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12.5: Small G proteins, GAPs and GEFs

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    G proteins: Cellular Switch for Kinases

    In preceding chapter sections we discussed two types of small G proteins, Gα, part of the heterotrimeric Gαβγ complex linked to GPCR signaling, and Ras (H, K, and N). Both bind GTP and GDP and have GTPase activity. When bound to GTP they are active while the GDP bound form is inactive. What a perfect molecular switch to turn on signaling and with a built in off switch (the GTPase activity). It turns out that his simple on/off switch is too simple. For example, a single mutation that inhibits the GTPase activity would leave the protein on continually which could (and does) lead to unregulated growth and tumor formation.

    Two new set of proteins that regulate the on-off activity of small G proteins are found abundantly in cells:

    • GTPase activating protein or GAPs: As they name implies that enhance the GTPase activity of the small G proteins, which would decrease G protein signaling;
    • Guanine nucleotide exchange proteins or GEFs: These lead to the dissociation of bound GDP and its replacement with GTP, which would increase G protein signaling.

    In the previous sections , the small GDP/GTP binding protein Ras was introduced. Mammalian cells contain 3 variants of Ras: H, K, and N. They all bind GDP/GTP and have GTPase activity. Ras is targeted to the cell membrane through the post-translational addition of a hydrophobic farnesyl group. When activated by binding to GTP, it can bind to and activate a protein call Raf-1, which is on binding become an active tyrosine kinase. Ras has intrinsic GTPase activity, so eventually active Ras will deactivate itself.

    Ras is just one member of a large superfamily of small G protein, which all have GTPase activity. However, they are poor GTPases, so they need help to autocatalytically cleave the GTP to GDP. GAPs and GEFs evolved to regulate their activity by modulating the balance of bound GTP (active form of the protein) and GDP (inactive form of the small G protein).

    Before describing these proteins, we need to have a better understanding of the family of small G proteins.

    Small G proteins

    The superfamily of small G proteins have a common 20 K molecular weight catalytic (GTPase) domain with 5 alpha helices, 6 beta strands and connecting loops. The small G proteins are "active" in the GTP bound form. Hydrolysis of GTP to GDP causes the protein to become inactive. Figure \(\PageIndex{1}\) below shows the domain structures of small G proteins.

    GEFS_Fig1.svg
    Figure \(\PageIndex{1}\): Domain Structure of small G proteins. Toma-Fukai et al. Molecules 2019, 24, 3308; doi:10.3390/molecules24183308. Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

    G boxes of the G domain are highlighted with orange boxes. The hyper variable region, including a polybasic region and a CAAX motif, is highlighted with pink boxes. The P-loop, switch I, and switch II are shown as bars colored colored green, red, and blue, respectively. The bottom structures shown H-Ras bound to GDP and GTP. The P-loop, switch I, and switch II are colored green, red, and blue, respectively.

    Figure \(\PageIndex{2}\) below show these key structural features of Ras.

    RasGTPlabels
    Figure \(\PageIndex{2}\): Key structural features of Ras

    Important parts of Ras necessary for GTP binding include the phosphate-binding (P loop), residues 10 to 16 (dark blue trace below), switch regions I (30 to 37, light blue trace) and II (60 to 76, green trace), which are flexible loops which sandwiches GTP.

    Figure \(\PageIndex{3}\) below is an animation showing structural differences between the GTP bound form (blue, pdb id 5p21) and GDP form (red, pdb id 4q21) of the H-Ras protein. One helix and nearby loops are perturbed.

    HRasbluewithredwithoutGTP
    Figure \(\PageIndex{3}\): Structural differences between the GTP bound form (blue, pdb id 5p21) and GDP form (red, pdb id 4q21) of Ras

    There are about 150 members of the human Ras superfamily as shown in Figure \(\PageIndex{4}\) and Table 1 below.

    Human Ras Superfamily.svg
    Figure \(\PageIndex{4}\): Members of the human Ras superfamily

    Table \(\PageIndex{1}\) shows common Ras superfamily functions

    Ras regulation of gene expression, cell proliferation, survival and differentation
    Rho regulation of actin cytoskeleton, cell shape and movement, cell interactions the extracellular matrix
    Rab vesicle trafficking, endocytosis, secretion
    Arf vesicle trafficking, endocytosis, secretion, microtuble assembly
    Ran nuclear cytoplasm transport, mitotic spindle

    We have discussed Ran before as a mediator of protein movement across the nuclear membrane in Chapter 11.5. It's mainly in the GDP bound form in the cytoplasm and the GTP bound form in the nucleus. Switches between a cytoplasmic GDP- and a nuclear GTP-bound state by nucleotide exchange and GTP hydrolysis. Nuclear import receptors with bound cargo protein containing a nuclear import signal bind RAN-GTP in the nucleus, leading to the release of the importin and the cargo protein. In contrast, cargo protein with a nuclear export signal bind exportins and RAN-GTP in the nucleus and move into the cytoplasm, where the RAN bound GTP is hydrolysed on binding a RAN-GAP. This cycle is illustrated in Figure \(\PageIndex{5}\) below.

    nucleoporinsRANFig2.svg
    Figure \(\PageIndex{5}\): Model of nuclear import and export. Cargo containing NLS (Nuclear localization signal) is imported with the help of Importin α and importin β heterodimer. Nuclear export of cargo having NES (nuclear export signal) is carried out with the help of exportins. Ran GTP is also required during that process. Khan Asmat Ullah, Qu Rongmei, Ouyang Jun, Dai Jingxing. Front. Physiol., 03 April 2020 | https://doi.org/10.3389/fphys.2020.00239. Creative Commons Attribution License (CC BY).

    Small G proteins are a fundamental form of molecular switch. They are simply too important to not be regulated. Probably the most common mutation in human cancer cells involved a single amino acid changes in Ras (H, K and N form). If the GTPase activity is inhibited with mutation, the protein may be constitutively active. Such a Ras mutation is found in almost 90% of pancreatic cancers. Hence researchers have been trying to design drugs that inhibit its GTPase activity. This has proven difficult since it has very few targetable pockets that could bind a drug.

    Regulation of small G proteins: GAPs and GEFs

    Given the critical importance of small G proteins, it makes biological sense that their on/off activity would be exquisitely regulated. Indeed, they are. Two families of protein have evolved to regulate them by determining whether GTP or GDP is bound to the protein (leading to an active, and inactive small G protein respectively). One family, GTPase activating proteins (GAPs) facilitate the hydrolysis of bound GTP, leading to the inhibition of the protein. The other proteins are GTP exchange proteins (GEFs), which facilitate the exchange of GTP for GDP, activating the protein.

    The activity of Ras GAPs and GEFs, as well as various proteins interacting with Ras, are depicted in Figure \(\PageIndex{6}\) below.

    RAS_GAP_GEF_EFFECTORS

    Figure \(\PageIndex{6}\): The activity of Ras GAPs and GEFs, as well as various proteins interacting with Ras

    It may seem crazy but the number of GEFs and GAPs is greater than the number of G proteins with which they interact. As shown in Figure \(\PageIndex{4}\), there are 20 Rho G proteins but about 80 GEFs and 70 GAPs for them. This number presumably allows greater control of the specificity of the reactions controled by Rho G protein..

    GAPs - GTPase Activating Proteins

    The hydrolysis of the gamma phosphate of GTP by water in Ras proceeds by a pentavalent transition state with two axial and three equatorial ligands to the P. Developing charge in the transition state would usually be stabilized by catalytic residues in the catalytic domain of Ras. However, Ras is a poor GTPase. There's were GAP comes in. In the Ras/GAP complex, GAP positions its Arg 789 on the GAP in a position to stabilize the transition state for Ras-bound GTP cleavage. This Arg 789 is almost in the same position as Arg 178 in the Galpha inhibitor subunit of a heterotrimeric G protein which inhibits GPCR signaling. Both of these arginines have similar catalytic function.

    Ras-GAP complex (1WQ1).png
    Figure \(\PageIndex{7}\): Ras-GAP complex (1WQ1) (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...MJiLGpT4w6kP69

    Ras is shown in secondary structure colors, while GAP is shown in gray.. GDP-AlF3, a GTP analog, is shown in color spacefill. Arginine 789 in GAP is shown in spacefill with CPK colors and labled R789. It is a clear position to stabilize the bound GTP in the complex and in its cleavage transition state.

    GEFs - GTP Exchange Factor

    Once bound to Ras, GDP dissociates very slowly. Values of 10-5 sec-1 have been reported for first order rate constant of the dissociation of GDP from a small G protein. Assuming that the diffusion controlled on rate constant for the complex, the KD for the G protein:GDP complex would be 0.1 pM and the half life would be 0.8 days, similar to the lac repressor:DNA operator complex. Hence the protein, when bound to GTP, is essentially locked in the off position. What if it needs to be reactivated in a quick fashion? How can the rate at which GDP dissociates be increased so that GTP could replace it? If it were to dissociate, GTP could quickly replace it since from an equilibrium point of view, Ras and other small G proteins would favor GTP binding since its concentration in the cell is higher.

    One could envision a number of ways to change the rate at which GDP dissociates. In organic chemistry, a favorite students answer to many questions is to envoke steric effects. In biochemistry, the analog is often conformational changes. How could you change the conformation of Ras such that it might favor GTP binding? That could occur by ligand binding or more likely by a post-translation modification such as phosphorylation as part of a signaling process. It turns out that for the case of small G proteins, another mechanism is evoked: the binding of another protein, a GTP Exchange Factor or GEF, which promotes GTP exchange for the bound GDP. If the Ras:GEF:GDP complex has a 10,000 increase in koff for GTP, the half life of the bound GDP is 7 seconds. There are 80 GEFs in the human genome. If you think about it, in GPCR coupled signaling, the ligand-bound GPCR is a GEF for the Gαsubunit of the heterotrimeric Gαβγ protein.

    The crystal structure of a Ras GEF, SOS, in complex with Ras allows a detailed understanding of the mechanism. SOS, a cytoplasmic protein, is recruited to the cell membrane where active Ras is found, teathered to the membrane with a fanesyl hydrophobic attachment.

    Ras and SOS (a GEF) complex (1bkd).png
    Figure \(\PageIndex{8}\): Ras and SOS (a GEF) complex (1bkd) (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...XJP4Fajqc9eEL6

    The actual biological unit (functional structure) is an hetero 8-mer (A4B4) with C4 symmetry. The iCn3D model shows just a heterodimer for clarity. Ras is shown in cyan and SOS in dark blue. SOS as a GEF affects nucleotide binding to SOS in two essential ways. An alpha helix from SOS displaces Switch 1 (amino acids 30-38, shown in red) in Ras, which opens the binding site for the guanine nucleotides open. Additional conformational changes in Switch II (59–72, green) in Ras and interference from the side chains form the SOS alpha helix interferes with the binding of the phosphates on the bound nucleotide. This promotes dissociation of the bound nucleotide and Mg2+. Now GTP can preferentially rebind. How?

    Before we answer that question, lets explore the conformational differences just in the structure of Ras in the Ras:GDP complex (4q21) and Ras in the Ras:SOS (1bkd) complex.These differences are shown in Figure \(\PageIndex{9}\) below. The green structure is the Ras:GDP. They cyan structure is Ras without bound GDP but bound to SOS.

    RasGTP RasinRasGEF
    Figure \(\PageIndex{9}\): Structure difference in Ras in the Ras:GDP complex (4q21) and Ras in the Ras:SOS (1bkd) complex

    Note the large shift in Switch 1 in the Ras structure from the Ras:SOS complex. This leaves a "gaping" hole from which GDP can "escape". Now how does the opening of the active site and release of bound GDP facilitate GTP binding?

    Once bound just to Ras, GDP dissociates very slowly. Values of 10-5 sec-1 have been reported for first order rate constant of the dissociation of GDP from a small G protein. Assuming that the diffusion controlled on rate constant for the complex, the KD for the G protein:GDP complex would be 0.1 pM and the half life would be 0.8 days, similar to the lac repressor:DNA operator complex. Hence the protein, when bound to GDP, is essentially locked in the off position.

    The conformational changes on RAS binding to SOS open up the active site, allowing GDP dissociation. GTP can now replace it since from an equilibrium point of view, Ras and other small G proteins favor GTP binding since the concentration of GTP in the cell is higher than that of GDP. In addition, some additional noncovalent interactions with the extra phosphate on GTP probably help.

    Figure \(\PageIndex{10}\) below shows a cartoon showing the changes in Ras on GEF binding as illustrated in these coupled chemical equilibria:

    GEFloose:Ras:GDPtight ↔ GEFtight:Ras:GDPloose ↔ GEFtight:Ras

    Ras_GEF_28pcent
    Figure \(\PageIndex{10}\): Chemical equation for Ras:GEF:GDP interactions

    12.5: Small G proteins, GAPs and GEFs is shared under a not declared license and was authored, remixed, and/or curated by Henry Jakubowski and Patricia Flatt.

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