In Vitro Assembly, Purification, and Crystallization of the Rab Geranylgeranyl Transferase:Substrate Complex

Rak, Alexey; Niculae, Anca; Kalinin, Alexandr; Thomä, Nicolas H.; Sidorovitch, Vadim; Goody, Roger S.; Alexandrov, Kirill

doi:10.1006/prep.2001.1605

Cited by 18 publications

(7 citation statements)

References 20 publications

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“…Further reduction in the sequence length completely removes the ability of Rab7 to act as the RabGGTase substrate. The inferred flexibility of the complex may explain why Rab7⅐REP-1⅐RabGGTase complex crystals diffract only to low resolution (24). The requirement for appropriate length of the linker peptide between CIM and the prenylation motif also finds its support in earlier in vivo data, where a truncation of 10 amino acid residues linking putative CIM (VDL) and prenylation motif (CC) of Rab5 rendered the protein cytosolic (28).…”

Section: Effects Of Rab C-terminal Sequence On the Prenylation And Thmentioning

confidence: 81%

“…Although we previously reported crystallization of such a complex, it has remained refractory to structure solution due to its poor x-ray diffraction properties (24). This motivated us to seek an alternative approach for analysis of the structural details of RabGGTase-mediated protein prenylation reaction.…”

Section: Simulation Of the Rab⅐rep⅐rabggtase Ternary Proteinmentioning

confidence: 99%

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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

Goody

Abagyan

et al. 2009

Journal of Biological Chemistry

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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 subcomplexes, 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.

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Section: Effects Of Rab C-terminal Sequence On the Prenylation And Thmentioning

confidence: 81%

Section: Simulation Of the Rab⅐rep⅐rabggtase Ternary Proteinmentioning

confidence: 99%

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

Goody

Abagyan

et al. 2009

Journal of Biological Chemistry

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“…Citrine-Rab7a and Citrine Rab27a were prenylated in vitro and purified as a complex with REP1 as described previously [36]. …”

Section: Methodsmentioning

confidence: 99%

Rab GTPase Prenylation Hierarchy and Its Potential Role in Choroideremia Disease

et al. 2013

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Protein prenylation is a widespread post-translational modification in eukaryotes that plays a crucial role in membrane targeting and signal transduction. RabGTPases is the largest group of post-translationally C-terminally geranylgeranylated. All Rabs are processed by Rab geranylgeranyl-transferase and Rab escort protein (REP). Human genetic defects resulting in the loss one of two REP isoforms REP-1, lead to underprenylation of RabGTPases that manifests in retinal degradation and blindness known as choroideremia. In this study we used a combination of microinjections and chemo-enzymatic tagging to establish whether Rab GTPases are prenylated and delivered to their target cellular membranes with the same rate. We demonstrate that although all tested Rab GTPases display the same rate of membrane delivery, the extent of Rab prenylation in 5 hour time window vary by more than an order of magnitude. We found that Rab27a, Rab27b, Rab38 and Rab42 display the slowest prenylation in vivo and in the cell. Our work points to possible contribution of Rab38 to the emergence of choroideremia in addition to Rab27a and Rab27b.

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“…Not shown in the scheme is the effect of binding of lipid substrate to this complex, which results in a decrease in affinity of RabGGTase to the Rab:REP complex and frees the RabGGTase for further cycles of activity and releases the prenylated Rab as a soluble complex with REP. be generated). The ability to generate stable binary complexes in the laboratory was of importance, since the ternary protein complex has so far proven refractory to structural analysis, although it can be generated and crystallized [12].…”

Section: Introductionmentioning

confidence: 99%

The structural and mechanistic basis for recycling of Rab proteins between membrane compartments

Goody

Rak

Alexandrov

2005

CMLS, Cell. Mol. Life Sci.

135

109

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Rab proteins are members of the Ras superfamily of GTPases and are key regulators of intracellular vesicular transport. They undergo a cycle of GTPase activity, and this activity is interconnected to a cycle of reversible attachment to membranes. This cycle is mediated by geranylgeranylation of (usually) two C-terminal cysteines, which in turn is effected by Rab geranylgeranyltransferase in concert with REP (Rab escort protein). After delivery to their respective membranes, Rabs are activated by replacement of GDP by GTP, allowing interaction with a wide variety of effector molecules involved in vesicular transport, in particular with docking of transport vesicles to their specific target membranes. After completion of these events and GTP hydrolysis, Rabs are retrieved by GDI (GDP dissociation inhibitor) and delivered to their starting compartment. Here, the structural and mechanistic basis of events occurring in Rab delivery and cycling, and the differences between REP and GDI are discussed on the basis of recent advances in the field.

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In Vitro Assembly, Purification, and Crystallization of the Rab Geranylgeranyl Transferase:Substrate Complex

Cited by 18 publications

References 20 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

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

Rab GTPase Prenylation Hierarchy and Its Potential Role in Choroideremia Disease

The structural and mechanistic basis for recycling of Rab proteins between membrane compartments

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