Sandbox Reserved 1706

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This Sandbox is Reserved from February 28 through September 1, 2022 for use in the course CH462 Biochemistry II taught by R. Jeremy Johnson at the Butler University, Indianapolis, USA. This reservation includes Sandbox Reserved 1700 through Sandbox Reserved 1729.

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Neurofibromin 1

1 Introduction
2 Function
3 Structure
4 Domains
5 Arginine 1276
6 SPRED 1
7 References placeholders
8 References

Introduction

Function

Neurofibromin is a GTPase-activating protein that binds to Ras, a GTPase, to increase the hydrolysis of GTP to GDP. This inactivates the cell signaling of Ras until another GTP can replace the GDP from the cytosol. Neurofibromin and Ras binding is possible in only the of Neurofibromin. The mechanism is shown in figure 1 and displays the slow hydrolysis of GTP bound to Ras and the fast hydrolysis of GTP when bound to Neurofibromin.

Structure

Neurofibromin is a protein dimer that exists in the and conformation. Each protomer contains a GRD, Sec14-PH, and a GAPex domain located on a HEAT N-C arm. Ras binds to the GRD site with Arg1276 being the critical residue for binding.

Closed conformation

In the, one protomer has its domains shifted by a 130 degree rotation of three separate linkers. That rotation places in an orientation that in the GRD site (Figure 3). Making sterically impossible in the . The can exist naturally without any form of stabilization but will also fall back to the .

Zinc Stabilized

The of Neurofibromin can be stabilized by a zinc ion to prevent the shift back to an . This binding is done between C1032, H1558, and H1576 within the N-HEAT domain, GRD-Sec14-PH linker L2 and is shown in figure #. When zinc stabilizes Neurofibromin, it will stay in the and continue to inhibit the binding of RAS.

Open conformation

In the one protomer is shifted due to a 90 rotation. This rotation allows for binding between RAS and the in the GRD site while in the . Allowing for the to occur without any steric hindrance as shown in the .

Conformational Change Linkers

The rotation of the domains between the and of Neurofibromin are conducted by three helical linkers named L1, L2, and L3. The and the undergo rotations to relocate the GRD and Sec14-PH sites. Linker 1 (L1) consists of a loop connected by two helices from L1173-M1215 and is the main contributor in rotation of the GRD domain. The rotation of L1 to the causes N-HEAT ARM alpha helix 48 and GRD helix 49 to extend out, aligning to form a hinge point at G1190. The GRD relocation is assisted by Sec14-PH relocation, which is initiated by Linker 3(L3) from Q1835 to G1852 where the proline rich section of the C-HEAT ARM changes conformation.L1 and L3 move closer to each other in the . Linker 2 (L2) consists of residues G1547-T1565 and begins at helix 63, the final helix of the GRD site, and connects into the short loop of alpha helix 65 of the Sec14-PH domain. The combination of these three linkers are responsible for the conformational shift of the and .

Domains

GRD

The GRD site is represented in cyan in all models. Neurofibromin’s main catalytic domain is the GRD active site. Linked structurally to both HEAT ARM’s, it consists of mainly loops and helices. Per protomer, there is one single GRD binding site. In the closed state Ras cannot bind due to a steric hindrance in which Ras clashes with the N-HEAT ARM upon attempting to bind to the GRD site. In its active state GRD can bind Ras. The critical residue within the GRD site is Arg1276.

GAPex-Subdomain

The GAPex subdomain is represented in magenta in all models. The GAPex subdomain of the GRD site lies between the Sec14-PH and GRD catalytic sites. This domain is non-catalytic and structurally consists of various loops and helices. Its main function is to bind SPRED-1, which is a recruiter protein that binds to this subdomain in the cytosol to recruit Neurofibromin to the plasma membrane.

Sec14-PH

The Sec14-PH domain is represented in yellow in all models. The Sec14-PH domain is linked and extends out from the HEAT ARM’s and consists of largely various helices and loops. Its function is a membrane associated domain and holds a largely hydrophobic cavity allowing for binding to the plasma membrane. In the it is blocked by the GRD and is inaccessible to the lipid membrane. In the it becomes exposed and can access the lipid membrane for interaction.

Arginine 1276

Arg1276 is the critical residue within the GRD site needed for proper binding to Ras. The interaction between is only possible in the open confirmation. has making binding of Ras impossible (Figure #).

SPRED 1

SPRED 1 is a protein that binds to the GAPex domain of Neurofibromin. Its function recruits the Neurofibromin protein when bound from the cytosol to the plasma membrane. SPRED 1 will bind to the GAPex domain of Neurofibromin in the in the cytosol to recruit Neurofibromin to the plasma membrane. Unlike Ras, SPRED-1 does show the ability to . When bound to the open conformation of Neurofibromin in the cytosol, it may present a different orientation that impacts the recruitment to the plasma membrane. Further research is needed to assess the impact of the function and the changes it may present.

References placeholders

^[1] ^[2] ^[3] ^[4] ^[5] ^[6]

References

↑ Bergoug M, Doudeau M, Godin F, Mosrin C, Vallee B, Benedetti H. Neurofibromin Structure, Functions and Regulation. Cells. 2020 Oct 27;9(11). pii: cells9112365. doi: 10.3390/cells9112365. PMID:33121128 doi:http://dx.doi.org/10.3390/cells9112365
↑ Bourne HR. G proteins. The arginine finger strikes again. Nature. 1997 Oct 16;389(6652):673-4. doi: 10.1038/39470. PMID:9338774 doi:http://dx.doi.org/10.1038/39470
↑ Kiuru M, Busam KJ. The NF1 gene in tumor syndromes and melanoma. Lab Invest. 2017 Feb;97(2):146-157. doi: 10.1038/labinvest.2016.142. Epub 2017, Jan 9. PMID:28067895 doi:http://dx.doi.org/10.1038/labinvest.2016.142
↑ Lupton CJ, Bayly-Jones C, D'Andrea L, Huang C, Schittenhelm RB, Venugopal H, Whisstock JC, Halls ML, Ellisdon AM. The cryo-EM structure of the human neurofibromin dimer reveals the molecular basis for neurofibromatosis type 1. Nat Struct Mol Biol. 2021 Dec;28(12):982-988. doi: 10.1038/s41594-021-00687-2., Epub 2021 Dec 9. PMID:34887559 doi:http://dx.doi.org/10.1038/s41594-021-00687-2
↑ Naschberger A, Baradaran R, Rupp B, Carroni M. The structure of neurofibromin isoform 2 reveals different functional states. Nature. 2021 Nov;599(7884):315-319. doi: 10.1038/s41586-021-04024-x. Epub 2021, Oct 27. PMID:34707296 doi:http://dx.doi.org/10.1038/s41586-021-04024-x
↑ Sabatini C, Milani D, Menni F, Tadini G, Esposito S. Treatment of neurofibromatosis type 1. Curr Treat Options Neurol. 2015 Jun;17(6):355. doi: 10.1007/s11940-015-0355-4. PMID:25917340 doi:http://dx.doi.org/10.1007/s11940-015-0355-4

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@@ Line 3: / Line 3: @@
 <StructureSection load='7PGR' size='350' frame='true'
-side='right' caption='NF1 Closed Conformation 7PGR' scene='90/904311/Open_conformation/2'>
+side='right' caption='Neurofibromin Closed Conformation 7PGR' scene='90/904311/Open_conformation/2'>
 ==Introduction==
-==History==
 ==Function==
-NF1 is a [https://en.wikipedia.org/wiki/GTPase-activating_protein GTPase-activating protein] that binds to [https://en.wikipedia.org/wiki/Ras_GTPase Ras], a [https://en.wikipedia.org/wiki/GTPase GTPase], to increase the hydrolysis of GTP to GDP. This inactivates the cell signaling of Ras until another GTP can replace the GDP from the cytosol. NF1 and Ras binding is possible in only the <scene name='90/904311/Open_conformation/2'>open conformation</scene> of NF1. The mechanism is shown in figure 1 and displays the slow hydrolysis of GTP bound to Ras and the fast hydrolysis of GTP when bound to NF1.
+Neurofibromin is a [https://en.wikipedia.org/wiki/GTPase-activating_protein GTPase-activating protein] that binds to [https://en.wikipedia.org/wiki/Ras_GTPase Ras], a [https://en.wikipedia.org/wiki/GTPase GTPase], to increase the hydrolysis of GTP to GDP. This inactivates the cell signaling of Ras until another GTP can replace the GDP from the cytosol. Neurofibromin and Ras binding is possible in only the <scene name='90/904311/Open_conformation/2'>open conformation</scene> of Neurofibromin. The mechanism is shown in figure 1 and displays the slow hydrolysis of GTP bound to Ras and the fast hydrolysis of GTP when bound to Neurofibromin.
 ==Structure==
-NF1 is a [https://en.wikipedia.org/wiki/Protein_dimer protein dimer] that exists in the<scene name='90/904311/Closed_conformation/3'>closed</scene> and <scene name='90/904311/Open_conformation/2'>open</scene> conformation. Each [https://en.wikipedia.org/wiki/Protomer protomer] contains a GRD, Sec14-PH, and a GAPex domain located on a HEAT N-C arm. Ras binds to the GRD site with Arg1276 being the critical residue for binding.
+Neurofibromin is a [https://en.wikipedia.org/wiki/Protein_dimer protein dimer] that exists in the<scene name='90/904311/Closed_conformation/3'>closed</scene> and <scene name='90/904311/Open_conformation/2'>open</scene> conformation. Each [https://en.wikipedia.org/wiki/Protomer protomer] contains a GRD, Sec14-PH, and a GAPex domain located on a HEAT N-C arm. Ras binds to the GRD site with Arg1276 being the critical residue for binding.
 ===Closed conformation===
 In the<scene name='90/904311/Closed_conformation/3'>closed conformation</scene>, one protomer has its domains shifted by a 130 degree rotation of three separate linkers. That rotation places <scene name='90/904312/Closed_arg/1'>Arg1276 in the closed conformation</scene> in an orientation that <scene name='90/904312/Closed_zoom/1'>sterical hinders the binding between Ras and Arg1276</scene> in the GRD site (Figure 3). Making <scene name='90/904312/Closed_with_ras/1'>Ras binding in closed conformation</scene> sterically impossible in the <scene name='90/904311/Closed_conformation/3'>closed conformation</scene>
 . The <scene name='90/904311/Closed_conformation/3'>closed conformation</scene> can exist naturally without any form of stabilization but will also fall back to the <scene name='90/904311/Open_conformation/2'>open conformation</scene>.
 ====Zinc Stabilized====
-The <scene name='90/904311/Closed_conformation/3'>closed conformation</scene> of NF1 can be stabilized by a zinc ion to prevent the shift back to an <scene name='90/904311/Open_conformation/2'>open conformation</scene>. This binding is done between C1032, H1558, and H1576 within the N-HEAT domain, GRD-Sec14-PH linker L2 and is shown in figure #. When zinc stabilizes NF1, it will stay in the <scene name='90/904311/Closed_conformation/3'>closed conformation</scene> and continue to inhibit the binding of RAS.
+The <scene name='90/904311/Closed_conformation/3'>closed conformation</scene> of Neurofibromin can be stabilized by a zinc ion to prevent the shift back to an <scene name='90/904311/Open_conformation/2'>open conformation</scene>. This binding is done between C1032, H1558, and H1576 within the N-HEAT domain, GRD-Sec14-PH linker L2 and is shown in figure #. When zinc stabilizes Neurofibromin, it will stay in the <scene name='90/904311/Closed_conformation/3'>closed conformation</scene> and continue to inhibit the binding of RAS.
 ===Open conformation===
 In the <scene name='90/904311/Open_conformation/2'>open conformation</scene> one protomer is shifted due to a 90 rotation. This rotation allows for binding between RAS and the <scene name='90/904312/Arg_1276_open/10'>Arg1276</scene>
 in the GRD site while in the <scene name='90/904311/Open_conformation/2'>open conformation</scene>. Allowing for the <scene name='90/904312/Arg_1276_open/11'>Arg1276 interaction with Ras in the open conformation</scene> to occur without any steric hindrance as shown in the <scene name='90/904311/Closed_conformation/3'>closed conformation</scene>.
 ===Conformational Change Linkers===
-The rotation of the domains between the <scene name='90/904311/Open_conformation/2'>open conformation</scene> and <scene name='90/904311/Closed_conformation/3'>closed conformation</scene> of NF1 are conducted by three helical linkers named L1, L2, and L3. The <scene name='90/904312/Linker_closed/1'>linkers in the closed conformation</scene>
+The rotation of the domains between the <scene name='90/904311/Open_conformation/2'>open conformation</scene> and <scene name='90/904311/Closed_conformation/3'>closed conformation</scene> of Neurofibromin are conducted by three helical linkers named L1, L2, and L3. The <scene name='90/904312/Linker_closed/1'>linkers in the closed conformation</scene>
 <scene name='90/904312/Linker_closed/2'>(zoomed in)'</scene> and the <scene name='90/904312/Linkers_open/2'>linkers in the open conformation</scene>
 <scene name='90/904312/Zoomed_lo/1'>(zoomed in)'</scene> undergo rotations to relocate the GRD and Sec14-PH sites. Linker 1 (L1) consists of a loop connected by two helices from L1173-M1215 and is the main contributor in rotation of the GRD domain. The rotation of L1 to the <scene name='90/904312/Linkers_open/2'>open conformation</scene>  causes N-HEAT ARM [https://en.wikipedia.org/wiki/Alpha_helix alpha helix] 48 and GRD helix 49 to extend out, aligning to form a hinge point at G1190. The GRD relocation is assisted by Sec14-PH relocation, which is initiated by Linker 3(L3) from Q1835 to G1852 where the proline rich section of the C-HEAT ARM changes conformation.L1 and L3 move closer to each other in the <scene name='90/904312/Linkers_open/2'>open conformation</scene>. Linker 2 (L2) consists of residues G1547-T1565 and begins at helix 63, the final helix of the GRD site, and connects into the short loop of [https://en.wikipedia.org/wiki/Alpha_helix alpha helix] 65 of the Sec14-PH domain. The combination of these three linkers are responsible for the conformational shift of the <scene name='90/904312/Linker_closed/1'> closed conformation</scene>
@@ Line 27: / Line 27: @@
 ==Domains==
 ===GRD===
-The GRD site is represented in cyan in all models. NF1’s main catalytic domain is the GRD active site. Linked structurally to both HEAT ARM’s, it consists of mainly loops and helices. Per protomer, there is one single GRD binding site. In the closed state Ras cannot bind due to a steric hindrance in which Ras clashes with the N-HEAT ARM upon attempting to bind to the GRD site. In its active state GRD can bind Ras. The critical residue within the GRD site is Arg1276.
+The GRD site is represented in cyan in all models. Neurofibromin’s main catalytic domain is the GRD active site. Linked structurally to both HEAT ARM’s, it consists of mainly loops and helices. Per protomer, there is one single GRD binding site. In the closed state Ras cannot bind due to a steric hindrance in which Ras clashes with the N-HEAT ARM upon attempting to bind to the GRD site. In its active state GRD can bind Ras. The critical residue within the GRD site is Arg1276.
 ===GAPex-Subdomain===
-The GAPex subdomain is represented in magenta in all models. The GAPex subdomain of the GRD site lies between the Sec14-PH and GRD catalytic sites. This domain is non-catalytic and structurally consists of various loops and helices. Its main function is to bind SPRED-1, which is a recruiter protein that binds to this subdomain in the cytosol to recruit NF1 to the plasma membrane.
+The GAPex subdomain is represented in magenta in all models. The GAPex subdomain of the GRD site lies between the Sec14-PH and GRD catalytic sites. This domain is non-catalytic and structurally consists of various loops and helices. Its main function is to bind SPRED-1, which is a recruiter protein that binds to this subdomain in the cytosol to recruit Neurofibromin to the plasma membrane.
 ===Sec14-PH===
 The Sec14-PH domain is represented in yellow in all models. The Sec14-PH domain is linked and extends out from the HEAT ARM’s and consists of largely various helices and loops. Its function is a membrane associated domain and holds a largely hydrophobic cavity allowing for binding to the plasma membrane. In the <scene name='90/904311/Closed_conformation/3'>closed conformation</scene> it is blocked by the GRD and is inaccessible to the lipid membrane. In the <scene name='90/904311/Open_conformation/2'>open conformation</scene> it becomes exposed and can access the lipid membrane for interaction.
@@ Line 37: / Line 37: @@
 <scene name='90/904312/Closed_zoom/1'>steric clashing with Ras</scene> making binding of Ras impossible (Figure #).
 ==SPRED 1==
-SPRED 1 is a protein that binds to the GAPex domain of NF1. Its function recruits the NF1 protein when bound from the cytosol to the plasma membrane. SPRED 1 will bind to the GAPex domain of NF1 in the <scene name='90/904312/Sped-1_closed/7'>closed conformation'</scene> in the cytosol to recruit NF1 to the plasma membrane. Unlike Ras, SPRED-1 does show the ability to <scene name='90/904312/Spred-1_open/1'>bind to the open conformation</scene>. When bound to the open conformation of NF1 in the cytosol, it may present a different orientation that impacts the recruitment to the plasma membrane. Further research is needed to assess the impact of the function and the changes it may present.
+SPRED 1 is a protein that binds to the GAPex domain of Neurofibromin. Its function recruits the Neurofibromin protein when bound from the cytosol to the plasma membrane. SPRED 1 will bind to the GAPex domain of Neurofibromin in the <scene name='90/904312/Sped-1_closed/7'>closed conformation'</scene> in the cytosol to recruit Neurofibromin to the plasma membrane. Unlike Ras, SPRED-1 does show the ability to <scene name='90/904312/Spred-1_open/1'>bind to the open conformation</scene>. When bound to the open conformation of Neurofibromin in the cytosol, it may present a different orientation that impacts the recruitment to the plasma membrane. Further research is needed to assess the impact of the function and the changes it may present.
 ==References placeholders==
 <ref name="Bergoug"> DOI:10.3390/cells9112365</ref>