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J. Biol. Chem., Vol. 276, Issue 26, 23873-23880, June 29, 2001

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The Function of Interdomain Interactions in Controlling Nucleotide Exchange Rates in Transducin^*

Ethan P. Marin, A. Gopala Krishna^§, Vincent Archambault, Eugene Simuni^§¶, Wing-Yee Fu^§, and Thomas P. Sakmar^§**

From the ^§ Howard Hughes Medical Institute,  Laboratory of Molecular Biology and Biochemistry, The Rockefeller University, New York, New York 10021

Received for publication, February 7, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The intramolecular contacts in heterotrimeric G proteins that determine the rates of basal and receptor-stimulated nucleotide exchange are not fully understood. The  subunit of heterotrimeric G proteins consists of two domains: a Ras-like domain with structural homology to the monomeric G protein Ras and a helical domain comprised of six -helices. The bound nucleotide lies in a deep cleft between the two domains. Exchange of the bound nucleotide may involve opening of this cleft. Thus interactions between the domains may affect the rate of nucleotide exchange in G proteins. We have tested this hypothesis in the  subunit of the rod cell G protein transducin (G_t). Site-directed mutations were prepared in a series of residues located at the interdomain interface. The proteins were expressed in vitro in a reticulocyte lysate system. The rates of basal and rhodopsin-catalyzed nucleotide exchange were determined using a trypsin digestion assay specifically adapted for kinetic measurements. Charge-altering substitutions of two residues at the interdomain interface, Lys²⁷³ and Lys²⁷⁶, increased basal nucleotide exchange rates modestly (5-10-fold). However, we found no evidence that interactions spanning the two domains in G_t significantly affected either basal or rhodopsin-catalyzed nucleotide exchange rates. These results suggest that opening of the interdomain cleft is not an energetic barrier to nucleotide exchange in G_t. Experiments with G_i1 suggest by comparison that the organization and function of the interdomain region differ among various G protein subtypes.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Transducin (G_t)¹ is the heterotrimeric guanine-nucleotide binding regulatory protein (G protein) of the rod cell. In the GDP-bound state, the  subunit of transducin (G_t) does not signal. Following exchange of GDP for GTP, which is catalyzed by photoactivated rhodopsin (R*), G_t signals to its downstream effector. Physiologically, the detection of dim light requires that the basal nucleotide exchange rates of G_t be very low to prevent background noise and that R*-catalyzed exchange be very efficient, to ensure consistent detection and amplification of light signals.

G_t consists of two domains: a Ras-like domain, which is structurally similar to the monomeric G protein p21^ras (Ras), and a helical domain, which is unique to the heterotrimeric G proteins (1). The bound nucleotide lies in a deep cleft between the two domains (Fig. 1A). Although the discovery of this arrangement initially prompted speculation that nucleotide exchange would involve opening of the interdomain cleft (1), and that interactions between the domains might affect the rate of nucleotide exchange, the intramolecular contacts in G_t that determine the rates of nucleotide exchange remain to be elucidated.

There is evidence that structures that do not directly interact with the nucleotide can modulate both the basal and the receptor-catalyzed rates of nucleotide exchange. For example, although the direct contacts between the protein and the nucleotide are virtually the same in closely related subtypes of G protein, the rates of basal nucleotide exchange vary widely. Furthermore, R* tremendously accelerates nucleotide exchange, yet available evidence indicates that it does not directly contact the nucleotide binding site (2).

One of the regions of the G protein hypothesized to control nucleotide release rates without directly contacting the nucleotide is the interdomain interface. The interface is composed of contacts adjacent to the nucleotide and also interactions that are distant from the nucleotide (Fig. 1). These latter interactions involve residues located on the D-E loop (amino acid residues 139-147 of G_t) of the helical domain, the Switch III region (residues 227-238), and the G region (residues 269-277) of the Ras-like domain (Fig. 1B). These interactions have been implicated in mediating the lower rate of dissociation of GTPS relative to GDP in G_i1 (3) and in affecting the basal nucleotide exchange rates in G_i1 (4) and G_s (5, 6). Additionally, studies in G_s have suggested that interdomain interactions are involved in mediating rapid nucleotide exchange catalyzed by the ₂-adrenergic receptor (7, 8).

We have studied the function of several residues of G_t that are located at the interdomain interface but do not contact the nucleotide. A number of site-directed mutations of these residues were constructed. The well documented difficulties in expressing and purifying recombinant G_t were overcome by expressing the mutant proteins in vitro in a rabbit reticulocyte lysate system. The rates of basal and R*-catalyzed nucleotide exchange were measured using a trypsin digestion assay specifically adapted for kinetic measurements. Alteration of two conserved lysine residues, Lys²⁷³ and Lys²⁷⁶, increased the rate of spontaneous nucleotide exchange 5-10-fold. However, in contrast to what would be predicted based on published structural and biochemical studies, we found no evidence that interactions that span the domain interface were important in either maintaining the low rate of basal nucleotide exchange or in supporting the high rate of R*-catalyzed exchange in G_t. Experiments with G_i1 demonstrated that conserved lysine residues serve different roles in G_i1 than in G_t. In general, the function of the interdomain region appears to differ among various G protein subtypes.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Reagents-- Buffers, nucleotides, protease inhibitors, and salts were from Sigma or Roche Molecular Biochemicals. [³⁵S]Methionine was purchased from PerkinElmer Life Sciences. TPCK-treated trypsin was from Worthington Biochemicals. Synthetic oligonucleotides were purchased from Genelink, Inc. DNA sequencing was carried out using BigDye Terminator Cycle sequencing in the DNA sequencing core facility at the Rockefeller University.

Site-directed Mutagenesis of G_t and G_i1-- The parent for all G_t constructs was pGEM2sT, the synthetic bovine G_t gene (9) cloned into the pGEM2 plasmid under control of a SP6 promoter. Site-directed point mutations were prepared using the QuikChange method (Stratagene). For each mutant, two complementary primers were designed that coded for the desired mutation as well as 10-15 bases of complementary sequence on either side of the mutation site. Most amino acid substitutions could be accomplished with two or less nucleotide changes. The total length of each primer was 20-30 bases. The mutagenesis reaction (50 µl final) consisted of 5 ng of template plasmid, 1 µl of cloned Pfu polymerase (2.5 units/µl) (Stratagene), 5 µl of 10× Pfu buffer (Stratagene), 250 nM concentration of each primer, 800 µM concentration dNTP mix. The reactions were thermocycled in a GeneAmp 9600 (PerkinElmer Life Sciences) thermocycler with the following program: 3 min at 95 °C, 14 cycles of 30 s at 95 °C, 1 min at 55 °C, 8 min at 68 °C, and then 10 min at 72 °C. Amplification of the mutated plasmid was verified by running 1 µl of the reaction on a 1% agarose gel. The parental plasmid was selectively digested using the restriction enzyme DpnI (New England Biolabs) for 1.5 h at 37 °C. Since DpnI digests only methylated DNA, the wild-type template plasmid was restricted. The DpnI-treated mutant plasmid was transformed into chemically competent bacteria (subcloning efficiency DH-5 (Life Technologies, Inc.) or OneShot TOP10 (Invitrogen)). Generally the ratio of recombinant transformants following transformations with QuikChange reactions done with primers compared with control reactions run without primers was >50:1. All constructs were verified by automated DNA sequencing of the entire coding region of G_t.

The parent for all G_i1 constructs was pGEM2G_i1, the G_i1 gene cloned into the pGEM2 plasmid under control of a SP6 promoter. To improve expression, a 217-base pair segment of the 5'-untranslated region was removed by QuikChange mutagenesis.

Transcription and Translation of G_t in Vitro-- Recombinant G_t subunits were prepared using the TNT Quick Coupled rabbit reticulocyte lysate transcription/translation kit (Promega). For each translation, 20 µl of lysate mix was combined with 4 µl of DNA (0.5 µg total) and 1 µl of ~9 µM [³⁵S]methionine (PerkinElmer Life Sciences) at a specific activity of approximately ~1250 Ci/mmol. The reactions were incubated at 30 °C for 90 min. Subsequent manipulations were performed on ice or at 4 °C. The translated products were passed over BioSpin 6 gel filtration spin columns (Bio-Rad) twice consecutively to remove excess nucleotides and [³⁵S]methionine. The volume of each reaction was then adjusted to 100 µl in Buffer A (5 mM Tris-HCl, pH 7.5, 150 mM NaCl, 2 mM MgCl₂, 1 mM dithiothreitol, 0.01% (w/v) n-dodecyl--D-maltoside (DM)). If the reaction was to be studied in a R*-catalyzed assay, G_t was added to a final concentration of 30 nM. Every experiment was performed using freshly translated material.

Trypsin Proteolysis and Analysis-- The digestion procedure was adapted from Garcia et al. (10). Aliquots (8 µl) of the reaction mix were withdrawn and mixed with 1.5 µl of digest buffer (5% Lubrol, 2 mM GDP, 1 mg/ml TPCK trypsin) or digest control buffer (5% Lubrol, 2 mM GDP). The digests were incubated on ice for 30 min (G_t samples) or for 5 min (G_i1 samples). Digestion was terminated by the addition of 2.5 µl of termination solution (10 mg/ml aprotinin, 10 µM phenylmethylsulfonyl fluoride), followed by 6 µl of 3× SDS sample buffer (New England Biolabs). Samples were frequently stored at 20° for up to 48 h. Subsequently, the samples were boiled for 3 min, and the protein fragments were resolved by SDS-PAGE on 15% pre-cast Tris-HCl minigels (Bio-Rad). Following electrophoresis, the gels were fixed for >30 min in 50% MeOH/10% acetic acid and then soaked for 5 min in 7% methanol/7% acetic acid/2% glycerol. The gels were vacuum-dried onto filter paper (Whatman), and the radiolabeled G_t bands were visualized by exposing the gels to a storage phosphor screen (Molecular Dynamics) for 1-7 days.

Data Analysis of Trypsin Proteolyis Patterns-- The phosphor storage screens were scanned using a Storm Imager machine (Molecular Dynamics) at 200-micron resolution. The resulting images of the gels were analyzed using ImageQuant software (Molecular Dynamics). Lines were drawn down the center of each lane on the gel image, perpendicular to the bands. The intensity of each pixel along the line was determined as the average of 5-10 pixels on either side of the line. In this way, most of the width of each lane was considered. Any defect in the gel was avoided by careful placement of the lines. The intensity of each pixel was plotted as a function of position along the line so that the bands on the gel were represented as peaks on the graph. The base line of the graph was set so as to exclude nonspecific background intensity. GDP-bound G_t yielded a ~23-kDa band, whereas GTPS-bound or GDP/AlF-bound Gt yielded a ~34-kDa band. The areas of the peaks corresponding to the ~23- and ~34-kDa bands were then calculated and recorded in an Excel spreadsheet. The fraction of functional G_t in the GTPS-bound state in a given lane was determined by the following formula: (area of ~34-kDa peak)/((1.4 × area of ~23-kDa peak) + (area of ~34-kDa peak)). The area of the ~23-kDa peak was multiplied by 1.4 to normalize for the smaller number of methionines in the smaller fragment relative to those in the ~34-kDa fragment. This coefficient was adjusted (to 1.3) for analysis of samples in which Met²²⁸ was replaced. Activation kinetics were analyzed by plotting the fraction of G_t activated as a function of time. In the basal exchange rate assays, the data were fit to a single exponential rise to a maximum equation of the form: percent activated = c + 100(1  exp(kt)). The apparent rate constants derived from the fits are presented in Table I.

For experiments conducted with G_i1, the intensity of the GDP-dependent band could not be determined reliably due to nonspecific background intensities in the region of the gel where the GDP band migrated. This background was present even in undigested samples of G_i1. Since the ratio of the GTPS-dependent band to the sum of the GTPS and GDP bands could not be determined, the intensity of the GTPS band was expressed as a fraction of the total intensity in each lane. Activation time courses were plotted as the change in this intensity over time. For fully activated G_i1, the GTPS band accounted for roughly 25-30% of the intensity in the lane.

Control Reactions for Trypsin Proteolysis of G_t-- Control reactions were performed on each sample to check the quantity and apparent molecular mass of the expressed protein, as well as the digest patterns following incubation with GDP and GDP/AlF. From each 100-µl sample of translated Gt, 30 µl was removed and combined with 100 µM GDP. Of this 30 µl, one 8-µl aliquot was digested ("+GDP"); another 8-µl aliquot was mock-digested with digest control buffer ("undigested"). The remaining 14 µl was combined with a final concentration of 0.17 mM AlCl₃ and 10 mM NaF added from separate 30× stock solutions. Following a 10-min incubation at room temperature, an 8-µl aliquot was removed and digested ("+GDP/AlF").

Basal Nucleotide Exchange Time Course of G_t-- Samples (70 µl each) of translated G_t or mutant G_t in Buffer A were quickly warmed to room temperature in a water bath, and GTPS was added to a final concentration of 100 µM. Aliquots (8 µl) were withdrawn at 1, 2, 3, 4, and 6 h following GTPS addition and digested. The activity of the protein following 6-h incubation at room temperature was investigated by addition of R* and G_t (30 nM each, see below) and incubation under room light for 20 min. A final 8-µl aliquot was then removed and digested. For G_i1 samples, which activated very quickly, aliquots were taken for a 180-min time course, and the R*/G_t mix was not added.

R*/G_t-catalyzed Activation Time Course of G_t-- Samples (70 µl each) of translated G_t or mutant G_t in Buffer A were quickly warmed to room temperature in a water bath. A mixture of R* and GTPS (4 µl) was added to the sample yielding a final concentration of 30 nM R* and 14 µM GTPS. Immediately before addition to the reaction, the rhodopsin was photolyzed by illumination for 15 s with a fiber optic cable connected to a Dolan Jenner lamp equipped with a >495-nm long-pass filter. The samples were incubated at room temperature under illumination. Aliquots (8 µl) were withdrawn and digested at 1, 2, 3, 5, 10, and 20 min following addition of the R*/GTPS mix.

Preparation of G_t, G_t, G_t, and Rhodopsin from Bovine Retinas-- G_t was prepared from frozen bovine retinas (Lawson, Inc., Lincoln, NE) using standard techniques (11, 12) as described previously (13). G_t and G_t were isolated from holo-G_t as described previously (13) using a Hitachi LC-organizer high performance liquid chromatography system with a 1-ml Hi-Trap Blue-Sepharose column (Amersham Pharmacia Biotech). The proteins were eluted from the column by applying a 0-2 M NaCl gradient. G_t concentrations were determined using the Bio-Rad protein assay reagent according to manufacturer's instructions. Concentrations of G_t and G_t samples were determined by spectrofluorometric titration as described previously (14). The proteins were stored at 20 °C in a 50% glycerol buffer until use. Purified recombinant G_i1 expressed in Sf9 cells was provided by Dr. S. Graber (West Virginia University) (15). Urea-washed disc membranes, the gift of Dr. K. C. Min, were prepared as described elsewhere (16). The membranes were solubilized in 1% DM, and insoluble material was removed by centrifugation. The resulting solubilized rhodopsin displayed a A₂₈₀/A₅₀₀ spectral ratio of <1.8 and a concentration of ~8 µM.

Fluorescence Activation Traces of G_t and G_i1-- The assay was performed essentially as described previously (14) using a Spex Fluorolog spectrofluorometer equipped with a 150-watt xenon arc lamp. A solution of 250 nM concentration of G_t or G_i1 was prepared in fluorescence buffer (100 mM NaCl, 10 mM Tris-HCl, pH 6.9, 2 mM MgCl₂, 1 mM dithiothreitol, 0.01% (w/v) DM). Solution (0.5 ml) was placed in a quartz microcuvette and loaded into the thermojacketed cuvette holder equipped with a magnetic stirrer at 25 °C. Protein fluorescence was excited at 300 nm with 2-nm slit width, and emission intensity was collected at 345 nm with a 12-nm slit width. The activation reaction was initiated by injecting 15 µl of GTPS solution to a final concentration of 5 µM. Nucleotide exchange was observed as an increase in the intensity of tryptophan fluorescence emission that results from conformational changes in Trp²⁰⁷ that occur upon binding GTP (17).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

A series of site-directed mutants of G_t with replacements of residues located at the interdomain interface was prepared. Sites were selected for mutation based on their position in the crystal structure of GDP-bound G_t (18) (Fig. 1), as well as their importance in G_i1 and G_s suggested by published studies (see below). Selected residues were replaced with alanine, or with the amino acid present in the homologous position of either G_i1 or G_s, and/or with amino acids reported to cause altered phenotypes in G_i1 or G_s. For each mutant G_t, the rates of both basal (i.e. uncatalyzed) and R*-catalyzed nucleotide exchange were measured.

View this table: [in this window] [in a new window]   Table I Rate constants for basal nucleotide exchange measured for Gt and Gt mutants

View larger version (60K): [in this window] [in a new window]   Fig. 1.   Crystal structures of the interdomain interface of GDP-bound Gt (Protein Data Bank number 1tag) and Gi1 (Protein Data Bank number 1gdd). Side chains of residues that have been mutated in this study are shown in ball-and-stick representation. Side chains of residues in the helical domain are yellow, those from Switch III are green, and those from the G region are orange. The figures were prepared with Molscript (32) and Raster3D (33). A, overall view of the Gt protein. The Ras-like domain is blue, the helical domain is red, and the GDP is purple. The nucleotide resides in a cleft between the two domains. The side chains of many of the residues studied in this report potentially interact with each other across the interdomain interface, but do not directly contact the nucleotide. B, the protein has been rotated 90° about the horizontal axis relative to A, and the interdomain region is enlarged. Hydrogen bonds are shown as dotted lines. The position of Lys276 in the GTPS-bound Gt structure is shown as an outline. C, the structure of GDP-bound Gi1 from a similar viewpoint as in B. The corresponding amino acid number in Gt is given in parentheses.

Expression of G_t in Vitro and Trypsin Digestion Assay of Nucleotide Binding and Exchange-- All G_t constructs were expressed in vitro in a coupled transcription/translation rabbit reticulocyte lysate system. Time course experiments confirmed that in vitro expression was maximal in 90 min (not shown). Typical reactions with plasmids encoding G_t or G_t mutant genes yielded one major protein band at the expected molecular mass of ~40-kDa (Fig. 2). Generally 80-90% of the total intensity in the lane was in the one band. Expressed G_t was digested with trypsin following various treatments. Inactive, GDP-bound G_t was prepared by incubating in vitro translated G_t with 100 µM GDP. Trypsin proteolysis of this sample resulted in the formation of a ~23-kDa fragment (Fig. 2). The active conformation was prepared by incubating G_t with GDP and AlF. AlF is known to bind to Gt-GDP and simulate the presence of the -phosphate of GTP. Therefore, a conformation nearly identical to the activated GTP-bound conformation is induced (19). Digestion of the AlF-activated Gt yielded a ~34-kDa band and no ~23-kDa band. Similarly, activation of G_t with GTPS yielded an identical ~34-kDa band following trypsin digestion (Fig. 2).

View larger version (67K): [in this window] [in a new window]   Fig. 2.   Determination of nucleotide exchange rates by analysis of trypsin digestion patterns. Gt was expressed in vitro in a rabbit reticulocyte lysate system and metabolically labeled with [35S]methionine. Translated material was passed twice over gel filtration spin columns to remove free nucleotides and then treated with various conditions as indicated. Aliquots were removed and digested with trypsin for 30 min on ice, except where indicated otherwise. The resulting fragments were analyzed by SDS-PAGE and visualized by phosphorimaging. A, basal nucleotide exchange. Translated Gt was mixed with 100 µM GTPS. Aliquots were removed and digested at 1, 2, 3, 4, and 6 h. Following collection of the 6-h aliquot, R* and Gt were added to a final concentration of 30 nM each. After a 20-min incubation, an aliquot was removed and digested (+rho/). Control reactions show undigested Gt and Gt digested following incubation with 100 µM GDP or GDP/AlF. B, R*/Gt-catalyzed nucleotide exchange. Translated Gt was mixed with 30 nM R*, 30 nM Gt, and 14 µM GTPS. Aliquots were removed and digested at 1, 2, 3, 5, 10, and 20 min. Control reactions were performed as in A.

In all cases, trypsin proteolysis produced a variety of lower molecular weight fragments. Some of these were the smaller polypeptides that were cleaved to produced the ~23- and ~34-kDa fragments. Others likely resulted from extensive proteolysis of protein that was not properly folded. This is consistent with the large number of potential trypsin sites present in the primary structure of G_t and the relatively small number of accessible sites in the properly folded tertiary structure. The fraction of the total pool of translated G_t that was properly folded and functional was estimated from the ratio of the intensities of the ~34-kDa band following GDP/AlF treatment (i.e. the properly folded, activable pool) to that of the ~40-kDa band in the undigested sample (i.e. the total pool of translated full-length protein). This ratio was generally about 0.15 for wild-type G_t and severalfold higher for G_i1. This result parallels the functional expression levels of G_t and G_i1 that have been observed in other heterologous expression systems. Thus, in vitro expression might serve as a rapid and useful predictor of the expression level of a given G protein construct in other systems.

The rate of trypsin proteolysis of GDP- and GDP/AlF-treated Gt and the stabilities of the resulting fragments were investigated by digestion time course experiments. Both the ~23- and ~34-kDa bands formed completely by 20 min and remained stable until at least 40 min in the presence of trypsin (data not shown). We therefore chose to stop the digestion after 30 min in all experiments. Similar experiments with G_i1 indicated that digestion occurred more quickly. Accordingly, digests were run for 5 min with G_i1 samples.

Three major sites of trypsin proteolysis in properly folded G_t have been identified: Lys¹⁸, Arg²⁰⁴, and Arg³¹⁰ (20). Using site-directed mutagenesis, we investigated which of these sites were contributing to the fragments produced under the digestion conditions used in this study. The mutants K18A, R204H, and R310A were prepared, expressed in vitro, and digested following treatment with either GDP or GDP/AlF.

Formation of the ~23-kDa band following treatment with GDP was altered only by the R204H mutation (not shown). Arg²⁰⁴ is the site at which cleavage occurred in the GDP form to yield the ~23-kDa fragment, but which was protected from digestion in the GTPS-bound conformation. Arg²⁰⁴ is located in the Switch II region of Gt, and crystal structures confirm that it moves from a surface exposed to a buried position upon activation (1, 18). Formation of the ~34-kDa band following activation with GDP/AlF was affected only by the R310A mutation (not shown). Thus Arg³¹⁰ appears to be the site at which trypsin proteolysis occurred to yield the ~34-kDa fragment. The K18A mutant was indistinguishable from wild-type; digestion at Lys¹⁸ does not appear to contribute to the formation of either fragment under the conditions used (not shown). Using these results, the ratio of the number of methionines in the ~34-kDa band relative to the ~23-kDa band was determined to be 1.4. This factor was used to normalize the intensity of the ~23-kDa band in calculating the fraction of activated Gt in each aliquot.

The fraction of G_t in the active conformation in a partially activated sample was calculated as the intensity of the ~34-kDa band (i.e. the activated G_t) divided by the sum of the intensities of the ~23- and ~34-kDa bands. The sum of the intensities of the ~23- and ~34-kDa bands is indicative of the total pool of functional G_t in the sample. This calculation is therefore internally normalized to the total amount of functional G_t in each aliquot and does not require comparison with the ~34-kDa band of a separate sample (such as one in a completely activated lane).

The rate of nucleotide exchange of each sample was determined by monitoring the fraction of G_t in the active conformation at specific times following addition of GTPS. In the basal exchange rate assay, G_t was 31% activated at 6 h following GTPS addition (Fig. 2A). The activity of G_t following the 6-h incubation was confirmed by demonstrating that addition of rhodopsin and G_t could fully activate the remaining G_t (Fig. 2). In comparison, G_t A322S (a mutant known to display high nucleotide exchange rates in G_i1 (21) and G_s (22)) was 100% activated in less than 1 h.²

In R*-catalyzed assays, G_t was nearly 100% activated in 20 min (Fig. 2B). Under the conditions of the assay, the rate of G_t activation was found to be dependent on the concentration of rhodopsin from 0-100 nM and sensitive to the presence of added G_t (not shown). Some residual activation was observed in the absence of added G_t, probably as a result of small quantities of G present in the reticulocyte lysate or the rhodopsin preparations. No rhodopsin-catalyzed activation was observed in the dark (data not shown). Additionally, mutant G348P, which was previously reported to be unable to bind rhodopsin (23), was not activated by R* in this assay (not shown). To our knowledge, this is the first report in which trypsin proteolysis of in vitro translated G_t has been used to measure the kinetics of R*-catalyzed nucleotide exchange.

Analysis of Single Amino Acid Replacements in the Interdomain Interface of G_t-- Two residues in the helical domain, Ser¹⁴⁰ and Gln¹⁴³, extend toward and interact with residues from the Ras-like domain. Ser¹⁴⁰ was replaced with alanine, arginine (the homologous residue in G_i1), and asparagine (the homologous residue in G_s). Gln¹⁴³ was mutated to alanine and was also combined with S140A in a S140A/Q143A double mutant. None of these mutations substantially altered the rate of basal (Fig. 3A, Table I) or R*-catalyzed nucleotide exchange (not shown).

View larger version (33K): [in this window] [in a new window]   Fig. 3.   Basal nucleotide exchange time courses of Gt and Gt mutants. The percent activation at each time point was determined by analysis of trypsin digestion patterns as described under "Experimental Procedures." The time 0 data point is calculated from protein mixed with 100 µM GDP for 10 min. Each data point is the average of three to five independent experiments; wild-type Gt was assayed 26 times. Error bars depict ± 2 × S.E. The solid lines represent fits to a one-component exponential rise function. A, mutations of residues located on the helical domain. B and C, mutations of residues located on Switch III. D, mutations of residues located in the G region.

Several residues in the Switch III region of G_t were altered by site-directed mutagenesis. The crystal structure suggests that Asp²²⁷ participates in hydrogen bonds with both Ser¹⁴⁰ and Lys²⁷⁶ (Fig. 1). This residue was replaced with both asparagine (the homologous residue in G_s) and with alanine. The adjacent Met²²⁸ was replaced with alanine, leucine (the equivalent in G_i1), and glutamine (the equivalent of a mutation in G_i1 reported to increase the GDP release rate (4)). Val²³¹ was replaced with alanine and with tryptophan. The V231W mutant was prepared to simulate a naturally occurring mutation in the homologous residue of G_s, Arg²⁵⁸, which was found in a patient with Albright's hereditary osteodystrophy (5). None of these mutations was found to substantially alter either the basal (Fig. 3, B and C, and Table I) or the R*-catalyzed activation rates (not shown). However, several mutations caused slight (~2-fold), but reproducible reductions in the basal nucleotide exchange rate. These mutations include D227A, D227N, V231A, and M228L (Table I).

Three lysine residues in the G region of G_t were studied. Lys²⁷³ and Lys²⁷⁶ are oriented toward Asp²²⁷ of Switch III (Fig. 1). In addition, the crystal structure of G_t-GDP indicates that Lys²⁷⁶ forms hydrogen bonds with Ser¹⁴⁰ of the helical domain (Fig. 1). Lys²⁷⁵ extends out toward the solvent (Fig. 1). Lys²⁷³, Lys²⁷⁵, and Lys²⁷⁶ were each replaced with alanine. In addition, Lys²⁷⁶ was replaced with glutamic acid and with arginine. The K273A, K276A, and K276E mutations all resulted in significantly increased rates of basal nucleotide exchange, from 5-10-fold above wild-type (Fig. 3D). The K276R mutation did not affect nucleotide exchange rates, nor did mutation of Lys²⁷⁵. None of the mutations substantially altered the rate of R*-catalyzed nucleotide exchange (not shown).

Analysis of Double Amino Acid Replacements-- To probe for functional interactions between pairs of residues, a series of double amino acid replacements was prepared by site-directed mutagenesis (Table I). The K276A/D227N, K276A/D227A, and K273A/D227N double replacements all displayed slower basal rates of nucleotide exchange than the corresponding single replacements of Lys²⁷⁶ or Lys²⁷³ (Fig. 4). The double mutants displayed faster exchange kinetics than wild-type G_t, however. Combining amino acid replacements at positions Lys²⁷⁶ and Ser¹⁴⁰ revealed that the effects on basal exchange rates of each individual mutation were roughly additive (Fig. 4). A combination of amino acid replacements at positions 140 and 227, S140A/D227N, had similar exchange kinetics to that of wild-type G_t (Table I).

View larger version (27K): [in this window] [in a new window]   Fig. 4.   Time courses of basal nucleotide exchange of double amino acid replacement mutants in Gt. The experiments were conducted as described in the legends to Figs. 2 and 3. A, the K276A replacement was combined with mutations of residues that are indicated in the crystal structure to interact with Lys276. The basal nucleotide exchange rates were determined. Data for K276A and D227N are replotted from Fig. 3 for comparison with the double mutants. B, the K273A mutation was combined with the D227N mutation and the basal activation rate determined. Data for K273A and D227N are re-plotted from Fig. 3 for comparison.

Analysis of G_i1 Mutants-- G_i1 is 66% identical to G_t at the primary structure level and very similar at the tertiary structure level (Fig. 1C). However, the basal nucleotide exchange rate of G_i1 has been reported to be significantly higher than that of G_t (24). We confirmed this observation in studies with recombinant G_i1 and retinal G_t in a fluorescence activation assay (Fig. 5A). The rate of basal nucleotide exchange as monitored by increases in fluorescence was much greater in G_i1 than in G_t at 25 °C. However, the rate and the magnitude of fluorescence change were comparable when each protein was fully activated with excess AlF.

View larger version (26K): [in this window] [in a new window]   Fig. 5.   Basal nucleotide exchange time course of Gi1 and mutants of Gi1. A, fluorescence activation traces of Gi1 (expressed and purified from Sf9 cells) and Gt (purified from bovine retinas) are superimposed. Fluorescence intensity change is plotted as a function of time. At 200 s, GTPS was added to the cuvette to start the reaction. At the times indicated by the arrows, AlCl3 and NaF were added from separate stock solutions to form AlF. B, basal nucleotide exchange rates of Gi1 and mutants expressed in vitro and assayed by analysis of trypsin digestion products. The intensity of the GTP-dependent band is expressed as a percent of the total intensity in the lane. Each point represents the mean of at least three experiments ± 2 × S.E.

G_i1 was expressed in vitro and studied by trypsin digestion. SDS-PAGE analysis indicated that a ~38-kDa band was produced following full activation with either GTPS or GDP/AlF and that a smaller fragment resulted from digestion in the presence of GDP. The intensity of the smaller GDP-dependent band of G_i1 could not be accurately quantitated due to reproducible nonspecific background in that portion of the gel. Therefore, the method for determining the fraction of in vitro translated G_i1 activated in an aliquot was modified from the "ratio" method used for G_t. In each aliquot, the intensity of the ~38-kDa band was determined as a fraction of the total intensity in the lane, and the time course of activation was plotted as a change in this fraction over time (Fig. 5B). This analysis suggests that basal nucleotide exchange of GDP for GTPS by G_i1 was complete in 1 h, significantly faster than that by G_t, which was only 32% complete in 6 h. The t_1/2 for activation of G_i1 (~20 min) was comparable in both the fluorescence and the trypsin protection assays (Fig. 5).

A series of site-directed mutants was prepared in G_i1 to probe for similarities between the functions of interdomain residues in G_i1 and G_t. The equivalent of Lys²⁷³ and Lys²⁷⁶ of G_t are conserved in G_i1 as Lys²⁷⁷ and Lys²⁸⁰, respectively (Fig. 1). Both were replaced with alanine, expressed in vitro, and the basal nucleotide exchange rates of the resulting mutants were determined. Neither of these mutations (K277A and K280A) increased the basal activation rate of G_i1 appreciably (Fig. 5B). Additionally Arg¹⁴⁴ of G_i1, the homolog of Ser¹⁴⁰ in G_t, was replaced with serine. Since the Ser¹⁴⁰ of G_t forms hydrogen bonds with Lys²⁷⁶, it was hypothesized that in G_i1 the replacement of Arg¹⁴⁴ with serine might alter the position of Lys²⁸⁰ (Lys²⁷⁶ in G_t) to resemble the G_t conformation and lower the basal rate of nucleotide exchange. However, the opposite was observed. The G_i1 R144S mutant exhibited accelerated nucleotide exchange rates (Fig. 5B), consistent with previous reports of mutagenesis at the Arg¹⁴⁴ position (4).

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Analysis of Trypsin Digest Products of in Vitro Translated G_t to Evaluate Nucleotide Exchange Kinetics-- Since G_t is refractory to expression in bacteria (24) and is cumbersome to express in insect cells (16), we chose to express G_t in vitro and analyze nucleotide exchange rates using trypsin digestion. The pattern of proteolytic fragments resulting from trypsin digestion directly reflects the conformation of G_t and, therefore, the identity of the bound nucleotide (25, 26). Trypsin proteolysis of expressed G_t yielded ~23-kDa fragments following incubation with GDP and ~34-kDa fragments following activation with either AlF or GTPS (Fig. 2). These observations are consistent with previously published results (23, 27).

Trypsin proteolysis of in vitro translated G_t allowed for the precise quantitation of basal and R*-catalyzed nucleotide exchange rates. By using both the ~23- and the ~34-kDa bands, the calculation of nucleotide exchange rates was internally normalized and took into consideration both the GDP- and the GTPS-bound fractions. As a result, the data were very reproducible, and samples in which only a small fraction of the total expressed protein was functional could be analyzed. Additionally, the methodology was rapid enough to allow for the characterization of relatively large numbers of mutants in parallel.

The kinetic parameters for G_t activation derived from the trypsin digestion assay are consistent with data reported using other traditional methodologies (24, 28) as well as with analysis of retinal G_t studied with the fluorescence activation assay (Fig. 5). Additionally, analysis of the mutant A322S by trypsin proteolysis indicated that the basal rate of activation was >60-fold greater than that of wild-type G_t (not shown). This result is also in agreement with published data using different methodologies on the analogous mutation in G_s and in G_i1 (21, 22).

Several control reactions demonstrated the fidelity of the R*-catalyzed assay. The rate of rhodopsin-dependent activation was sensitive to light (not shown), to the concentration of rhodopsin from 0-100 nM, and to the presence of added G_t. A mutation near the carboxyl terminus of G_t (G348P) that was previously reported to disrupt rhodopsin-transducin interactions (23) was not activated (not shown).

This expression and assay system offers additional flexibility not explored in the present work. Expressed recombinant rhodopsin could be used in place of retinal rhodopsin to test the combined effects of mutations of rhodopsin and transducin.³ Other proteins (e.g. G_t mutants, regulator of G protein signaling proteins, etc.), could be co-translated with G_t in the in vitro system (25). Furthermore, this system will likely prove useful in the kinetic characterization of other G protein subtypes, such as cone transducin, which are difficult to express heterologously.

Site-directed Mutation of Lys²⁷³ or Lys²⁷⁶ Increases Basal Nucleotide Exchange Rates-- Replacement of Lys²⁷⁶ or Lys²⁷³ with alanine increased the basal rate of nucleotide exchange ~5-fold in G_t (Fig. 3D, Table I). Replacement of the adjacent Lys²⁷⁵, which in the crystal structure is oriented toward the solvent (Fig. 1), had no effect. Replacement of Lys²⁷⁶ with a negatively charged glutamic acid increased basal nucleotide exchange rates more dramatically (~10-fold) than the neutral alanine replacement mutant. However, mutation to a positively charged arginine did not alter the activation rate. Together, these results suggest that the function of Lys²⁷⁶ and Lys²⁷³ is dependent on positive charge and orientation toward the interior of the protein. Interestingly, in the activated, GTPS-bound structure of G_t, Lys²⁷⁶ is rotated outward toward the solvent relative to the GDP-bound conformation (Fig. 1B). The K276A mutant may anticipate this active conformation.

Lys²⁷⁶ and Lys²⁷³ both lie in the third of four regions of G_t for which there is no homologous sequence in the monomeric G protein Ras. The four regions are known as Insert 1 through Insert 4 (1). Specific functions have been attributed to Insert 1 (the helical domain), Insert 2 (the Switch III region), and Insert 4 (which may interact with heptahelical receptors). The present work is the first to identify a functional role for residues in Insert 3 of G_t.

The structure of GDP-bound G_t reveals that Lys²⁷⁶ and Lys²⁷³ lie near to and possibly form ionic interactions with Asp²²⁷. This observation suggests that the reason for accelerated nucleotide exchange rates caused by mutations of Lys²⁷⁶ and Lys²⁷³ might involve disruption of interactions with Asp²²⁷. To test this hypothesis, the K276A and K273A mutations were combined with mutation of D227N to produce the K276A/D227N and K273A/D227N double mutants. If the effects of K276A were due solely to breaking of an interaction with Asp²²⁷, then the Lys²⁷⁶ mutation should not increase the rate of nucleotide exchange in the context of D227N or D227A mutants. Indeed, the increase in the basal rate of activation caused by the K276A and K273A mutations was reduced (from ~5-fold to ~2-fold relative to wild-type) when combined with D227N (Fig. 4). However, since the rate of the D227N mutant alone is 2-fold slower than that of wild-type, the effect of the K276A mutation is roughly the same (i.e. a ~5-fold increase in rate) whether introduced into a wild-type or a D227N background. Similar results were obtained with a D227A mutation. Thus, the origin of the increase in basal nucleotide exchange rates by the K276A and K273A mutation is not merely due to disruption of interactions with Asp²²⁷.

Lys²⁷⁶ also appears to interact across the interdomain interface with S140. Replacement of Ser¹⁴⁰ with alanine, which would have disrupted hydrogen bonding to K276A, did not affect the rate of activation. Thus, breaking of the putative Ser¹⁴⁰-Lys²⁷⁶ interaction does not fully explain the effects of the K276A mutation. There appear to be other unidentified requirements for positively charged side chains in the G region to maintain low basal rates of nucleotide exchange.

Mutation of Asp²²⁷ slows the rate of basal nucleotide exchange, both in the context of the wild-type protein as well as in the K276A mutant (Fig. 4). This is surprising since G_t has an extremely low rate of basal nucleotide exchange, as is demanded by the low background noise required for sensitive light detection by photoreceptors. Other mutations in the Switch III regions also appear to slightly reduce the basal rate of nucleotide exchange, including M228L and V231A (Fig. 3; Table I). The origins of these effects, which are relatively small, are unclear. Previously, it has been demonstrated that the entire Switch III region can be deleted from G_t without disrupting the ability to bind nucleotides (29). The rates of nucleotide exchange in these Switch III-deleted constructs were not characterized, but such studies might illuminate the role of Switch III in facilitating or impeding nucleotide exchange.

Interdomain Interactions in G_t Do Not Affect Basal or Rhodopsin-catalyzed Nucleotide Exchange Rates-- When the structure of G_t was determined, the nucleotide was found to reside in a deep cleft between the Ras-like domain and the helical domain (1). It was proposed that rhodopsin might accelerate the nucleotide exchange rate by opening the cleft (1, 30). Similarly, interactions between these domains could control the rate of basal nucleotide exchange. G_t has a very low rate of basal nucleotide exchange as compared with related G proteins and a very high rate of R*-catalyzed exchange. If interdomain interactions were important mediators of either of these processes, one might expect nucleotide exchange rates in G_t to be particularly sensitive to mutation of residues involved in those interactions. However, none of the G_t mutants characterized in this report significantly affected either basal or R*-catalyzed rates.

Close analyses of G protein structures indicate that opening of the interdomain cleft is not necessarily an energetic barrier to nucleotide release. The helical domain does not contribute many contacts to the nucleotide binding pocket, and certain monomeric G proteins, which do not have a helical domain at all, release GDP more slowly than some heterotrimeric G proteins subtypes (31). A crystal structure of the G_i1 mutant A326S, which releases GDP ~250-fold faster than wild-type G_i1, does not reveal any alteration in the interdomain interactions, suggesting that an open cleft is not a prerequisite of fast nucleotide exchange (21). In addition, the reported increases in nucleotide release rates resulting from mutations at the interdomain interface of G_i1 and G_s are relatively modest (~5-10-fold) as compared with mutations in other regions of G proteins hypothesized to be involved in regulating nucleotide exchange rates. For example, we observed >150-fold increases in nucleotide exchange rates in mutations of certain residues of the 5-helix of G_t, a structure implicated in the mechanism of rhodopsin-catalyzed activation (34). In summary, the opening of the interdomain cleft may not necessarily be a rate-determining step in nucleotide exchange in G proteins.

The Function of Residues at the Interdomain Interface Differs among G_t, G_i1, and G_s-- Many of the residues mutated in this study, such as Ser¹⁴⁰, Gln¹⁴³, Met²²⁸, and Val²³¹, have been previously found to alter basal or receptor-catalyzed nucleotide exchange rates in the related G proteins, G_s and G_i1. In G_s, a mutation in the Switch III region, R258W (corresponding to Val²³¹ in G_t), was found in a patient with Albright's hereditary osteodystrophy (5). Biochemical studies indicated that replacement of Arg²⁵⁸ to tryptophan and to alanine, as well as alteration of a proposed interacting residue, Gln¹⁷⁰ of the helical domain (corresponding to Gln¹⁴³ in G_t), led to increases in the basal nucleotide exchange rate (5, 6). These mutations were hypothesized to widen the interdomain cleft. Other studies in G_s found that mutation of Arg²⁵⁸ or Asn¹⁶⁷ (corresponding to Ser¹⁴⁰ in G_t) disrupted receptor-catalyzed activation (8), suggesting that the receptor induces structural changes that are communicated across the interdomain interface. In G_i1, mutation of either Leu²³² or Arg¹⁴⁴ (corresponding to Met²²⁸ and Ser¹⁴⁰, respectively, in G_t) increased the basal nucleotide exchange rate by disrupting a proposed interdomain hydrophobic interaction (4). The effects of mutating Arg¹⁴⁴ were corroborated by the results of the R144S mutant in the current work (Fig. 5B).

The residues analogous to those proposed to interact with each other across the interdomain interface in G_s and G_i1 are also potentially interacting in G_t (Fig. 1B). In many cases, however, the amino acids are not conserved. For example, Val²³¹ and Gln¹⁴³ of G_t, which correspond to the proposed interaction between Arg²⁵⁸ and Gln¹⁷⁰ in G_s, are adjacent. Ser¹⁴⁰ and Met²²⁸ (corresponding to Arg¹⁴⁴ and Leu²³² of G_i1) and Ser¹⁴⁰ and Asp²²⁷ (corresponding to the proposed interaction of Asn¹⁶⁷ and Asn²⁵⁴ of G_s (8)) are similarly adjacent. However, in contrast to the results with G_i1 and G_s, replacement of these residues in G_t did not affect nucleotide exchange rates. These results suggest that the interdomain interface residues are functionally different in G_t than in G_i1 and G_s. Counterintuitively, G_i1 and G_s, which exchange nucleotides faster than G_t, appear to have tighter and more sensitive interactions across the interdomain interface than those of G_t.

Both Lys²⁷³ and Lys²⁷⁶ of G_t are conserved in G_i1. However, the structure of GDP-bound G_i1 reveals that Lys²⁸⁰ (cognate to Lys²⁷⁶ of G_t) is oriented toward the solvent instead of toward the Switch III region as in G_t (Fig. 1C). Functionally, mutation of Lys²⁸⁰ and Lys²⁷⁷ to alanine did not lead to increases in nucleotide exchange rates in G_i1, as was observed in G_t. Thus, conserved residues, Lys²⁷⁶ and Lys²⁷³ of G_t, are found to serve different roles and to assume different structures in closely related G proteins.

In conclusion, the data in this report ascribe a role to Lys²⁷³ and Lys²⁷⁶ in the G region of G_t in maintaining low basal rates of nucleotide exchange. However, unlike in G_i1 and G_s, interactions that span the interdomain interface do not appear to be important in regulating either basal or rhodopsin-catalyzed nucleotide exchange rates. Differences exist in the organization of the interdomain interface among G_t, G_i1, and G_s, and even between conserved residues in G_i1 and G_t.

	FOOTNOTES

^* This work was supported in part by National Institutes of Health Training Grants GM07739 and GM07982 and by the Allene Reuss Memorial Trust.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ Current address: Harvard College, 2351 Harvard Yard Mail Center, Cambridge, MA 02138.

Current address: Regeneron Pharmaceuticals, 777 Old Saw Mill River Rd., Tarrytown, NY 10591.

^** Associate Investigator of the Howard Hughes Medical Institute. To whom correspondence should be addressed: Box 284, Rockefeller University, 1230 York Ave., New York, NY, 10021. Tel.: 212-327-8288; Fax: 212-327-7904; E-mail: sakmar@rockvax.rockefeller.edu.

Published, JBC Papers in Press, April 4, 2001, DOI 10.1074/jbc.M101197200

² E. P. Marin, unpublished observation.

³ E. P. Marin, W.-Y. Fu, and T. P. Sakmar, unpublished observation.

	ABBREVIATIONS

The abbreviations used are: G_t, transducin; G_t, subunit of transducin; G_t, subunits of transducin; DM, n-dodecyl--D-maltoside; G protein, guanine nucleotide-binding regulatory protein; R*, signaling active state of rhodopsin; Ras, p21^ras; GTPS, guanosine 5'-O-(thiotriphosphate); TPCK, L-1-tosylamido-2-phenylethyl chloromethyl ketone; PAGE, polyacrylamide gel electrophoresis.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES