Limits...
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.


Related in: MedlinePlus

Structure and sequence of the hN2 NRR. (A) (i) hN2-NRR comprising three LNR repeats (cartoon and surface representation) and the HD domain (cartoon representation). S1 cleavage site, which separates the HD domain into the HD-N (turquoise) and HD-C (blue) is shown as a black arrowhead. S2 cleavage site which activates the signalling pathway, is highlighted by a red arrowhead. Black box highlights region enlarged to show mutation sites in (ii) and rotated 90° left in (iii). Wild type residues shown as sticks in domain colours, variant residue shown as sticks in red. (B) Alignment of hN1 and hN2 HD domain sequences, highlighting mutations (red) in residues that are identical (yellow). Conserved residues shown as grey and not conserved residues are shown as white. Diagram created using PyMol 1.3, sequence alignment performed using ClustalW. (C) Table detailing the hN2 mutations analysed within this study, alongside the equivalent mutation found in hN1 T-ALL samples.
© Copyright Policy - CC BY
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4537882&req=5

f0005: Structure and sequence of the hN2 NRR. (A) (i) hN2-NRR comprising three LNR repeats (cartoon and surface representation) and the HD domain (cartoon representation). S1 cleavage site, which separates the HD domain into the HD-N (turquoise) and HD-C (blue) is shown as a black arrowhead. S2 cleavage site which activates the signalling pathway, is highlighted by a red arrowhead. Black box highlights region enlarged to show mutation sites in (ii) and rotated 90° left in (iii). Wild type residues shown as sticks in domain colours, variant residue shown as sticks in red. (B) Alignment of hN1 and hN2 HD domain sequences, highlighting mutations (red) in residues that are identical (yellow). Conserved residues shown as grey and not conserved residues are shown as white. Diagram created using PyMol 1.3, sequence alignment performed using ClustalW. (C) Table detailing the hN2 mutations analysed within this study, alongside the equivalent mutation found in hN1 T-ALL samples.

Mentions: Given the increasing evidence for a role for mechanical force in the Notch signal activation mechanism, we investigate the effect of six mutations, related to those shown previously to destabilise the HD domain within hN1, [6,39] on the mechanical stability of recombinant hN2-NRR (hN2 residues 1425–1672). Five of the six mutations are within the core region of the HD domain (Fig. 1A; made up of the β4 strand and α1 helix) which has previously been shown to be a critical region for HD domain stability [27] and at the interface between the HD-N and HD-C. Since our model is the hN2 protein, mutations were chosen based on the sequence homology between hN1 and hN2, ensuring mutation at conserved positions (Fig. 1B). The mutated recombinant hN2-NRR proteins generated are hence: F1565S, L1566P, L1573P, V1623D, I1627N and A1647P (equivalent to the F1593S, L1594P, L1601P, V1677D, I1681N and A1702P in hN1; Fig. 1C). Three of these mutations (L1566P, V1623D and I1627N) have recently been biochemically analysed, revealing fundamental differences between the N1 and N2 NRR [40]. Interestingly, contrary to observations in hN1, none of these three mutations increased HD domain dissociation following EDTA treatment [40], therefore altering ligand independent signalling within hN1 but not in hN2. Here, these mutations will be analysed within hN2 to determine the effect they have on forced unfolding, and therefore potentially ligand induced activation. To do this, we subjected these recombinant hN2-NRR protein constructs with destabilising HD domain mutations to forced unfolding, both in an atomic force microscope and in molecular dynamics (MD) simulations, in order to examine their mechanical properties relative to the wild-type (WT) NRR. The results identify key contributing regions within the HD domain for governing mechanical stability, which could suggest these mutations still affect ligand dependent signalling of hN2, without affecting ligand independent signalling.


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

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

Structure and sequence of the hN2 NRR. (A) (i) hN2-NRR comprising three LNR repeats (cartoon and surface representation) and the HD domain (cartoon representation). S1 cleavage site, which separates the HD domain into the HD-N (turquoise) and HD-C (blue) is shown as a black arrowhead. S2 cleavage site which activates the signalling pathway, is highlighted by a red arrowhead. Black box highlights region enlarged to show mutation sites in (ii) and rotated 90° left in (iii). Wild type residues shown as sticks in domain colours, variant residue shown as sticks in red. (B) Alignment of hN1 and hN2 HD domain sequences, highlighting mutations (red) in residues that are identical (yellow). Conserved residues shown as grey and not conserved residues are shown as white. Diagram created using PyMol 1.3, sequence alignment performed using ClustalW. (C) Table detailing the hN2 mutations analysed within this study, alongside the equivalent mutation found in hN1 T-ALL samples.
© Copyright Policy - CC BY
Related In: Results  -  Collection

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

f0005: Structure and sequence of the hN2 NRR. (A) (i) hN2-NRR comprising three LNR repeats (cartoon and surface representation) and the HD domain (cartoon representation). S1 cleavage site, which separates the HD domain into the HD-N (turquoise) and HD-C (blue) is shown as a black arrowhead. S2 cleavage site which activates the signalling pathway, is highlighted by a red arrowhead. Black box highlights region enlarged to show mutation sites in (ii) and rotated 90° left in (iii). Wild type residues shown as sticks in domain colours, variant residue shown as sticks in red. (B) Alignment of hN1 and hN2 HD domain sequences, highlighting mutations (red) in residues that are identical (yellow). Conserved residues shown as grey and not conserved residues are shown as white. Diagram created using PyMol 1.3, sequence alignment performed using ClustalW. (C) Table detailing the hN2 mutations analysed within this study, alongside the equivalent mutation found in hN1 T-ALL samples.
Mentions: Given the increasing evidence for a role for mechanical force in the Notch signal activation mechanism, we investigate the effect of six mutations, related to those shown previously to destabilise the HD domain within hN1, [6,39] on the mechanical stability of recombinant hN2-NRR (hN2 residues 1425–1672). Five of the six mutations are within the core region of the HD domain (Fig. 1A; made up of the β4 strand and α1 helix) which has previously been shown to be a critical region for HD domain stability [27] and at the interface between the HD-N and HD-C. Since our model is the hN2 protein, mutations were chosen based on the sequence homology between hN1 and hN2, ensuring mutation at conserved positions (Fig. 1B). The mutated recombinant hN2-NRR proteins generated are hence: F1565S, L1566P, L1573P, V1623D, I1627N and A1647P (equivalent to the F1593S, L1594P, L1601P, V1677D, I1681N and A1702P in hN1; Fig. 1C). Three of these mutations (L1566P, V1623D and I1627N) have recently been biochemically analysed, revealing fundamental differences between the N1 and N2 NRR [40]. Interestingly, contrary to observations in hN1, none of these three mutations increased HD domain dissociation following EDTA treatment [40], therefore altering ligand independent signalling within hN1 but not in hN2. Here, these mutations will be analysed within hN2 to determine the effect they have on forced unfolding, and therefore potentially ligand induced activation. To do this, we subjected these recombinant hN2-NRR protein constructs with destabilising HD domain mutations to forced unfolding, both in an atomic force microscope and in molecular dynamics (MD) simulations, in order to examine their mechanical properties relative to the wild-type (WT) NRR. The results identify key contributing regions within the HD domain for governing mechanical stability, which could suggest these mutations still affect ligand dependent signalling of hN2, without affecting ligand independent signalling.

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.


Related in: MedlinePlus