Introduction

Structural Introduction

Surface representation of SHOC2-PP1C-MRAS designed in PyMol from PDB.Blue is SHOC2, Orange is PP1C and green is MRAS.

The enzyme requires 3 domains (SHOC-2(blue), PP1C(coral), and MRAS (green)) to form the active enzyme (SMP Complex), also known as a holoenzyme^[1]. SHOC-2 is a that holds the other subunits in the correct orientation, allowing for the holoenzyme to be functional. PP1C is a catalytic domain of a phosphatase enzyme PP1[1], which cleaves a phosphate. MRAS is a GTPase protein and is located near (typically just below) the cell membrane. When MRAS binds GTP, it becomes active and triggers the assembly of the active holoenzyme^[1]. The SMP complex was determined via cryo-electron microscopy as well as x-ray diffraction. These studies found that PP1C and MRAS occupy the concave surface of SHOC2, leaving the catalytic site of PP1C and the substrate binding cleft in MRAS exposed.

Biological Introduction

SHOC2-PP1C-MRAS is a human enzyme that is involved in regulating cell proliferation and division^[2]. The enzyme is involved in the vast RAS-MAPK pathway, which is initially activated by an extracellular growth factor binding to a membrane bound RAS GTPase[2] such as HRAS, NRAS, or KRAS. RAS-GTPases are a family of proteins that work by functioning as molecular switches[3]. This occurs from the protein alternating between binding GTP to be active and GDP to be inactive ^[2]. After activation via an extracellular growth factor, the RAS-GTPase enzyme binds GTP, which activates RAF^[3].

Structure

Special Interactions

SHOC2 is a domain which acts as a cradle to bind PP1C and MRAS. The is a leucine rich repeat (LRR) protein that consists of 20 consecutive . This motif results in an extended beta sheet on the inner concavity of the protein surface with alpha helices facing outward. This results in a largely hydrophobic core. binds to SHOC2 exclusively through this concaved region ^[4], primarily by the descending loop and strands of each LRR domains 2-10. This reaction is stabilized through . Protein phosphatase 1 catalytic subunit ( ) is a highly characterized serine/ threonine phosphatase. PP1C with the ascending loops of the SHOC2 LRR regions, and is further engaged through the N-terminal region containing the motif (cite, AACR). The initial forming of the complex begins with SHOC2:PP1C engagement, then is completed and stabilized by the GTP-loaded MRAS binding (cite, AACR). Once MRAS has associated with SHOC2, it to PP1C and guides the holoenzyme complex to the cell membrane to begin signaling^[4].

Switch I and II

Switch I and II are located on MRAS. The switches determine whether MRAS can bind to SHOC2-PP1C. The switches have to go through a conformational change to allow binding of SHOC2-PP1C to MRAS. This conformational change is caused by GTP replacing GDP. Once GTP is added MRAS shifts and binds with SHOC2. When GDP is bound to MRAS switch II is moved outward which causes a steric clash with SHOC2 . When GTP is bound, switch II can form various hydrophobic interactions with SHOC2^[5] . Interactions are strengthened with hydrogen bonding and pi stacking. When MRAS is bound to SHOC2-PP1C, switch I has an important role in making interactions with PP1C.

Binding Pocket

Once all the domains are bound NTpS binds to PP1C in the active site. Once the NTpS is bound it becomes dephosphorylated and the complex falls apart. NTpS is dephosphorylated to prevent the active dimeric RAF from inactivating and changing into its monomeric structure. The is surrounded by hydrophobic and acidic regions along with C-terminal. These regions are located on the surface of PP1C whereas the active site is placed further into the structure. It is thought that these regions help the ligand bind to the active site by making interactions that will lead NTpS into the protein. There is still some uncertainty as to how the substrate selectivity works but these regions could play an essential role in it. Specifically, the C-terminal of the pS from NTpS would bind to the hydrophobic region on PP1C^[6].

NTpS can bind to the PP1C active site without PP1C also being bound to SHOC2 and MRAS, however the catalytic activity is much slower and the reaction is less efficient. Also for this event to occur NTpS would need to be exposed from its binding site in the inactive RAF complex. A RAS has to bind to RAF to expose the NTpS allowing PP1C and NTpS to bind.

Mechanism

Once the SMP complex comes together, it plays a key role in regulating the activation of the RAF and MAPK pathway. To do so, PP1C, enhanced through the interactions with SHOC2 and MRAS, dephosphorylating a specific phosphoserine on RAF kinases. Doing so regulates cell growth, survival, proliferation, and differentiation (cite, bR).

Implications

Because of this complex's key role in the regulation of the MAPK-RAF pathway, minor changes can be deleterious. Unregulated activation of the MAPK pathway is known to be one of the most common causes of human cancer. This is due to unchecked cell division and proliferation which are the common trademarks of cancer. Cancer is thought to be so prevalent from this pathway because it stabilizes the interactions of the SMP complex members and consequently enhances holophosphatase activity (cite, AACR).

The unregulated MAPK pathway is also thought to be responsible for a multitude of developmental disorders commonly known as RASopathies (cite, bR). One such RASopathy caused by the mutation of a RAF kinase is known as Noonan Syndrome (NS), which enhances complex formation. NS is a genetic disorder that can prevent normal development in different parts of the body in a variety of ways. Though specifics are related to the gene carrying the mutation, it can be characterized as having unusual facial characteristics, short stature, a variety of heart defects and disease, and other physical problems and developmental delays (cite, may0).