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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.


Comparison of the simulated forced unfolding of the hN2-NRR mutated constructs with wild type. Force-extension outputs from the molecular dynamics simulations are shown as follows: F1565S (red), L1566P (purple), L1573P (light blue), V1623D (orange), I1627N (green), A1647P (dark blue), wild type (black). Each graph is staggered by 300 pN with a horizontal line dissecting the plots at 200 pN. Raw data shown (semi-transparent) in addition to a running average (period: 50). Data generated from Gromacs 4.5.3. Peaks in the force-extension outputs are numbered [1–4] according to structural transitions identified from simulation video outputs (Movies S1–S7) and described in the main text and previously (18).
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f0010: Comparison of the simulated forced unfolding of the hN2-NRR mutated constructs with wild type. Force-extension outputs from the molecular dynamics simulations are shown as follows: F1565S (red), L1566P (purple), L1573P (light blue), V1623D (orange), I1627N (green), A1647P (dark blue), wild type (black). Each graph is staggered by 300 pN with a horizontal line dissecting the plots at 200 pN. Raw data shown (semi-transparent) in addition to a running average (period: 50). Data generated from Gromacs 4.5.3. Peaks in the force-extension outputs are numbered [1–4] according to structural transitions identified from simulation video outputs (Movies S1–S7) and described in the main text and previously (18).

Mentions: The first key region to be affected by three out of the six mutations investigated is the LNRA:B linker region/LNRB. Force-extension profiles from the MD simulations of mutated constructs compared to the wild-type construct are shown in Fig. 2. NRR-WT shows four distinctive peaks (numbered) corresponding to the unfolding of the LNRA:B linker [1] and LNRB [2], the removal of the β5 strand from the surrounding HD domain [3] and unfolding of the α3 helix and some LNRC unfolding [4], as previously described in detail [18]. A1647P, L1573P and V1623D demonstrate clear loss of force required for the unfolding of the LNRA:B linker and LNRB (peaks 1 and 2, Fig. 2). Crucially, removing the LNRA:B linker and LNRB from their contacts with the HD domain has previously been shown to be sufficient to alter the auto inhibited conformation of the NRR such that the S2 site, within the β5 strand, is accessible to metalloprotease allowing signal activation [18,21]. Thus, these observations indicate that these three mutations within the core region of stability cause destabilisation of regions distant to the mutation site, removing the mechanical resistance to unravelling of this key autoinhibitory region.


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

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

Comparison of the simulated forced unfolding of the hN2-NRR mutated constructs with wild type. Force-extension outputs from the molecular dynamics simulations are shown as follows: F1565S (red), L1566P (purple), L1573P (light blue), V1623D (orange), I1627N (green), A1647P (dark blue), wild type (black). Each graph is staggered by 300 pN with a horizontal line dissecting the plots at 200 pN. Raw data shown (semi-transparent) in addition to a running average (period: 50). Data generated from Gromacs 4.5.3. Peaks in the force-extension outputs are numbered [1–4] according to structural transitions identified from simulation video outputs (Movies S1–S7) and described in the main text and previously (18).
© Copyright Policy - CC BY
Related In: Results  -  Collection

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

f0010: Comparison of the simulated forced unfolding of the hN2-NRR mutated constructs with wild type. Force-extension outputs from the molecular dynamics simulations are shown as follows: F1565S (red), L1566P (purple), L1573P (light blue), V1623D (orange), I1627N (green), A1647P (dark blue), wild type (black). Each graph is staggered by 300 pN with a horizontal line dissecting the plots at 200 pN. Raw data shown (semi-transparent) in addition to a running average (period: 50). Data generated from Gromacs 4.5.3. Peaks in the force-extension outputs are numbered [1–4] according to structural transitions identified from simulation video outputs (Movies S1–S7) and described in the main text and previously (18).
Mentions: The first key region to be affected by three out of the six mutations investigated is the LNRA:B linker region/LNRB. Force-extension profiles from the MD simulations of mutated constructs compared to the wild-type construct are shown in Fig. 2. NRR-WT shows four distinctive peaks (numbered) corresponding to the unfolding of the LNRA:B linker [1] and LNRB [2], the removal of the β5 strand from the surrounding HD domain [3] and unfolding of the α3 helix and some LNRC unfolding [4], as previously described in detail [18]. A1647P, L1573P and V1623D demonstrate clear loss of force required for the unfolding of the LNRA:B linker and LNRB (peaks 1 and 2, Fig. 2). Crucially, removing the LNRA:B linker and LNRB from their contacts with the HD domain has previously been shown to be sufficient to alter the auto inhibited conformation of the NRR such that the S2 site, within the β5 strand, is accessible to metalloprotease allowing signal activation [18,21]. Thus, these observations indicate that these three mutations within the core region of stability cause destabilisation of regions distant to the mutation site, removing the mechanical resistance to unravelling of this key autoinhibitory region.

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.