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.

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<StructureSection load='7PGR' size='350' frame='true' side='right' caption='NF1 Closed Confirmation 7PGR' scene=’’>

Function

NF1 is a GTPase-activation protein that binds to RAS 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 open 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 a closed and open conformation. Each protomer contains a GRD, Sec14-PH, and a Gpex 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 closed conformation, one protomer has its domains shifted due to a 130 degree rotation. That rotation places Arg1276 in 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 closed conformation . The closed conformation can exist naturally without any form of stabilization but will also fall back to the open conformation.

Zinc Stabilized

The closed conformation of NF1 can be stabilized by a zinc ion to prevent the shift back to an open 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 2. When zinc stabilizes, NF1 will stay in the closed conformation and continue to inhibit the binding of RAS.

Open conformation

In the open 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 open conformation. Allowing for the Arg1276 interaction with Ras in the open conformation to occur without any steric hindrance as shown in the closed conformation.

Conformational Change Linkers

The rotation of the domains between the open conformation and closed 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)'.

Arginine 1276

Arg1276 in open conformation Arg1276 interaction with Ras in open conformation