Molecular Biology: Protein Structure and Function - Stepanov V.M. 2005

G proteins
c-H-ras protein

A vast Class of GTP-binding Proteins, or G proteins, plays an exceptionally vital role in both intercellular and intracellular signal Transduction. Their functional state—in other words, their ability to transmit signals to other proteins—is determined by whether they carry a bound molecule of guanosine triphosphate (GTP) or its Hydrolysis product, guanosine diphosphate (GDP). These proteins possess intrinsic GTPase activity; consequently, a G protein activated by the non-covalent binding of GTP gradually hydrolyzes this effector into GDP, thereby returning to its inactive form:

Thus, a crucial feature of G proteins is their ability to maintain an active conformation and participate in signal transmission for a specific duration. This depends on their GTPase activity as well as the Action of Protein factors that either promote the exchange of GDP for GTP—thus switching the protein to its active state—or, conversely, enhance the GTPase activity of the G protein, thereby accelerating its transition into the inactive GDP-bound complex. The structural and Functional Characteristics of G proteins are illustrated by the following Examples.

Certain oncoproteins belong to the G protein family, notably the human c-H-ras oncoprotein. G proteins encoded by mutant GENES OF THE ras family are frequently detected in tumors. The normal c-H-ras Gene encodes the so-called p21 protein with a Molecular Weight of 21 kDa, which is localized in the cytoplasmic membrane and acts as a potential oncoprotein precursor, or proto-oncoprotein. Its function in a normal Cell apparently consists in receiving cell-growth-stimulating signals from the exterior and relaying them to target proteins inside The Cell, thereby initiating Cell Growth and Division.

The p21–GTP complex associates with the GAP (GTPase-Activating Protein), which enhances the intrinsic GTPase activity of this complex. It is specifically the ternary p21–GTP–GAP complex that serves as the signal transducer. Simultaneously, hydrolysis of GTP to GDP takes place within this complex, catalyzed by the p21 protein itself. The latter acts inherently as a very slow-acting GTPase (accelerating guanosine triphosphate hydrolysis approximately 1,000-fold, with a half-life of the p21–GTP complex of about 1 hour). The GTPase activity increases dramatically (by an estimated 100,000-fold) upon Formation of the ternary complex with the activating protein GAP. Crucially, As a result of GTP hydrolysis to GDP, the complex with GAP dissociates, and signal transmission ceases.

Thus, the duration of the signal depends on the efficiency of GTP hydrolysis; the G protein essentially incorporates an internal timer regulated through its interaction with GAP. Understandably, regarding a single isolated molecule, one can only speak of a greater or lesser probability of GTP Cleavage within a given time interval. However, because numerous such molecules participate in signal transduction within a specific biological system, GTP hydrolysis effectively dictates the duration of G protein action—that is, the length of the signal.

The p21 protein consists of 189 amino acid residues in a single peptide chain. This chain forms six ß-strands and four a-helices (Fig. 12.1), with the C-terminal half of the protein characterized by an alternating Secondary Structure packing motif of ß—a-ß-a—ß, resembling the structural fold of the nucleotide-binding domain in dehydrogenases. Of the ten loops connecting the a-helical and ß-strand regions, five (L1, L2, L4, L9, and L10) form a pocket that binds GDP and GTP, with the protein interacting with both the guanine base, the ribose, and the phosphate groups.

Fig. 12.1. STRUCTURE OF THE human c-H-ras p21 protein (residues 1–171). Four a-helices, ß-strands ß₁–ß₆, and loops L₁–L₈ are visible. THE POSITION OF the bound GDP is indicated by a rectangle, a pentagon, and two circles.

Loop L1, which connects the N-terminal ß-strand and the first a-helix, contains the sequence:

It effectively "saddles" the phosphoester bond between the ß- and a-phosphate groups of the substrate, evidently forming part of the GTPase catalytic center. In viral proteins belonging to the same family and structured similarly, the terminal (y) phosphate of GTP is intramolecularly transferred to the hydroxyl group of Thr-59 rather than to Water (a process known as autophosphorylation). It should be emphasized that the binding site for the activating protein, GAP, is located in close proximity to the catalytic center.

The hydrolysis of guanosine triphosphate involves a water molecule that forms a Hydrogen bond with the carbonyl group of Thr-35 and is activated through interaction with other Functional groups of the catalytic center. However, in the free G protein, these Regions of the active center exist in multiple conformers, of which only one is capable of activating the specified water molecule, which explains the very low catalytic efficiency. Apparently, the complex of the ras protein with the activating protein (GAP) stabilizes precisely this conformation, leading to a dramatic acceleration of GTP hydrolysis.

The hydrolysis of GTP bound in the active center of the G protein leads to local structural Changes in the vicinity of the contact with the ß- and y-phosphate groups of GTP (Fig. 12.2). Apparently, the Cleavage of the y-phosphate group of GTP is followed by the movement of mobile elements of the Protein Structure closer to the ß-phosphate of GDP, effectively filling the resulting "gap." Notably, the folding of loops L2 (residues 26–36) and L4 (residues 59–65) changes noticeably, as does the mode of coordination of the magnesium ion that interacts with the active center of the p21 protein and GTP or GDP. Because these same loops mediate the interaction with the activating protein GAP, such a conformational transition disrupts the p21–GAP contact, thereby terminating signal transduction.

Fig. 12.2. Changes in the interaction between the nucleotide and the G protein accompanying the hydrolysis of GTP to GDP.

Only the triphosphate and diphosphate fragments of the NUCLEOTIDES are shown. A water molecule, activated by interactions with the glutamine residue Gln-61 and its environment, attacks the protein-bound GTP molecule, whose terminal y-phosphate residue is linked to the NH groups of the Threonine residue Thr-35 in loop L2 and the Glycine residue Gly-60 in loop L4. Cleavage of the terminal y-phosphate residue results in The formation of GDP. As a result, Thr-35 and Gly-60, and consequently loops L2 and L4, lose direct contact with the Ligand—the GDP molecule. This induces a conformational change in the loops that is unfavorable for the interaction of the G protein with the signal-transducing protein.

The C-terminal fragment of the c-H-ras protein, which is hypothesized to interact with a membrane receptor, is connected to the aforementioned "catalytic" domain by a long a-helix that presumably participates in signal transduction. All Proteins of the ras family feature a characteristic CAAX sequence at their carboxyl terminus (where C is Cysteine and A is an aliphatic amino acid), which serves as a signal for the prenylation of the Cys-186 thiol group followed by cleavage of the C-terminal tripeptide. Such post-translational hydrophobic Modification of the C-terminus of the ras protein (see Chapter 11) is essential for its attachment to the cytoplasmic face of The cell membrane. ras proteins lacking this structure exhibit no transforming activity on Cells, although they retain The ability to bind and hydrolyze GTP.

The proteins responsible for inputting and outputting the signal transmitted by the p21 protein have not yet been strictly identified, although there is reason to believe that one of them is the GAP protein, which in turn interacts with the growth factor receptor. Apparently, another protein catalyzes the exchange of GDP for GTP—a process that proceeds very slowly in the p21–GDP complex.

Thus, the relatively small c-H-ras protein is involved in a complex system of intermolecular interactions, the execution of which depends critically on the presence of its bound GTP molecule and The rate of its hydrolysis. It is easy to see that such a complex protein function can be readily disrupted by Mutations affecting individual amino acid residues in the normal protein, or proto-oncogene. As a consequence of such an impairment in functional properties, the mutant protein can convert into an actual oncogene.

For instance, the substitution of the Gly-12 residue in the loop belonging to the catalytic center by any amino acid other than Proline drastically reduces GTPase activity, and this activity can no longer be stimulated by interaction with the GAP protein. As X-ray crystallographic analysis using a technique that monitors GTP hydrolysis in real time has shown, the mutation does not cause major Conformational Changes in the GTP-binding region. For example, the Gly-12 → Val substitution affects only the positions of the residues

Gly-60, Thr-38, and Gln-61, which surround the GTP y-phosphate. As a result, however, the mutant protein hydrolyzes GTP extremely slowly and essentially fails to "turn off," continuing to send stimulating signals into the cell even when they are not needed, in the absence of an external signal. Thus, the c-H-ras protein acquires the ability to transform normal cells into tumor cells—that is, it gains transforming activity.

Transforming activity, and consequently The conversion of a proto-oncogene into an oncogene, is also triggered by Amino Acid Substitutions at positions 116 and 119, which are involved in binding the guanine moiety of GTP, as well as residues 59, 61, and 63, which can influence the conformation of the loop containing the Gly-12 residue, thereby reducing GTPase activity.

At the same time, amino acid substitutions in the 35–40 region reduce the ability of the activated c-H-ras protein to effect cell transformation, although they do not alter GTPase activity or GTP/GDP binding. Apparently, this region is involved in the interaction with the target protein to which the signal is transmitted. Disruption of this interaction renders the activation of the c-H-ras protein upon GTP binding meaningless, thereby interrupting signal transduction.

The main structural features and MECHANISM OF ACTION of the ras p21 protein are presumably typical of other G proteins, at least concerning their G domains. In particular, the G domain of the elongation factor EF–Tu has a closely similar structure.