Active site of an aminoacyl-tRNA synthetase dissected by energy-transfer-dependent fluorescence.

Aminoacyl-tRNA synthetases establish the rules of the genetic code by catalyzing attachment of amino acids to specific transfer RNAs (tRNAs) that bear the anticodon triplets of the code. Each of the 20 amino acids has its own distinct aminoacyl-tRNA synthetase. Here we use energy-transfer-dependent fluorescence from the nucleotide probe N-methylanthraniloyl dATP (mdATP) to investigate the active site of a specific aminoacyl-tRNA synthetase. Interaction of the enzyme with the cognate amino acid and formation of the aminoacyl adenylate intermediate were detected. In addition to providing a convenient tool to characterize enzymatic parameters, the probe allowed investigation of the role of conserved residues within the active site. Specifically, a residue that is critical for binding could be distinguished from one that is important for the transition state of adenylate formation. Amino acid binding and adenylate synthesis by two other aminoacyl-tRNA synthetases was also investigated with mdATP. Thus, a key step in the synthesis of aminoacyl-tRNA can in general be dissected with this probe.

Errors from selective disruption of the editing center in a tRNA synthetase.

Some aminoacyl-tRNA synthetases have two catalytic centers that together achieve fine-structure discrimination of closely similar amino acids. The role of tRNA is to stimulate translocation of a misactivated amino acid from the active site to the editing site where the misactivated substrate is eliminated by hydrolysis. Using isoleucyl-tRNA synthetase as an example, we placed mutations in the catalytic center for editing at residues strongly conserved from bacteria to humans. A particular single substitution and one double substitution resulted in production of mischarged tRNA, by interfering specifically with the chemical step of hydrolytic editing. The substitutions affected neither amino acid activation nor aminoacylation, with the cognate amino acid. Thus, because of the demonstrated functional independence of the two catalytic sites, errors of aminoacylation can be generated by selective mutations in the center for editing.

Misactivated amino acids translocate at similar rates across surface of a tRNA synthetase.

Certain aminoacyl-tRNA synthetases have a second active site that destroys (by hydrolysis) errors of amino acid activation. For example, isoleucyl-tRNA synthetase misactivates valine (to produce valyl adenylate or Val-tRNA(Ile)) at its active site. The misactivated amino acid is then translocated to an editing site located >25 A away. The role of the misactivated amino acid in determining the rate of translocation is not known. Valyl-tRNA synthetase, a close homolog of isoleucyl-tRNA synthetase, misactivates threonine, alpha-aminobutyrate, and cysteine. In this paper, we use a recently developed fluorescence-energy-transfer assay to study translocation of misactivated threonine, alpha-aminobutyrate, and cysteine. Although their rates of misactivation are clearly distinct, their rates of translocation are similar. Thus, the rate of translocation is independent of the nature of the misactivated amino acid. This result suggests that the misactivated amino acid per se has little or no role in directing translocation.

Transfer RNA-dependent translocation of misactivated amino acids to prevent errors in protein synthesis.

Misactivation of amino acids by aminoacyl-tRNA synthetases can lead to significant errors in protein synthesis that are prevented by editing reactions. As an example, discrete sites in isoleucyl-tRNA synthetase for amino acid activation and editing are about 25 A apart. The details of how misactivated valine is translocated from one site to the other are unknown. Here, we present a kinetic study in which a fluorescent probe is used to monitor translocation of misactivated valine from the active site to the editing site. Isoleucine-specific tRNA, and not other tRNAs, is essential for translocation of misactivated valine. Misactivation and translocation occur on the same enzyme molecule, with translocation being rate limiting for editing. These results illustrate a remarkable capacity for a specific tRNA to enhance amino acid fine structure recognition by triggering a unimolecular translocation event.

Kinetics of Cdc42 membrane extraction by Rho-GDI monitored by real-time fluorescence resonance energy transfer.

The mechanisms underlying the ability of the Rho-GDP dissociation inhibitor (RhoGDI) to elicit the release of Rho-related GTP-binding proteins from membranes is currently unknown. In this report, we have set out to address this issue by using fluorescence resonance energy transfer approaches to examine the functional interactions of the RhoGDI with membrane-associated Cdc42. Two fluorescence assays were developed to monitor the interactions between these proteins in real time. The first involved measurements of resonance energy transfer between N-methylanthraniloyl GDP (MantGDP) bound to Cdc42 and fluorescein maleimide covalently attached to cysteine 79 of RhoGDI (RhoGDI-FM). This assay allowed us to directly monitor the binding of RhoGDI to membrane-associated Cdc42. The second fluorescence assay involved measurements of resonance energy transfer between membrane-associated Cdc42-MantGDP and hexadecyl(amino) fluorescein that was randomly inserted into the membrane bilayer. This assay enabled us to directly monitor the (GDI-induced) release of Cdc42 from membranes. Analyses of the rates of change in the fluorescence of Cdc42-MantGDP, which serves as a resonance energy transfer donor in both of these assays, as a function of RhoGDI concentration suggests a two-step mechanism to explain the ability of RhoGDI to stimulate the release of Cdc42 from membranes. Specifically, we propose that the GDI first binds rapidly to membrane-associated Cdc42 and then a slower isomerization occurs which represents the rate-limiting step for the dissociation of the Cdc42-RhoGDI complex from membranes. We propose that this slow step in the observed kinetics reflects the time-course of translocation of the geranyl-geranyl lipid tail of Cdc42 from the outer leaflet of the membrane to the isoprenyl binding site observed in the previously reported NMR structure of the Cdc42-RhoGDI complex [Gosser et al. (1997) Nature 387, 814].

C-terminal binding domain of Rho GDP-dissociation inhibitor directs N-terminal inhibitory peptide to GTPases.

The Rho GDP-dissociation inhibitors (GDIs) negatively regulate Rho-family GTPases. The inhibitory activity of GDI derives both from an ability to bind the carboxy-terminal isoprene of Rho family members and extract them from membranes, and from inhibition of GTPase cycling between the GTP- and GDP-bound states. Here we demonstrate that these binding and inhibitory functions of rhoGDI can be attributed to two structurally distinct regions of the protein. A carboxy-terminal folded domain of relative molecular mass 16,000 (M[r] 16K) binds strongly to the Rho-family member Cdc42, yet has little effect on the rate of nucleotide dissociation from the GTPase. The solution structure of this domain shows a beta-sandwich motif with a narrow hydrophobic cleft that binds isoprenes, and an exposed surface that interacts with the protein portion of Cdc42. The amino-terminal region of rhoGDI is unstructured in the absence of target and contributes little to binding, but is necessary to inhibit nucleotide dissociation from Cdc42. These results lead to a model of rhoGDI function in which the carboxy-terminal binding domain targets the amino-terminal inhibitory region to GTPases, resulting in membrane extraction and inhibition of nucleotide cycling.

Characterization of the interaction between RhoGDI and Cdc42Hs using fluorescence spectroscopy.

The GDP-dissociation-inhibitor (GDI) for Rho-like GTP-binding proteins is capable of three different biochemical activities. These are the inhibition of GDP dissociation, the inhibition of GTP hydrolysis, and the stimulation of the release of GTP-binding proteins from membranes. In order to better understand how GDI interactions with Rho-like proteins mediate these different effects, we have set out to develop a direct fluorescence spectroscopic assay for the binding of the GDI to the Rho-like protein, Cdc42Hs. We show here that when the GDI interacts with Cdc42Hs that contains bound N-methylanthraniloyl GDP (Mant-GDP), there is an approximately 20% quenching of the Mant fluorescence. The GDI-induced quenching is only observed when Mant-GDP is bound to Spodoptera frugiperda-expressed Cdc42Hs and is not detected when the Mant nucleotide is bound to Escherichia coli-expressed Cdc42Hs and thus shows the same requirement for isoprenylated GTP-binding protein as that observed when assaying GDI activity. A truncated Cdc42Hs mutant that lacks 8 amino acids from the carboxyl terminus and is insensitive to GDI regulation also does not show changes in the fluorescence of its bound Mant-GDP upon GDI addition. Thus, the GDI-induced quenching of Mant-GDP provides a direct read-out for the binding of the GDI to Cdc42Hs. Titration profiles of the GDI-induced quenching of the Mant-GDP fluorescence are saturable and are well fit to a simple 1:1 binding model for Cdc42Hs-GDI interactions with an apparent Kd value of 30 nM. A very similar Kd value (28 nM) is measured when titrating the GDI-induced quenching of the fluorescence of Mant-guanylyl imidotriphosphate, bound to Cdc42Hs. These results suggest that the GDI can bind to the GDP-bound and GTP-bound forms of Cdc42Hs equally well. We also have used the fluorescence assay for GDI interactions to demonstrate that the differences in functional potency observed between the GDI molecule and a related human leukemic protein, designated LD4, are due to differences in their binding affinities for Cdc42Hs. This, together with the results from studies using GDI/LD4 chimeras, allow us to conclude that a limit region within the carboxyl-terminal domain of the GDI molecule is responsible for its ability to bind with higher affinity (compared with LD4) to Cdc42Hs.

Investigation of the GTP-binding/GTPase cycle of Cdc42Hs using extrinsic reporter group fluorescence.

The overall goal of these studies was to examine the applicability of extrinsic reporter group fluorescence in monitoring the GTP-binding/GTPase cycle of a Ras-like GTP-binding protein. Toward this end, we have labeled the GTP-binding protein Cdc42Hs with the environmentally sensitive fluorophore succinimidyl 6-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]hexanoate (sNBD) at a single reactive lysine residue. We find that the sNBD-labeled Cdc42Hs undergoes a fluorescence enhancement at 545 nm when Cdc42Hs exchanges bound GDP for GTP. This enhancement is then fully reversed upon GTP hydrolysis. The specific GTPase-activating protein for Cdc42Hs, the Cdc42Hs-GAP, strongly stimulates the rate of reversal of the fluorescence enhancement at 545 nm, consistent with its ability to fully catalyze the GTPase reaction of Cdc42Hs. Conversely, the specific guanine nucleotide exchange factor (GEF), Cdc24, strongly stimulates the fluorescence enhancement that accompanies GTP binding, consistent with its ability to stimulate the GDP-GTP exchange reaction on Cdc42Hs. Resonance energy transfer measurements yielded a distance of approximately 32 A for the sNBD moiety and the guanine nucleotide binding site occupied with either N-methylanthraniloyl- (Mant) dGDP or MantdGTP. Taken together, these results identify a conformationally sensitive reporter site on the Cdc42Hs molecule that is located some distance away from the guanine nucleotide binding site but nonetheless provides a highly sensitive monitor for GTP-binding, GTPase activity, and the interactions of key regulatory proteins.