Tài liệu Báo cáo Y học: The solution structure and activation of visual arrestin studied by small-angle X-ray scattering pot

The solution structure and activation of visual arrestin studied by small-angle X-ray scattering Brian H. Shilton 1 , J. Hugh McDowell 2 , W. Clay Smith 2 and Paul A. Hargrave 2,3 1 Department of Biochemistry, University of Western Ontario, London, Ontario, Canada; 2 Departments of Ophthalmology and 3 Biochemistry and Molecular Biology University of Florida, Gainesville, Florida, USA Visual arrestin is converted from a ÔbasalÕ state to an ÔactivatedÕ state by interaction with the phosphorylated C-terminus of photoactivated rhodopsin (R*), but the conformational changes in arrestin that lead to activation are unknown. Small-angle X-ray scattering (SAXS) was used to investigate the solution structure of arrestin and characterize changes attendant upon activation. Wild-type arrestin forms dimers with a dissociation constant of 60 l M . Small conformational changes, consistent with local movements of loops or the mobile N- or C-termini of arrestin, were observed in the presence of a phospho- peptide corresponding to the C-terminus of rhodopsin, and with an R175Q mutant. Because both the phospho- peptide and the R175Q mutation promote binding to unphosphorylated R*, we conclude that arrestin is acti- vated by subtle conformational changes. Most of the arrestin will be in a dimeric state in vivo.Usingthe arrestin structure as a guide [Hirsch, J.A., Schubert, C., Gurevich, V.V. & Sigler, P.B. (1999) Cell 97, 257–269], we have identified a model for the arrestin dimer that is consistent with our SAXS data. In this model, dimeriza- tion is mediated by the C-terminal domain of arrestin, leaving the N-terminal domains free for interaction with phosphorylated R*. Keywords: visual arrestin; rhodopsin; G-protein coupled receptor signalling; small-angle X-ray scattering; solution structure. The first event in the visual cycle is activation of rhodopsin by light. Photoactivated rhodopsin (R*) initiates a signal transduction cascade that culminates in membrane hyper- polarization and the sensation of light (reviewed in [1]). The sensitivity of the system requires that the signal transmitted by R* be rapidly attenuated. This is accomplished by a two- step process involving phosphorylation of the C-terminus of R* and binding by arrestin. Phosphorylation somewhat decreases the ability of R* to signal transducin. Rapid shut- off of R* signalling is then accomplished by binding of arrestin to photoactivated phosphorylated rhodopsin (R*P [2–5]). Arrestin plays a critical role in visual signalling by completely blocking the ability of R*P to bind and activate transducin. Arrestin is present in rod cells at high concen- trations [6] and therefore a mechanism must exist that prevents arrestin from inappropriately associating with R*. In fact, arrestin shows very little propensity to bind to R* until the C-terminal region of R* becomes phosphorylated. Thus, the C-terminal peptide appears to act as a ÔswitchÕ that, once phosphorylated, converts arrestin into a state that is able to bind to R*. The effects of rhodopsin’s phospho- rylated C-terminal peptide can be mimicked by a synthetic phosphopeptide or even certain point mutations: both wild-type arrestin in the presence of the synthetic phospho- peptide [7], and arrestin-R175Q on its own [8] are able to bind to unphosphorylated R* and abrogate signalling to transducin. The crystal structure of arrestin is known [9,10], but it is not clear how binding of the phosphorylated C-terminal peptide of rhodopsin promotes tight complex formation between arrestin and R*. One possibility is that binding of phosphopeptide leads to a conformational change in arrestin that increases its affinity for R*. Conformational changes in arrestin can take place in solution, as demonstrated by changes in the proteolytic digestion pattern that result from phosphopeptide binding [7] or by heparin binding [11], and changes in cysteine reactivity due to phosphopeptide binding or the activating R175Q mutation [12]. The nature and extent of the conforma- tional change that leads to activation of arrestin is not known. The situation is complicated by the fact that visual arrestin participates in a monomer–dimer equilib- rium [13,14]. It has been suggested that the arrestin dimer may function as an inert storage form of the protein, which can be recruited by dissociation to terminate the visual signal [14]. To characterize further the mechanism and nature of arrestin’s activation, we conducted small-angle X-ray scat- tering (SAXS) studies of arrestin in solution to measure directly the quaternary structure and conformation of arrestin, and changes associated with phosphopeptide binding or the R175Q mutation. We demonstrate that the conformation and oligomeric structure of arrestin are not drastically altered by either phosphopeptide binding or by the R175Q mutation. The arrestin dimer will probably be Correspondence to B. H. Shilton, Department of Biochemistry, The University of Western Ontario, London ON, N6A 5C1 Canada. Fax: + 1 519 6613175, Tel: + 1 519 6614124, E-mail: bshilton@uwo.ca Abbreviations: R*, photoactivated rhodopsin; R*P, phosphorylated and photoactivated rhodopsin; R g , radius of gyration; S, momentum transfer equal to 2sinh/L; SAXS, small-angle X-ray scattering. Note: a web site is available at http://www.biochem.uwo.ca (Received 24 January 2002, revised 19 June 2002, accepted 25 June 2002) Eur. J. Biochem. 269, 3801–3809 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03071.x the major species in vivo [14], and because the changes attendant upon activation are relatively minor, it is conceivable that the dimer plays an active role in binding to R*. We have identified a model for the arrestin dimer in solution that is consistent with our SAXS data; in this model, dimerization is mediated by the C-terminal domains of arrestin, leaving each of the N-terminal domains free to interact with R*. This dimer could play an active role in attenuation of R* signalling. EXPERIMENTAL PROCEDURES Protein expression and purification Wild-type arrestin was prepared from bovine retina [7], while arrestin R175Q was expressed from yeast cells and purified as previously described [12]. In both cases, the arrestin yielded a single band when analysed by SDS/ PAGE. Protein preparations were dialysed against 10 m M Hepes, 400 m M NaCl, pH 7.5, and concentrated to approximately 0.13 m M by ultrafiltration; the ultrafiltrate was retained and used for buffer subtraction during the SAXS experiments. Following concentration, the protein was flash frozen and maintained at )80 °C. When required, additional concentration was carried out just prior to SAXS measurements using 0.5 mL centrifugal ultrafilters (Milli- pore Corp., Bedford, MA, USA). Protein concentration The concentration of BSA was measured using A 280 (1%) ¼ 6.14, while the concentration of arrestin was measured using A 278 (1%) ¼ 6.38 [15]. Preparation of the phosphopeptide The peptides used correspond to residues 330–348 from bovine rhodopsin, which comprises the carboxyl terminal phosphorylation site (DDEASTTVSKTETSQVAPA). Peptide synthesis, for both the unphosphorylated and fully phosphorylated versions, has been described previ- ously [7,16]. The lyophilized phosphopeptide was dis- solved in 500 m M Hepes, pH 7.6, to yield a final pH that was above 7. For arrestin in the concentration range of 30–150 l M (1.3–6.5 mgÆmL )1 ), 90 lL of arrestin solution was mixed with 10 lLof10m M peptide solution to yield a final peptide concentration of 1 m M . For higher concentrations of arrestin a 130 m M solution of peptide was used to yield a final peptide concentration of 13 m M . SAXS measurements All measurements were made at the European Molecular Biology Laboratory Outstation at the Deutsches Elektro- nen-Synchrotron (Hamburg, Germany), beamline X33 [17], at 15 °C using radiation with a wavelength of 0.15 nm. Measurements were made with either a position- sensitive linear detector or a Quadrant segment-shaped multiwire detector [18,19]. Sample–detector distances of 1.2 m (high angle) and 3 m (low angle) were used to cover therangeofmomentumtransfer(S ¼ 2sinh/k,where2h is the scattering angle) from 0.02 to 0.8 nm )1 . Fifteen successive 1-min exposures were recorded for each sample; there was no evidence of protein degradation over this time interval. Recording of each protein sample was preceded and followed by recording from the buffer alone; these buffer measurements were compared and provided a check on beam properties and the cleanliness of the cell between readings of protein solutions. Averaging of frames, corrections for detector response and beam intensity, and buffer subtraction, were performed using the programs SAPOKO (Svergun, D.I. & Koch, M.H.J., unpublished material) and OTOKO [20]. Phosphopeptide was added to the protein samples and matching buffer just prior to measurement. Determination of binding constants from forward scattering The intensity of Ôforward scatteringÕ,orI(0), is the X-ray solution scattering that is parallel to the incident beam, and was determined by extrapolation using Guinier curves [21] or from the distance distribution function, P(r), as evaluated by the indirect transform package GNOM [22,23]. Because of the changing oligomeric state of arrestin in these experiments, Guinier analysis was preferred over the use of the distance distribution function, which requires a prior estimation of the maximal dimension. The forward scattering is proportional to the product of the molecular mass and concentration of the scattering particle. For a mixture of particles, the total forward scattering, I(0) total is equal to the sum of contributions from individual species. Therefore, if I(0) M is the forward scattering of monomeric arrestin, the forward scattering from a mixture of monomers and dimers is: Ið0Þ Total ¼ f M Ið0Þ M þ 2f D Ið0Þ M ð1Þ where f M and f D are the mass fractions of monomer and dimer, respectively. Assuming that the monomer and dimer are the only species present, the expression for total forward scattering can be simplified as follows. f D ¼ 1 À f M Ið0Þ Total ¼ð2 À f M ÞIð0Þ M ð2Þ In the experiments, the total amount of protein is varied and the forward scattering is measured. The dissociation con- stant for dimerization, K d , is defined as: K d ¼ ½M 2 ½D ð3Þ where [M] and [D] are the molar concentrations of monomer and dimer, respectively. The K d canalsobe expressed in terms of the total mass concentration of protein, [arrestin] total (in mgÆmL )1 ), the mass fraction of monomer, f M , and the molecular mass of the monomer, W M in Daltons, as follows: K D ¼ 2f 2 M ½arrestin Total W M ð1 À f M Þ ð4Þ This expression for K d canbesolvedforthemassfractionof monomer, f M , which can then be used in the expression for I(0) total to yield the following equation: 3802 B. H. Shilton et al. (Eur. J. Biochem. 269) Ó FEBS 2002 Forward scattering, I(0) total , was plotted as a function of the mass concentration of arrestin, [arrestin] total , and the curve was fit to Eqn (5) using nonlinear regression as implemented in the program KALEIDAGRAPH (v3.08, Synergy Software), setting the molecular mass of the monomer equal to 45.3 kDa and the forward scattering of the monomer, I(0) M to the appropriate value, as determined using a BSA standard. The only variable fitted was the value for K d . To account for the formation of large aggregates, a linear term (k) was inserted into Eqn (5), to yield the following: In this case, the values of both K d and k were varied to fit the experimental data. Radius of gyration, model fitting and analysis For a mixture of monomeric and dimeric scattering species, the radius of gyration is given by: R 2 g ¼ f M R 2 gM þ f D R 2 gD ð7Þ where f M and f D are the mass fractions of monomer and dimer, respectively, and R gM and R gD are the radii of gyration for the monomer and dimer, respectively. Missing pieces of crystallographic models were built as extended polypeptide using the program O [24]. Crystallo- graphic models, which did not include crystallographic water molecules, were fit to experimental SAXS data using the program CRYSOL [25], with a solvent density of 0.36 electronsÆA ˚ )3 , using default limits for the contrast of the hydration shell and average displaced solvent volume per atomic group. The target function for CRYSOL is the v value, which is a measure of the agreement between the theoretical scattering from a model and the experimental data: v 2 ¼ 1 N À 1 X N j¼1 IðS j ÞÀI exp ðS j Þ rðS j Þ         2 ð8Þ where for each momentum transfer value, S j , I(S j )isthe theoretical scattering, I exp (S j ) is the experimentally observed scattering, and r(S j )istheerrorfortheexperimental measurement. RESULTS AND DISCUSSION Arrestin participates in a monomer–dimer equilibrium in solution Dimers of arrestin have been detected by sedimentation velocity [13] and sedimentation equilibrium [14]; in addition to the dimer, an arrestin tetramer was taken to be the predominant species when the protein was at high concen- tration (220 l M or 10 mgÆmL )1 ), even though the tetramer constituted only a minor component at 62 l M [14]. Our first task in understanding the solution structure and activation of visual arrestin was to ascertain its oligomeric state in our preparations. The X-ray solution scattering from particles at an angle of 0° with respect to the direct beam, the Ôforward scatteringÕ, is directly proportional to the molecular mass of the scattering species. The forward scattering values for various concentrations of wild-type arrestin were measured by extrapolation of Guinier curves [21] to an S value of 0nm )1 . With a 3-m sample–detector distance, useful data began at an S value of 0.04 nm )1 and the linear Guinier region extended to approximately S ¼ 0.08 nm )1 (Fig. 1). Ið0Þ Total ¼ Ið0Þ M 2 þ K D W M À ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi K 2 D W 2 M þ 8½arrestin Total K D W M q 4½arrestin Total 0 @ 1 A ð5Þ Ið0Þ Total ¼ Ið0Þ M 2 þ K D W M À ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi K 2 D W 2 M þ 8½arrestin Total K D W M q 4½arrestin Total 0 @ 1 A þ k½arrestin ð6Þ Fig. 1. Guinier plot of wild-type arrestin. (A) Small-angle X-ray scat- tering from arrestin (110 l M )in10m M Hepes buffer, pH 7.5, con- taining 400 m M NaCl, was measured using a 3- M sample to detector distance. Complete scattering data are represented by a fine line con- necting data points, while data used for the Guinier analysis are highlighted as filled circles. For data analysis, Guinier curves (solid straight line) were fitted to the data using weighted linear least squares as implemented in the program OTOKO [20].(B)Identicalto(A),except expandedintheregion0<S <0.09nm )1 . Ó FEBS 2002 Activation of visual arrestin (Eur. J. Biochem. 269) 3803 One preparation of wild-type arrestin, with a concentration of 140 l M , was diluted with buffer to yield concentrations of 110, 86, 57 and 29 l M . These five points were plotted (Fig. 2A, circles) and it was clear that there was a concentration dependence for the I(0) values. The apparent molecular masses at the different protein concentra- tions were calculated based on a BSA standard, and ranged from 50 to 80 kDa, consistent with a monomer–dimer equilibrium. We sought to characterize further the arrestin self- association, and to this end measured SAXS from solutions with concentrations of arrestin from 180 to 1300 l M (Fig. 2A, squares). Some of these data were recorded using a 1.2 m sample–detector distance, and only the outer part of the Guinier region (S values from 0.06 to 0.08 nm )1 )was available for the analysis. The increase of molecular mass in response to increased protein concentration was much less dramatic through this concentration range, progressing from just over 80 kDa at 180 l M , to just under 100 kDa at 1300 l M . A qualitative analysis of these data indicates that the apparent molecular mass at low protein concentrations approaches that of a monomer, and increases with protein concentration to that of a dimer, with an equilibrium dissociation constant, K d , of approximately 100 l M (corre- sponding to 4–5 mgÆmL )1 ). In contrast to a previous study [14], there does not seem to be a significant concentration of tetrameric arrestin, even at very high protein concentrations. The arrestin tetramer present in the asymmetric unit of both crystal structures [9,10] has a radius of gyration of almost 4.3 nm, and would have a forward scattering approximately twice as large as that observed by us. Neither of these parameters are within the range of our experimental observations, and therefore the predominant species in solution are the monomer and dimer. Additional evidence that the crystallographic tetramer is not a major species in solution is provided by comparison of the theoretical scattering from the crystallographic tetramer with our experimental scattering (Fig. 2B), where it is quite clear that the overall shape of the tetramer does not match what we have observed in solution. These data were fit to a simple model that incorporates a monomer–dimer equilibrium, and relates the protein con- centration to the forward scattering using the K d as the single variable. Using this equation, it was found that the K d was 40 ± 20 l M . Other fits to the data were also tested. A monomer–tetramer equilibrium was found to be incompat- ible with the data because it requires a monomer molecular mass of 30 kDa. Incorporation of a simple linear term in the monomer–dimer model, with the slope of the line as a second variable, results in a better overall fit to the data. In this case, the K d of the monomer–dimer interaction increases to 60 ± 25 l M . This linear term represents the formation of high molecular mass, irreversible arrestin aggregates, which have been observed in other studies [14,26,27]. Because the forward scattering is directly pro- portional to molecular mass, these high molecular mass species comprise only a minor component of the mixture and their effect is limited to the low angle Guinier region. Fig. 2. Self-association of wild-type arrestin. (A) Guinier analysis was used to calculate the I(0) value for arrestin solutions of varying con- centrations. The sample–detector distance was 3 m for the concen- trations from 1.3 to 260 l M (first seven points), allowing a momentum transfer range of 0.04 < S <0.08nm )1 for the Guinier curves. For the concentrations of 310, 500 and 1300 l M (last three points), a 1.2-m sample to detector distance was used, limiting the momentum transfer range available for Guinier analysis to 0.06 < S < 0.08. Data points obtained by dilution of a 140-l M stocksolutionareindicatedbycircles, while those obtained by further concentration of the stock solution are indicated by squares. All of the data points were fit to an equation that describes a monomer–dimer equilibrium in terms of the I(0) value and protein concentration, with the equilibrium dissociation constant, K d , as the sole variable (curve, short dashes; Eqn 5). To account for the formation of aggregates at higher protein concentrations, a linear term (single variable) was incorporated into the binding equation (Eqn 6), resulting in a better fit to the data (solid curve). The contribution from this Ôlinear componentÕ is illustrated by the dashed line at the bottom of the graph. (B) Calculated scattering from the crystallographic tetramer does not agree with experimental solution scattering. Data collected at two protein concentrations, 140 and 1300 l M , were merged to provide a representative scattering curve (dots) that covers a broad range of momentum transfer values. The structure of the crystallographic tetramer [9,10] was used to calculate the theoretical solution scattering for this particle (solid curve), which was fit to the experimental data using the program CRYSOL [25]. 3804 B. H. Shilton et al. (Eur. J. Biochem. 269) Ó FEBS 2002 Effect of rhodopsin C-terminal phosphopeptide on arrestin conformation Interaction between arrestin and the C-terminal phospho- rylated peptide of rhodopsin leads to tight binding of arrestin so that it is able to stop R* signalling [28]. It has been shown that the phosphopeptide does not have to be covalently bound to rhodopsin: a short, soluble phospho- peptide corresponding to the C-terminus of rhodopsin is sufficient to ÔactivateÕ arrestin and allow it to bind to photoactivated but not phosphorylated rhodopsin [7]. Given the structure of arrestin, it is conceivable that binding of the phosphopeptide could drive large conformational changes, such as a reorientation of the N- and C-terminal domains, that produce the dramatically increased affinity for R*; on the other hand, binding of the phosphopeptide may simply alter the surface features and cause relatively minor conformational changes. To distinguish between these two possibilities, we investigated the changes in arrestin structure and conformation produced by rhodop- sin’s C-terminal phosphopeptide. We first wanted to determine whether the peptide had any effect on the oligomeric state of arrestin. A peptide concentration of 1 m M was chosen because this had been shown previously to change the proteolytic digestion pattern of arrestin in solution [7]. We compared arrestin solutions with and without 1 m M phosphopeptide, at arrestin concentrations ranging from 20 to 130 l M monomer (1–6 mgÆmL )1 ), using the same protein preparation, buffer composition, and SAXS camera settings. Under these conditions, the phosphopeptide had a minor effect on forward scattering at low arrestin concentrations (up to 66 l M ), but no effect on arrestin from 66 to 130 l M (Fig. 3A). The phosphopeptide appears to produce a small increase in the amount of dimer at low arrestin concentrations, but has little effect at higher arrestin concentrations. To detect conformational changes in the arrestin mono- mers and dimers at a fixed arrestin concentration, the entire scattering curves for arrestin (110 l M ) in the presence and absence of phosphopeptide were compared (Fig. 3B), and it can be seen that the scattering curves are identical out to an S value of 0.2 nm )1 . If the phosphopeptide caused a reorientation of the N- and C-terminal domains, one would expect to see a change in this scattering curve: the absence of any such change indicates that the phosphopeptide has little effect on the gross conformation of arrestin. Because the effect of the phosphopeptide was not detectable at low angles, additional measurements were carried out at arrestin concentrations of 260 and 500 l M using a 1.2-m sample to detector distance to measure higher angle scattering, which is sensitive to more subtle changes in structure. The scattering in the presence and absence of approximately 13 m M (33 mgÆmL )1 ) phosphopeptide was Fig. 3. Effect of phosphopeptide on arrestin. (A) Dependence of I(0) on protein concentration for wild type arrestin in the presence (squares) or absence (circles) of phosphopeptide (1 m M ). (B) Comparison of low- angle scattering from arrestin (110 l M ) in the presence (solid curve) or absence (dashed curve) of 1 m M phosphopeptide. (C) Comparison of high-angle scattering from arrestin (500 l M ) in the presence (solid curve) or absence (dashed curve) of phosphopeptide (12 m M ). To aid visualization, curves were smoothed using a running five-point aver- age. The difference between free arrestin and arrestin in the presence of phosphopeptide is given at the top of the (B) and (C), expressed as a percentage of the total signal from the detector. Ó FEBS 2002 Activation of visual arrestin (Eur. J. Biochem. 269) 3805 compared. A very high concentration of peptide was used in these measurements to ensure that the arrestin was fully saturated. When the entire scattering curve for arrestin at 500 l M is inspected (Fig. 3C), it can be seen that there is no observable difference produced by the phosphopeptide in the S range from 0.07 to 0.2 nm )1 . The phosphopeptide does alter arrestin scattering at higher angles, particularly in region 0.2 < S <0.4nm )1 , where the two curves have different shapes. At S values above 0.4 nm )1 , the scattering signal is not sufficiently strong to determine whether the difference between the two curves is significant. The changes in scattering at S values greater than 0.2 nm )1 could be produced by the presence of the phosphopeptide on the surface of arrestin and/or by changes in ÔlocalÕ arrestin structure. These results are consistent with a model where binding of phosphopeptide causes a displacement of arres- tin’s C-terminus [29] and/or changes in the conformation of certain loops that facilitate R* binding [9]. The structure of arrestin R175Q resembles that of wild-type arrestin Replacement of arginine 175 with either glutamine or glutamic acid produces a constitutively activated arrestin molecule that binds photoactivated but unphosphorylated rhodopsin [8,30]. We used SAXS to elucidate the structural changes leading to activation of the R175Q mutant. Increases in the concentration of arrestin R175Q produce increases in forward scattering that are virtually identical to those observed for wild-type arrestin (data not shown), indicating that the R175Q mutation does not influence the monomer–dimer equilibrium. Comparison of arrestin- R175Q (110 l M ) with the wild-type protein (130 l M )further demonstrates that there is no change in low-angle scattering due to the R175Q mutation (Fig. 4A). Thus, the R175Q mutation does not affect the quaternary structure of arrestin, nor does it cause a large conformational change in arrestin. To define further any differences between wild-type and R175Q arrestin, scattering was measured from more con- centrated solutions (500 l M ;Fig.4B):uptoanS value of 0.2 nm )1 , X-ray scattering from the two proteins is identical, but there are small differences in the curves between S values of 0.2 and 0.4 nm )1 (Fig. 4B), similar to what was observed when phosphopeptide was bound to wild-type arrestin. The effect of the R175Q mutation on the properties of arrestin is consistent with the observed effect of the phosphopeptide: both cause activation of arrestin but produce very little change in arrestin’s solution structure and conformation. In summary, the transition in arrestin from a ÔbasalÕ state to an ÔactivatedÕ state that binds R* with high affinity involves relatively subtle structural changes. Models for the arrestin dimer in solution Guinier curves provide the radius of gyration (R g ), defined as the root mean square distance of all atoms from their common centre of mass. The R g canbeusedtoevaluatethe overall shape of a scattering particle. The R g for a mixture of scattering species depends on the R g for each species and their mass fraction according to Eqn (7). The radius of gyration of the monomer is 2.6 nm, determined using the crystallographic coordinates [10]. The measured R g for wild- type arrestin at 110 l M is 3.6 nm: at 110 l M the mass fraction of monomer is 0.35, and therefore the R g for the dimer is approximately 4.0 nm. Measurements at other protein concentrations also indicated an R g for the dimer of between 4.0 and 4.1. The dimer is therefore a highly elongated molecule. The only way to make such an elongated molecule from the monomeric species is to put two monomers together such that their long axes are arranged in tandem; that is, dimerization must be mediated by either the N-terminal or C-terminal domain to yield a dimer that is even more elongated than the monomer. The dimer in solution may be contained in the crystal structures of arrestin. Arrestin has been crystallized in two different space groups: P2 1 2 1 2 [9] and C222 1 [10]. In both cases, a tetramer of arrestin was present in the asymmetric unit. Referring to the C222 1 structure, the four polypeptide chains in each asymmetric unit have names A, B, C and D. Fig. 4. Effect of R175Q mutation on arrestin. (A) Comparison of low- angle scattering from arrestin R175Q (dashed curve) and wild-type arrestin (solid curve), both at 110 l M . (B) Comparison of high-angle scattering from arrestin R175Q (dashed curve) and wild-type arrestin (solid curve), both at 500 l M . The curves in graphs A and B were smoothed and difference curves plotted as in Fig. 3. 3806 B. H. Shilton et al. (Eur. J. Biochem. 269) Ó FEBS 2002 A twofold noncrystallographic symmetry axis runs through the tetramer, which means that it can be viewed as a dimer of dimers. The tetramer may be composed of either AB and CD dimers, or AD and CB dimers. The AB dimer has a radius of gyration of 4.0 nm, but has an extremely limited contact area (less than 1000 A ˚ 2 of buried surface), making it an unlikely candidate for the solution dimer, as noted previously [14]. The AD dimer is the most compact in the crystal, with an R g of 3.4 nm, yielding a relatively poor fit to the scattering data (not shown). On purely theoretical grounds, it is unlikely that either the AB or AD dimers exist to a significant degree in solution for the following reason: these dimers participate in Ôheterologous associationÕ [31]. That is, they do not have inherent two-fold symmetry, the surface used for the dimer interface is different for each protomer. Such an arrangement is unlikely to result in the formation of stable dimers as observed in solution; rather, the formation of such dimers in solution would be expected to progress to larger and larger polymers. In the C222 1 structure, there are two other dimers formed by crystallographic symmetry operations: one of these consists of two A chains interacting through their C-termi- nal domains (the ÔAAÕ dimer), while the other consists of a B chain and D chain interacting through their N-terminal domains (the ÔBDÕ dimer). These dimers possess twofold symmetry, and have similar R g values of approximately 3.6 nm and 3.5 nm, respectively, making them reasonable candidates for the solution dimer. How well do the various molecular models agree with solution scattering data? Low angle SAXS data collected at 140 l M arrestin were merged with high angle data collected with 1300 l M arrestin; in this way, the entire scattering curve could be used to evaluate models for the arrestin dimers and other species. We tested the agreement of the monomer, four dimers and tetramer against the merged SAXS data and found that neither the monomer nor tetramer yielded a reasonable fit to the data (Fig. 2B), in agreement with the results from analysis of forward scattering. Of the dimer models, the AA dimer yielded the best fit, with the BD dimer a close second (data not shown). The crystallographic models were missing regions of the protein that were disordered: residues 1–9, 363–378, and 393–404. These disordered elements in the crystal structure will also be disordered in solution, but they will contribute to the solution X-ray scattering. These pieces were modelled into the AA and BD dimers as extended polypeptide [26], and produced an improvement in the fit of the AA model at S values between 0.15 and 0.2 nm )1 (Fig. 5A; v ¼ 3.2) but only a slight improvement in the fit of the BD dimer model (Fig. 5B; v ¼ 4.4). The agreement between the theoretical scattering from the models and the experimental scattering Fig. 5. Models of the arrestin dimer in solution. Models of the arrestin dimer in solution. Structural models were constructed from dimers present in the crystal structure of arrestin [10] using the macromolecular modelling program O [24]. Approximately 3 kDa of polypeptide was missing from the N-terminus, C-terminus and two internal loops of the crystal structures; these missing pieces were modelled as extended polypeptide to improve the agreement with experimental SAXS data (dashed curves in both graphs). (A) Structure (ribbon diagram) and predicted scattering (solid curve) of the ÔAAÕ dimer formed by interac- tion between the C-terminal domains of arrestin monomers. (B) Structure (ribbon diagram) and predicted scattering (solid curve) of the ÔBDÕ dimer formed by interaction of N-terminal domains of arrestin monomers. Ribbon diagrams were drawn using the Swiss PDB Viewer [32]. (C) The agreement between theoretical and experimental scattering curves is indicated by the square of the weighted residual (summed term in Eqn 8) for each momentum transfer value; the solid line is for the AA dimer, and the dashed line is for the BD dimer. Ó FEBS 2002 Activation of visual arrestin (Eur. J. Biochem. 269) 3807 is indicated in Fig. 5(C), where the squared residuals are plotted as a function of the momentum transfer: the AA dimer has the best agreement in the lower angle region, up to S ¼ 0.3 nm )1 . Within the crystal structures of arrestin, the AA dimer is the most likely candidate for the major species in solution. CONCLUSION The activation of arrestin involves structural alterations that promote a high–affinity interaction with photoactivated rhodopsin (R*). Arrestin has been shown previously to form dimers in solution [13,14], and activation of arrestin could involve changes in the monomer–dimer equilibrium. A perturbation in the equilibrium would result in significant changes in the SAXS curve at a given protein concentration (Fig. 6A), which we did not observe. A second possibility is that the activation involves domain movements in either the monomer, dimer, or both. For example, a 15° rotation of the N-terminal domain relative to the C-terminal domain results in a subtle alteration of the arrestin dimer (Fig. 6B) which produces a small but detectable change in its solution scattering (Fig. 6C). We did not observe such a change in the solution structure of arrestin. We conclude that activation of arrestin in solution involves small and localized changes in conformation and no observable change in oligomeric structure. What is the biological role of the arrestin dimer? Studies of arrestin binding to R*P in vitro are conducted with dilute, monomeric arrestin, and therefore it is clear that the monomer is sufficient for binding to R*P. This does not preclude a biological role for the dimer. As noted by Schubert et al. [14] the fraction of dimeric arrestin found in vitro would be over 50% in the biologically relevant concentration range, an estimate that is a lower limit in vivo because of volume exclusion effects. Thus, dimeric arrestin is likely the major species in vivo. It has been proposed that the biological function of the arrestin dimer is to provide an inert ÔstorageÕ form of the protein [14]; the implication is that the dimer is not capable of binding to R*. However, the ÔAAÕ dimer identified in this study has the intriguing characteristic that the N-terminal domains are left open and available for interaction with rhodopsin. In fact, the structure of this dimer is such that both N-terminal domains could conceivably interact with two rhodopsin molecules simultaneously. It may be that only monomeric arrestin is able to effectively interact with R*P, and that the arrestin dimer is a storage form of the protein [14]. An alternative possibility raised by the present study is that dimeric arrestin has a more active role in attenuation of rhodopsin signalling or Fig. 6. Detection of structural changes in arrestin. To demonstrate how changes in oligomeric structure and/or conformational would affect scattering patterns, theoretical SAXS curves were calculated from model structures derived from the high resolution crystal structure of arrestin [10]. (A) The theoretical scattering from the putative arrestin dimer (dotted curve; see Fig. 5A) is compared to that of monomeric arrestin (solid curve). (B) The arrestin dimer (grey backbone) is superimposed over a possible alternative conformation (black backbone). In the alternative conformation, the N-terminal domains of each monomer are rotated approximately 15° with respect to the C-terminal domains; the overall effect is a slight ÔclosureÕ of the arrestin dimer. (C) The theoretical scattering curves for the two structures in (B) are compared: the dashed curve represents scattering from the grey structure, while the solid curve represents scattering from the black, structure. The difference between scattering from the two structures is given in the top of the graph, expressed as a per- centage of the total signal, as in Figs 3 and 4. 3808 B. H. Shilton et al. (Eur. J. Biochem. 269) Ó FEBS 2002 in postsignalling processes such as rhodopsin recycling. The proposed structure for the arrestin dimer allows us to design experiments to study the precise role of the C-terminal domain and the biological function of the arrestin dimer. ACKNOWLEDGEMENTS The assistance of Michel Koch at beamline X33 is gratefully acknowledged. Supported by Canadian Institutes for Health Research grant MT-15624 to BHS; and National Institutes of Health grants EY06225, EY06226, and EY08571 to PAH and a departmental award from Research to Prevent Blindness. PAH is a recipient of a Senior Investigator Award and WCS is recipient of a Research Career Development Award from Research to Prevent Blindness. REFERENCES 1. Yau, K.W. (1994) Phototransduction mechanism in retinal rods and cones. 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(1997) Swiss-model and the Swiss- PdbViewer: An environment for comparative protein modeling. Electrophoresis 18, 2714–2723. 33. Palczewski, K., Kumasaka, T., Hori, T., Behnke, C.A., Motoshima, H., Fox, B.A., Le Trong, I., Teller, D.C., Okada, T., Stenkamp, R.E., Yamamoto, M. & Miyano, M. (2000) Crystal structure of rhodopsin: a G protein-coupled receptor. Science 289, 739–745. Ó FEBS 2002 Activation of visual arrestin (Eur. J. Biochem. 269) 3809 [...]... polymers In the C2221 structure, there are two other dimers formed by crystallographic symmetry operations: one of these consists of two A chains interacting through their C-terminal domains (the ÔAAÕ dimer), while the other consists of a B chain and D chain interacting through their N-terminal domains (the ÔBDÕ dimer) These dimers possess twofold symmetry, and have similar Rg values of approximately... produced by the presence of the phosphopeptide on the surface of arrestin and/ or by changes in ÔlocalÕ arrestin structure These results are consistent with a model where binding of phosphopeptide causes a displacement of arrestin s C-terminus [29] and/ or changes in the conformation of certain loops that facilitate R* binding [9] The structure of arrestin R175Q resembles that of wild-type arrestin Replacement... very high protein concentrations The arrestin tetramer present in the asymmetric unit of both crystal structures [9,10] has a radius of gyration of almost 4.3 nm, and would have a forward scattering approximately twice as large as that observed by us Neither of these parameters are within the range of our experimental observations, and therefore the predominant species in solution are the monomer and. .. the solid line is for the AA dimer, and the dashed line is for the BD dimer Activation of visual arrestin (Eur J Biochem 269) 3807 to the solution X-ray scattering These pieces were modelled into the AA and BD dimers as extended polypeptide [26], and produced an improvement in the fit of the AA model at S values between 0.15 and 0.2 nm)1 (Fig 5A; v ¼ 3.2) but only a slight improvement in the fit of the. .. for the arrestin dimer in solution Guinier curves provide the radius of gyration (Rg), defined as the root mean square distance of all atoms from their common centre of mass The Rg can be used to evaluate the overall shape of a scattering particle The Rg for a mixture of scattering species depends on the Rg for each species and their mass fraction according to Eqn (7) The radius of gyration of the monomer... structure The difference between scattering from the two structures is given in the top of the graph, expressed as a percentage of the total signal, as in Figs 3 and 4 Ó FEBS 2002 in postsignalling processes such as rhodopsin recycling The proposed structure for the arrestin dimer allows us to design experiments to study the precise role of the C-terminal domain and the biological function of the arrestin. .. present in the asymmetric unit Referring to the C2221 structure, the four polypeptide chains in each asymmetric unit have names A, B, C and D Ó FEBS 2002 A twofold noncrystallographic symmetry axis runs through the tetramer, which means that it can be viewed as a dimer of dimers The tetramer may be composed of either AB and CD dimers, or AD and CB dimers The AB dimer has a radius of gyration of 4.0 nm,... tested the agreement of the monomer, four dimers and tetramer against the merged SAXS data and found that neither the monomer nor tetramer yielded a reasonable fit to the data (Fig 2B), in agreement with the results from analysis of forward scattering Of the dimer models, the AA dimer yielded the best fit, with the BD dimer a close second (data not shown) The crystallographic models were missing regions of. .. missing regions of the protein that were disordered: residues 1–9, 363–378, and 393–404 These disordered elements in the crystal structure will also be disordered in solution, but they will contribute Fig 5 Models of the arrestin dimer in solution Models of the arrestin dimer in solution Structural models were constructed from dimers present in the crystal structure of arrestin [10] using the macromolecular... wild-type arrestin The effect of the R175Q mutation on the properties of arrestin is consistent with the observed effect of the phosphopeptide: both cause activation of arrestin but produce very little change in arrestin s solution structure and conformation In summary, the transition in arrestin from a ÔbasalÕ state to an ÔactivatedÕ state that binds R* with high affinity involves relatively subtle . The solution structure and activation of visual arrestin studied by small-angle X-ray scattering Brian H. Shilton 1 , J. Hugh McDowell 2 , W. Clay Smith 2 and. Within the crystal structures of arrestin, the AA dimer is the most likely candidate for the major species in solution. CONCLUSION The activation of arrestin