Dataset Information

Structure of the disordered C terminus of Rab7 GTPase induced by binding to the Rab geranylgeranyl transferase catalytic complex reveals the mechanism of Rab prenylation.

ABSTRACT: Protein prenylation is a widespread process that involves the transfer of either a farnesyl or a geranylgeranyl moiety to one or more C-terminal cysteines of the target protein. Rab geranylgeranyl transferase (RabGGTase) is responsible for the largest number of individual protein prenylation events in the cell. A decade-long effort to crystallize the catalytic ternary complex of RabGGTase has remained fruitless, prompting us to use a computational approach to predict the structure of this 200-kDa assembly. On the basis of high resolution structures of two sub-complexes, we have generated a composite model where the rigid parts of the protein are represented by precomputed grid potentials, whereas the mobile parts are described in atomic details using Internal Coordinate Mechanics. Selection of the best docking solution of the flexible parts on the grid is followed by explicit atomistic refinement of the lowest energy conformations enabling realistic modeling of complex structures. Using this approach we demonstrate that the flexible C terminus of Rab7 substrate forms a series of progressively weaker and less specific interactions that channel it into the active site of RabGGTase. We have validated the computational model through biochemical experiments and demonstrated that to be prenylated RabGTPase must possess at least nine amino acids between the prenylation motif and the hydrophobic sequence anchoring the beginning of the Rab C terminus on the enzyme. This sequence, known as the C-terminal interacting motif is shown to play a dual role in Rab prenylation by contributing a significant fraction of binding energy to the catalytic complex assembly and by orienting the C terminus of RabGTPase in the vicinity of the active site of RabGGTase. This mechanism is unique to RabGGTase when compared with other prenyltransferases, which encode the specificity for their cognate substrates directly at their active site.

SUBMITTER: Wu YW

PROVIDER: S-EPMC2676050 | biostudies-literature | 2009 May

REPOSITORIES: biostudies-literature

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Structure of the disordered C terminus of Rab7 GTPase induced by binding to the Rab geranylgeranyl transferase catalytic complex reveals the mechanism of Rab prenylation.

Wu Yao-Wen YW Goody Roger S RS Abagyan Ruben R Alexandrov Kirill K

The Journal of biological chemistry 20090224 19

Protein prenylation is a widespread process that involves the transfer of either a farnesyl or a geranylgeranyl moiety to one or more C-terminal cysteines of the target protein. Rab geranylgeranyl transferase (RabGGTase) is responsible for the largest number of individual protein prenylation events in the cell. A decade-long effort to crystallize the catalytic ternary complex of RabGGTase has remained fruitless, prompting us to use a computational approach to predict the structure of this 200-kD ...[more]

PMID: 19240028

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Project description:Rab GTPases are post-translationally modified by addition of geranylgeranyl moieties to carboxyl-terminal cysteine residues. For Rab proteins ending with xxCC xCxC and CCxx motifs this modification is catalysed by geranylgeranyltransferase type II (GGTaseII), and is entirely dependent on the Rab substrate being bound to Rab escort protein (REP). Several Rab proteins contain carboxyl-terminal CaaL prenylation motifs typical of members of the Rho family, which are modified in a REP-independent manner by geranylgeranyltransferase type I (GGTaseI). The present studies show that one such Rab protein (Rab8), which ends with a CVLL motif, is uniquely able to serve as a substrate for either REP/GGTaseII or GGTaseI in cell-free assays. The modification of Rab8 by GGTaseI did not require REP, indicating that a REP-induced conformational change is not essential for exposure of the Rab carboxyl-terminal cysteine prenylation site. To determine whether one enzyme plays a predominant role in Rab8 prenylation in vivo, the incorporation of [3H]mevalonate into Rab8 was measured in human embryonal kidney 293 cells under conditions where the activity of GGTaseI, but not GGTaseII, was blocked by the peptidomimetic inhibitor GGTI-298. The GGTaseI inhibitor did not prevent prenylation of either overexpressed Myc-tagged Rab8 or endogenous Rab8, whereas prenylation of a known GGTaseI substrate with the same carboxyl-terminal motif, Cdc42Hs, was completely blocked. To rule out the possibility that the apparent prenylation of Rab8 by GGTaseII occurs only when GGTaseI activity is eliminated, metabolic labelling studies were carried out in the absence of the GGTaseI inhibitor, using a REP-binding-deficient Rab8 construct (Y78D) that cannot serve as a substrate for GGTaseII, but is indistinguishable from wild-type Rab8 as a substrate for GGTaseI. Prenylation of the Y78D mutant was reduced by 60-70% in intact cells, consistent with the conclusion that the majority of Rab8 is prenylated by the REP/GGTaseII system in vivo.

Project description:NphB is an aromatic prenyltransferase that catalyzes the attachment of a 10-carbon geranyl group to aromatic substrates. Importantly, NphB exhibits a rich substrate selectivity and product regioselectivity. A systematic computational study has been conducted in order to address several question associated with NphB-catalyzed geranylation. The reaction mechanism of the prenylation step has been characterized as a S(N)1 type dissociative mechanism with a weakly stable carbocation intermediate. A novel ?-chamber composed of Tyr121, Tyr216, and 1,6-DHN is found to be important in stabilizing the carbocation. The observed difference in the rates of product formation from 5- and 2-prenylation arises from the differing orientations of the aromatic substrate in the resting state. 4-Prenylation shares the same resting state with 5-prenylation, but the lower free energy barrier for carbocation formation makes the latter reaction more facile. The high free energy barrier associated with 7-prenylation is caused by the unfavorable orientation of 1,6-DHN in active site pocket, along with the difficulty of proton elimination after the prenylation step. A water-mediated proton transfer facilitates the loss of hydrogen at the prenylation site to form the final prenylated product. Interestingly, the same crystallographically observed water molecule has been found to be responsible for proton loss in all three experimentally identified products. After proton transfer, the relaxation of the final product from a sp(3) carbon center to a sp(2) center triggers a "spring-loaded" product release mechanism which pushes the final product out of the binding pocket toward the edge of the active site. The hydrogen bond interactions between the two hydroxyl groups of the aromatic product and the side chains of Ser214 and Tyr288 help to "steer" the movement of the product. In addition, mutagenesis studies identify these same two side chains as being responsible for the observed regioselectivity, particularly 2-prenylation. These observations provide valuable insights into NphB chemistry, offering an opportunity to better engineer the active site and to control the reactivity in order to obtain high yields of the desired product(s). Furthermore, the S(N)1 reaction mechanism observed for NphB differs from the prenylation reaction found in, for example, the farnesyltransferase, which proceeds via an S(N)2-like reaction pathway. The spring-loaded release mechanism highlighted herein also offers novel insights into how enzymes facilitate product release.

Dataset Information

Structure of the disordered C terminus of Rab7 GTPase induced by binding to the Rab geranylgeranyl transferase catalytic complex reveals the mechanism of Rab prenylation.

Publications

Structure of the disordered C terminus of Rab7 GTPase induced by binding to the Rab geranylgeranyl transferase catalytic complex reveals the mechanism of Rab prenylation.

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