HD domain mutations highlight key regions of the Notch2 NRR that are important for mechanical, but not chemical, stability. This research could also help determine the fundamental differences in the NRRs of Notch1 and Notch2.

No MeSH data available.


Changes in the angle and position of the α3-helix within variant constructs compared to the wild type during the unfolding simulation. (A) Comparison of the maximum angle across the α3-helix over the course of the pull simulation for each variant and the wild type construct (black). Each comparison is staggered by 50°. Data were generated using the Bendix plugin for VMD [46] from a simulation trajectory gained from Gromacs 4.5.3. (B) Comparison of (i) the wild type and L1566P structures at time 2000 ps; (ii) the wild type and L1573P structures at time 2000 ps. Data generated from Gromacs 4.5.3, images created in PyMol 1.3.
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f0025: Changes in the angle and position of the α3-helix within variant constructs compared to the wild type during the unfolding simulation. (A) Comparison of the maximum angle across the α3-helix over the course of the pull simulation for each variant and the wild type construct (black). Each comparison is staggered by 50°. Data were generated using the Bendix plugin for VMD [46] from a simulation trajectory gained from Gromacs 4.5.3. (B) Comparison of (i) the wild type and L1566P structures at time 2000 ps; (ii) the wild type and L1573P structures at time 2000 ps. Data generated from Gromacs 4.5.3, images created in PyMol 1.3.

Mentions: In addition to the observed changes in the LNRA:B linker unfolding we identify significant differences in the structure of the α3-helix of the HD domain during simulated forced unfolding of mutated hN2-NRR constructs compared to the WT (Fig. 5). To analyse the degree of bending occurring across the α3-helix during the simulations the maximum angle across the helix (where a straight helix would have an angle of 0°) was measured for all constructs (Fig. 5A). The WT construct shows minimal bending of the α3-helix during unfolding, with a relatively low angle and minimal changes throughout the simulation (Fig. 5A). In contrast, F1565S, I1627N and V1623D, show large increases in the angle across the α3-helix beginning relatively early in the unfolding process (around 800 ps, corresponding to the time at which the LNRA:B linker has been removed from its contacts with the S2 cleavage site). From structural analysis of these constructs the α3-helix can be observed either partially (F1565S and V1623D) or fully (I1627N) unfolding whilst the α3-helix within the WT NRR remains intact (Fig. S2B–D). This unfolding event occurs before unfolding of the HD domain and appears to be the result of the LNRB domain being pulled from the structure (Movie S1, S3, S4 and S7). A1647P shows a smaller general increase in the angle across the α3-helix throughout the simulation, compared to the other mutations discussed. Furthermore, the α3-helix of A1647P is observed unfolding in a similar manner to the WT construct (Fig. S2A and Movie S1 and S2). The difference in angle observed for A1647P is likely the result of the proline residue introduced within the α3-helix in this construct. Interestingly, in addition to the introduced kink in the α3-helix, this mutation also causes the helix to rotate upwards towards LNRB, which could cause wider destabilising effects. Finally, L1566P and L1573P show little change to the angle across the α3-helix during the unfolding simulation; however, structural analysis shows a positional shift of this α3-helix, in both constructs (Fig. 5B). The observed shift brings the α3-helix away from the β5-strand, which houses the S2 site, allowing greater exposure of this cleavage site. Overall, the α3-helix would thus appear to either be structurally destabilised or positionally changed during mechanical unfolding following mutation at all six HD domain sites, potentially resulting in greater access to the S2 cleavage site.


Mutational analysis of the Notch2 negative regulatory region identifies key structural elements for mechanical stability.

Stephenson NL, Avis JM - FEBS Open Bio (2015)

Changes in the angle and position of the α3-helix within variant constructs compared to the wild type during the unfolding simulation. (A) Comparison of the maximum angle across the α3-helix over the course of the pull simulation for each variant and the wild type construct (black). Each comparison is staggered by 50°. Data were generated using the Bendix plugin for VMD [46] from a simulation trajectory gained from Gromacs 4.5.3. (B) Comparison of (i) the wild type and L1566P structures at time 2000 ps; (ii) the wild type and L1573P structures at time 2000 ps. Data generated from Gromacs 4.5.3, images created in PyMol 1.3.
© Copyright Policy - CC BY
Related In: Results  -  Collection

License
Show All Figures
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f0025: Changes in the angle and position of the α3-helix within variant constructs compared to the wild type during the unfolding simulation. (A) Comparison of the maximum angle across the α3-helix over the course of the pull simulation for each variant and the wild type construct (black). Each comparison is staggered by 50°. Data were generated using the Bendix plugin for VMD [46] from a simulation trajectory gained from Gromacs 4.5.3. (B) Comparison of (i) the wild type and L1566P structures at time 2000 ps; (ii) the wild type and L1573P structures at time 2000 ps. Data generated from Gromacs 4.5.3, images created in PyMol 1.3.
Mentions: In addition to the observed changes in the LNRA:B linker unfolding we identify significant differences in the structure of the α3-helix of the HD domain during simulated forced unfolding of mutated hN2-NRR constructs compared to the WT (Fig. 5). To analyse the degree of bending occurring across the α3-helix during the simulations the maximum angle across the helix (where a straight helix would have an angle of 0°) was measured for all constructs (Fig. 5A). The WT construct shows minimal bending of the α3-helix during unfolding, with a relatively low angle and minimal changes throughout the simulation (Fig. 5A). In contrast, F1565S, I1627N and V1623D, show large increases in the angle across the α3-helix beginning relatively early in the unfolding process (around 800 ps, corresponding to the time at which the LNRA:B linker has been removed from its contacts with the S2 cleavage site). From structural analysis of these constructs the α3-helix can be observed either partially (F1565S and V1623D) or fully (I1627N) unfolding whilst the α3-helix within the WT NRR remains intact (Fig. S2B–D). This unfolding event occurs before unfolding of the HD domain and appears to be the result of the LNRB domain being pulled from the structure (Movie S1, S3, S4 and S7). A1647P shows a smaller general increase in the angle across the α3-helix throughout the simulation, compared to the other mutations discussed. Furthermore, the α3-helix of A1647P is observed unfolding in a similar manner to the WT construct (Fig. S2A and Movie S1 and S2). The difference in angle observed for A1647P is likely the result of the proline residue introduced within the α3-helix in this construct. Interestingly, in addition to the introduced kink in the α3-helix, this mutation also causes the helix to rotate upwards towards LNRB, which could cause wider destabilising effects. Finally, L1566P and L1573P show little change to the angle across the α3-helix during the unfolding simulation; however, structural analysis shows a positional shift of this α3-helix, in both constructs (Fig. 5B). The observed shift brings the α3-helix away from the β5-strand, which houses the S2 site, allowing greater exposure of this cleavage site. Overall, the α3-helix would thus appear to either be structurally destabilised or positionally changed during mechanical unfolding following mutation at all six HD domain sites, potentially resulting in greater access to the S2 cleavage site.

Bottom Line: Here, mutations are made within the heterodimerization (HD) domain of the NRR that are known to cause constitutive activation of Notch1 whilst having no effect on the chemical stability of Notch2.Comparison of the mechanical stability and simulated forced unfolding of recombinant Notch2 NRR proteins demonstrates a reduced stability following mutation and identifies two critical structural elements of the NRR in its response to force - the linker region between Lin12-Notch repeats LNRA and LNRB and the α3 helix within the HD domain - both of which mask the S2 cleavage site prior to Notch activation.In two mutated proteins, the LNRC:HD domain interaction is also reduced in stability.

View Article: PubMed Central - PubMed

Affiliation: Faculty of Life Sciences, Manchester Institute of Biotechnology, University of Manchester, 131 Princess Street, Manchester M1 7DN, United Kingdom.

ABSTRACT
The Notch signalling pathway is fundamental to cell differentiation in developing and self-renewing tissues. Notch is activated upon ligand-induced conformational change of the Notch negative regulatory region (NRR), unmasking a key proteolytic site (S2) and facilitating downstream events. The favoured model requires endocytosis of a tightly bound ligand to transmit force to the NRR region, sufficient to cause a structural change that exposes the S2 site. We have previously shown, using atomic force microscopy and molecular dynamics simulations, that application of force to the N-terminus of the Notch2 NRR facilitates metalloprotease cleavage at an early stage in the unfolding process. Here, mutations are made within the heterodimerization (HD) domain of the NRR that are known to cause constitutive activation of Notch1 whilst having no effect on the chemical stability of Notch2. Comparison of the mechanical stability and simulated forced unfolding of recombinant Notch2 NRR proteins demonstrates a reduced stability following mutation and identifies two critical structural elements of the NRR in its response to force - the linker region between Lin12-Notch repeats LNRA and LNRB and the α3 helix within the HD domain - both of which mask the S2 cleavage site prior to Notch activation. In two mutated proteins, the LNRC:HD domain interaction is also reduced in stability. The observed changes to mechanical stability following these HD domain mutations highlight key regions of the Notch2 NRR that are important for mechanical, but not chemical, stability. This research could also help determine the fundamental differences in the NRRs of Notch1 and Notch2.

No MeSH data available.