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The Crystal Structure of KRAS G12V Mutant in Complex with GDP from Homo sapiens

Created by Payal Panchal

KRAS G12V (PDB ID: 5UQW) is a mutated oncogenic protein found in Homo sapiens that is part of the RAS family. RAS is a GTPase protein involved in cellular differentiation and signal transduction. HRAS, KRAS and NRAS are the three RAS subfamilies most commonly involved with cancer. Certain mutations cause these RAS proteins to be constitutively active, causing the protein to continue cell division - a hallmark of cancer. Mutated RAS proteins have been commonly linked to pancreatic, colon, and lung cancers. Since oncogenic mutations are so heavily concentrated in the RAS protein community, researchers have been trying to develop methods that apply to all RAS proteins (Pan-RAS), and not just to RAS subfamilies (1, 4).   

KRAS G12V has a molecular weight of 21298.32 Da, an isoelectric point of 6.33 and is comprised of 189 residues (7, 9). KRAS G12V has a missense mutation in its primary structure causing a glycine to valine switch in the twelfth position. While both amino acids are nonpolar, the side chain of glycine is quite smaller than that of valine’s, as a result, bulky side chain of valine prevents the binding of the GAP protein. KRAS G12V is comprised of two subunits and it has two active sites called switch I and switch II (1). The switch I region begins at residue 30 and ends at residue 40, while switch II begins at residue 60 and ends at residue 70. These switch regions are fundamental to the KRAS G12V protein as they allow effector proteins to bind, which are needed in order to induce cell signaling. The switch regions are regulated by the protein’s ability to activate GTPase activity. Preventing GTPase activity causes the effector proteins to constitutively promote cell division (1). KRAS G12V is composed of six beta sheets, six alpha helices, and fifteen random coils that exhibit a general trend when binding to ligands. KRAS G12V has two ligands, one of which is Guanosine-5'-diphosphate (GDP) and the other which is a magnesium ion. GDP, when bound to KRAS, maintains the protein in its inactive conformation and the magnesium ion catalyzes the hydrolysis of GTP (1). Sixteen GDP molecules bind to random coils in the protein while magnesium binds specifically to Ser-17 in an alpha helix. Ionic interactions occur at multiple residues such as between isoleucine and leucine, lysine and Aspartic Acid, Aspartic Acid and Glutamic Acid, and lastly, between the protein and the magnesium ion ligand. Sixteen hydrogen bonds have been noted between GDP and KRAS G12V. Hydrogen bonding in the interior of a protein structure provides substantial stabilization energy, particularly as GDP maintains RAS in an inactive conformation (1). KRAS G12V significant residues that bind to GDP during inactivation and are also being researched include Tyr-32, Glu-31, Val-12, Ala-11, Val-14, Asp-57, Ser-17, Asp-30, Val-29, Gly-15, Ala-18, Phe-28, Leu-19, Lys-16, Ala-146, Lys-147, Asn-116, Lys-117, Thr-144, Ser-145, Leu-120, Asp-119, and Cys-118. Lastly, residues that bind specifically to the magnesium ion include Pro-34, Asp-33, Ile-36, Tyr-32, Thr-58, Asp-57, Lys-16, and Ser-17 (8).   

Generally, wild-type RAS proteins must first bind with GTP in order to be activated and change the conformation of the active sites (switch regions). As a result, there can be proper binding of the effector proteins, which are responsible for KRAS G12V sending signals for cell proliferation. GTP is already bound to KRAS and exhibits GTPase activity at a GTP-binding site once a GAP protein activates KRAS. Afterwards, GTP hydrolyzes to GDP and an inorganic phosphate, which causes the effector proteins to dissociate and the switch regions to return to their original conformations. GDP and KRAS bind again, repeating the cycle (1, 3). Typical oncogenic RAS mutations either prevent GAP from binding properly or prevent GTPase activity from occurring (3, 4).                 

In order to fully understand the physiological function of KRAS G12V, it is important to examine its interaction with a small drug molecule called 3144. 3144 was developed to prevent KRAS G12V from signaling of cell division. The mutation in KRAS G12V prevents GTPase activity from manifesting, locking effector proteins in place. Researchers have theorized that instead of effector proteins, an artificial small-molecule drug could bind to the switch regions in order to block cell signaling and senesce the cells. The protein can no longer release a signaling cascade that promotes cell growth, thus leading to cell death (1). This process would hypothetically prevent tumor growth and cancer from spreading. This is the theory behind the small-molecule drug 3144. Compound 3144 was noted to bind to sites such as Val-32 and Asp-38 via hydrogen bonding. It has proven difficult to find small molecules that exhibit this type of behavior because the switch regions are significantly shallow and possess weak electrostatic interactions. After significant research, an active site named Alanine 59 had been discovered. It is located between the two switch regions of KRAS G12V. Compound 3144 was then manipulated to have higher binding affinity for these regions. However, there is still significant research being conducted on this compound, such as how to make it physically large enough to bind to both regions simultaneously, and capable of binding to all RAS proteins, which was concluded from data collected from mice models (1).

PSI-BLAST searches for proteins with similar primary structure to a protein query. It provides an E value, which is determined by comparing the gaps, which are amino acids that exist in the subject protein but not in the query protein, between the query and subject proteins. The lower the E value, the more similar the proteins. An E value below 0.05 is considered significant and indicates high similarity between proteins. The E value obtained when comparing the crystal structure of human KRAS G12V in complex with GDP to the crystal structure of a GDP-bound G13D oncogenic mutant of human GTPase KRAS (PDB ID: 4TQA) is 5e-119, indicating high similarity between the primary structures of two proteins (9).

The Dali Server compares tertiary structures of proteins and calculates the differences in intramolecular distances using the sum-of-pairs method. It provides a Z-score, which indicates similarity in tertiary structure. The higher the Z-score, the more similar the folds of the query and subject proteins are. A Z-score above 2 is considered significant and indicates that the two proteins have similar folds. The Z-score obtained when comparing the crystal structure of human KRAS G12V in complex with GDP to the crystal structure of a GDP-bound G13D oncogenic mutant of human GTPase KRAS is 30.8, indicating a very similarity in the tertiary structures of both proteins (6). The crystal structure of a GDP-bound G13D oncogenic mutant of human GTPase KRAS, better known as KRAS G13D (PDB ID: 4TQA), is very similar in structure and function to KRAS G12V, as they are both linked to causing the same types of cancer (4). The molecular weight of KRAS G13D is 39450.35 Da with an isoelectric point of 5.01 (7). KRAS G13D is comprised of two subunits and 169 residues. KRAS G13D has the same ligands as KRAS G12V; GDP and the magnesium ion, both of which are located at the same sites on the two different proteins. KRAS proteins are categorized by where missense mutations occur. In KRAS G13D, the glycine at position 13 was substituted for an aspartic acid. This mutation occurred in the protein’s primary sequence, where in the thirteenth codon, the nucleotide in the second position has changed from a guanine to an adenine. Glycine’s side chain is significantly smaller than the side chain of aspartic acid, which is why the mutation prevents the binding of a GAP protein, which activates GTPase activity. KRAS G13D interferes with another protein called SOS, which collaborates with a GAP protein to initiate GTPase activity, which is different from KRAS G12V’s inhibitory process (5, 8). A key characteristic of the function of KRAS G13D is that it displays rapid nucleotide exchange kinetics compared with other mutants analyzed, meaning that KRAS G13D causes mutations faster than KRAS G12V, accelerating the potential spread of cancer in the body (4). This property can be explained by changes in the electrostatic charge distribution of the active site induced by the G13D mutation, which was shown by X-ray crystallography.  

In conclusion, KRAS G12V is a specific missense mutation of a RAS protein where the glycine at position 12 was switched for a valine. This mutation prevents the binding of a GAP protein to KRAS G12V, inhibiting GTPase activity and ultimately causing effector proteins to continue signaling to cells to replicate. Mutated RAS proteins have been linked to 20% of all human cancers, and specific mutations have been associated with the development of certain cancers, not just associated with exposure to certain mutagens. For example, KRAS G12V has been observed predominantly in colorectal cancer (2). Researchers are trying to develop methods that would ultimately block effector proteins from participating in signal transduction, which would in turn prevent excessive cell replication (4).