Sandbox Reserved 1790: Difference between revisions

<scene name='95/952717/Pp1c/1'>PP1C</scene> is a catalytic protein. After forming a ternary complex, the hydrophobic active site on the protein interacts with Raf to act as a phosphatase and dephosphorylate Ser 259. PP1C's active site is adjacent to a hydrophobic patch. It's theorized that the hydrophobic patch binds to the C-terminal of N-terminal phosphoserine of RAF, the target for dephosphorylation. PP1C can act as a phosphatase in the absence of SHOC2 but PP1C lasks intrinsic substrate selectively. So SMP complex formation is necessary for PP1C specificity to RAF <ref name="Hauseman" />.  

<scene name='95/952717/Shoc2_and_pp1c/1'>PP1C binds to SHOC2</scene> on its leucine rich region(LRR). Between LRR2 and LRR5 and between LRR7 and LRR11. Mutations between SHOC2 and PP1C to the LRR were shown to completely inhibit the binding of PP1C. Five main <scene name='95/952716/Shoc2_and_pp1c/3'>hydrogen and ionic bonds</scene> are made between PP1C and SHOC2 respectively: E56-R182, E167-R203, E54-K180, R187-H178, R188-E155 <ref name="Kwon">PMID:35831509</ref>. Reflecting this ionic character, the binding regions are contained within extensive acidic and basic patches on <scene name='95/952718/Acid_base_pp1c/1'>PP1C</scene> and <scene name='95/952718/Acid_base_shoc2/2'>SHOC2</scene>. The negative acidic patches of PP1C interact with the positive basic patches of SHOC2 and vice versa to form a <scene name='95/952718/Acid_base_shoc2pp1c/1'>binary complex</scene>. These interactions do not result in significant conformational changes to PP1C in comparison to other protein interactions that can be made with PP1C <ref name="Kwon">PMID:35831509</ref>.

<scene name='95/952717/Mras/2'>MRAS</scene> is a monomeric GTPase. MRAS is membrane-bound due to post-translational lipidation which allows the protein to interact with the inner membrane leaflet. <ref name="Seabra">PMID:9607139</ref> MRAS localizes the SMP complex near RAF and other components of downstream signaling. The region of MRAS not directly bound to the membrane binds SHOC2 and PP1C to orient the complex such that PP1C’s active site faces the serine that will get dephosphorylated on RAF. MRAS also controls SMP complex formation in connection with extracellular signaling based on its dualistic switching between its inactive and active state. In its inactive state, MRAS is bound to GDP. When signaled by growth factors, the GDP is exchanged for GTP when a ligand binds to the RTK <ref name="Hauseman" />. The now <scene name='95/952718/Zoom_in_gtp/1'>GTP bound MRAS</scene> undergoes a conformational change of the <scene name='95/952716/Ras-switch-zoomed/1'>switch I and switch II regions</scene>. These regions are the major binding sites with SHOC2. This conformational change activates MRAS allowing it to bind with the SHOC2-PP1C complex. In its inactive GDP-bound state, MRAS is sterically occluded from binding SHOC2. For example, R83 of GDP-bound MRAS directly clashes with SHOC2 as shown in figure 2. In comparison to other RAS proteins such as H/K/NRAS, MRAS has a greater affinity for the SHOC2-PP1C complex<ref name="Kubicek">Kubicek M, Pacher M, Abraham D, Podar K, Eulitz M, Baccarini M. Dephosphorylation of Ser-259 regulates Raf-1 membrane association. J Biol Chem. 2002 Mar 8;277(10):7913-9. [http://10.1074/jbc.M108733200 doi: 10.1074/jbc.M108733200.]</ref>. This indicates that the specific structure of MRAS is necessary for SMP function. While MRAS engages the SHOC2-PP1C complex to bring the complex to the membrane, an additional membrane-bound RAS binds RAF nearby. This binding is also stimulated by ligand binding to the RTK. This indicates that for full RAF activation and continuous signaling of Raf, two separate active RAS proteins are needed. Having two MRASs also help with the co-localization of PP1C to the NTpS region on RAF. To inactivate Raf signaling, MRAS uses its intrinsic GTPase to remove the activating gamma-phosphate on GTP. In the GDP-bound state, switch I and II move to the position shown in green in Figure 2. This inactivates SHOC2 binding due to steric clashing which causes the SMP structure to dissociate.

MRAS is initially bound to GDP causing it to be in its inactive state. Inactive GDP-MRAS cannot bind to the SHOC2-PP1C complex due to steric clashing of the switch I and II regions of MRAS and its binding zone on SHOC2. Once GDP is exchanged for GTP when signaled by growth factors, MRAS is activated and conformational changes occur within the switch I and switch II regions to allow <scene name='95/952716/Scho2-mras-interactions/2'>MRAS to interact with SHOC2</scene>. These <scene name='95/952716/Scho2-mras-interactions/1'>interactions</scene> between SHOC2 and the switch I and II regions of MRAS include hydrogen bonds, ionic interactions, and π stacking. There is a hydrogen bond at R288-Q71 and ionic interaction at R177-E47. π staking occurs at R104-R83. These interactions occur between SHOC2 and MRAS respectively <ref name="Lavoie">Lavoie H, Therrien M. Structural keys unlock RAS-MAPK cellular signaling pathway. Nature. 2022 Sep;609(7926):248-249. [http://dx.doi.org/10.1038/d41586-022-02189-7 doi: 10.1038/d41586-022-02189-7. PMID: 35970881.]</ref>.

[[Image:Switches.png|500 px|thumb|'''Figure 2:'''Steric clashing of Switch I and II of GDP bound MRAS, in green, with the surface of SHOC2, in magenta. GTP-bound MRAS, in white, shows no steric clashing with SHOC2s surface.</div></font>]]

The interactions between <scene name='95/952718/Mras_and_pp1c/1'>PP1C and MRAS</scene> are respectively mediated by four main <scene name='95/952716/Mras_and_pp1c/3'>polar interactions</scene>: ionic interactions are between D48-R188 and H53-D197, hydrogen bonds are between K36-Q198 and Q35-M190. As the complex forms, the active site for the dephosphorylation of RAF's S259 is oriented such that it remains accessible for RAF <ref name="Hauseman" />. The relative order of complex ordering is still an area of debate. Some experiments indicate that PP1C must bind to SHOC2 before MRAS binds<ref name="Lavoie" /> but others indicated that PP1C and MRAS can bind to SHOC2 at the same time <ref name="Hauseman" />.