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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.


Observed differences in unfolding of LNRC for hN2-NRR A1647P and L1566P. (A) Comparison of distances between the LNRC and HD domain between wild type and variant NRR constructs. (B) Comparison of the wild type and (i) A1647P NRR structure at time 4000 ps and (ii) L1566P NRR structures at time 5000 ps.
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f0030: Observed differences in unfolding of LNRC for hN2-NRR A1647P and L1566P. (A) Comparison of distances between the LNRC and HD domain between wild type and variant NRR constructs. (B) Comparison of the wild type and (i) A1647P NRR structure at time 4000 ps and (ii) L1566P NRR structures at time 5000 ps.

Mentions: The final change observed during simulated unfolding following HD domain mutation is within the LNR:HD domain interaction. During simulated forced unfolding, the WT hN2-NRR shows a steady increase in distance between the LNRC and HD domain (using the starting centre-of-mass for each domain) from 2800 ps until 4400 ps, where it plateaus at 1.5 nm (Fig. 6A), however, changes observed within the structure are minimal (Fig. S3). The same data recorded for the six mutated protein constructs draws attention to two constructs in particular that have an apparent decreased stability for the LNRC:HD association (A1647P and L1566P) with increases observed in the LNRC:HD domain distances. Upon structure analysis of the LNRC:HD domain contacts within A1647P and L1566P large differences are observed relative to WT (Fig. 6B). The loss of stability of the LNRC:HD domain contact results in unfolding of LNRC, an event not observed within simulations of equivalent duration in all other constructs, including WT (Movies S1–S7). Interestingly, calculated distances between the HD and LNRC domain of L1566P (Fig. 6A) results in a final distance equivalent to that of the WT construct, despite the observable differences in the structures (Fig. 6B(ii)). This appears to be the result of a shift in part of the HD domain structure causing a shift in the centre-of-mass. As the LNRC of L1566P unravels and dissociates from the HD domain, part of the HD domain also raises up away from the HD domain structure, altering the position of the centre-of-mass in this domain and resulting in the reduced final distance measurement. This paradox illustrates the difficulty in using distances between two centres-of-mass within the protein structure, as regions respond to force differently. F1565S, I1627N, L1573P and V1623D exhibit a decrease in the distance between the two regions over the unfolding time course which corresponds to minimal structural changes similar to WT (Suppl. Movies S1, S3, S6 and S7). The differences in distance observed are likely due to changes in the centre of mass within the HD domain as other unfolding events occur (e.g. β5 strand removal from the main HD domain structure).


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

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

Observed differences in unfolding of LNRC for hN2-NRR A1647P and L1566P. (A) Comparison of distances between the LNRC and HD domain between wild type and variant NRR constructs. (B) Comparison of the wild type and (i) A1647P NRR structure at time 4000 ps and (ii) L1566P NRR structures at time 5000 ps.
© Copyright Policy - CC BY
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4537882&req=5

f0030: Observed differences in unfolding of LNRC for hN2-NRR A1647P and L1566P. (A) Comparison of distances between the LNRC and HD domain between wild type and variant NRR constructs. (B) Comparison of the wild type and (i) A1647P NRR structure at time 4000 ps and (ii) L1566P NRR structures at time 5000 ps.
Mentions: The final change observed during simulated unfolding following HD domain mutation is within the LNR:HD domain interaction. During simulated forced unfolding, the WT hN2-NRR shows a steady increase in distance between the LNRC and HD domain (using the starting centre-of-mass for each domain) from 2800 ps until 4400 ps, where it plateaus at 1.5 nm (Fig. 6A), however, changes observed within the structure are minimal (Fig. S3). The same data recorded for the six mutated protein constructs draws attention to two constructs in particular that have an apparent decreased stability for the LNRC:HD association (A1647P and L1566P) with increases observed in the LNRC:HD domain distances. Upon structure analysis of the LNRC:HD domain contacts within A1647P and L1566P large differences are observed relative to WT (Fig. 6B). The loss of stability of the LNRC:HD domain contact results in unfolding of LNRC, an event not observed within simulations of equivalent duration in all other constructs, including WT (Movies S1–S7). Interestingly, calculated distances between the HD and LNRC domain of L1566P (Fig. 6A) results in a final distance equivalent to that of the WT construct, despite the observable differences in the structures (Fig. 6B(ii)). This appears to be the result of a shift in part of the HD domain structure causing a shift in the centre-of-mass. As the LNRC of L1566P unravels and dissociates from the HD domain, part of the HD domain also raises up away from the HD domain structure, altering the position of the centre-of-mass in this domain and resulting in the reduced final distance measurement. This paradox illustrates the difficulty in using distances between two centres-of-mass within the protein structure, as regions respond to force differently. F1565S, I1627N, L1573P and V1623D exhibit a decrease in the distance between the two regions over the unfolding time course which corresponds to minimal structural changes similar to WT (Suppl. Movies S1, S3, S6 and S7). The differences in distance observed are likely due to changes in the centre of mass within the HD domain as other unfolding events occur (e.g. β5 strand removal from the main HD domain structure).

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.