Sandbox Reserved 1706

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.

To get started: Click the edit this page tab at the top. Save the page after each step, then edit it again. show the Scene authoring tools, create a molecular scene, and save it. Copy the green link into the page. Add a description of your scene. Use the buttons above the wikitext box for bold, italics, links, headlines, etc. More help: Help:Editing

Neurofibromin 1

<StructureSection load='7PGR' size='350' frame='true' side='right' caption='Placeholder' scene='90/904311/Open_conformation/1'>

Function

NF1 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. NF1 and Ras binding is possible in only the conformationopen conformation 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.

Structure

NF1 is a protein dimer that exists in the conformationclosed and conformationopen 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 conformationclosed conformation, one protomer has its domains shifted by a 130 degree rotation of three separate linkers. That rotation places Arg1276 in the closed conformation in an orientation that sterical hinders the binding between Ras and Arg1276 in the GRD site (Figure 3). Making Ras binding in closed conformation sterically impossible in the conformationclosed conformation . The conformationclosed conformation can exist naturally without any form of stabilization but will also fall back to the conformationopen conformation.

Zinc Stabilized

The conformationclosed conformation of NF1 can be stabilized by a zinc ion to prevent the shift back to an conformationopen conformation. 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 conformationclosed conformation and continue to inhibit the binding of RAS.

Open conformation

In the conformationopen conformation one protomer is shifted due to a 90 rotation. This rotation allows for binding between RAS and the Arg1276 in the GRD site while in the conformationopen conformation. Allowing for the Arg1276 interaction with Ras in the open conformation to occur without any steric hindrance as shown in the conformationclosed conformation.

Conformational Change Linkers

The rotation of the domains between the conformationopen conformation and conformationclosed conformation of NF1 are conducted by three helical linkers named L1, L2, and L3. The linkers in the closed conformation (zoomed in)' and the linkers in the open conformation (zoomed in)' 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 open conformation 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 open conformation. 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 closed conformation (zoomed in)' and open conformation (zoomed in)'.

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.

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.

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 conformationclosed conformation it is blocked by the GRD and is inaccessible to the lipid membrane. In the conformationopen conformation 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 Arg1276 and Ras in the open conformation (zoomed in) is only possible in the open confirmation. Arg1276 in closed conformation has steric clashing with Ras 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 closed conformation' in the cytosol to recruit NF1 to the plasma membrane. Unlike Ras, SPRED-1 does show the ability to bind to the open conformation. 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.