Recognizing and Defining True Ras Binding Domains I: Biochemical Analysis

doi:10.1016/j.jmb.2005.02.048

J. Mol. Biol. (2005) 348, 741–758

Recognizing and Defining True Ras Binding Domains I: Biochemical Analysis Sabine Wohlgemuth1†, Christina Kiel1,2†, Astrid Kra¨mer1, Luis Serrano2 Fred Wittinghofer1* and Christian Herrmann1 1

Max-Planck-Institut fu¨r Molekulare Physiologie Abteilung Strukturelle Biologie Otto-Hahn Str. 11, Dortmund 44227, Germany 2

European Molecular Biology Laboratory, Meyerhofstrasse 1 69117 Heidelberg, Germany

Common domain databases contain sequence motifs which belong to the ubiquitin fold family and are called Ras binding (RB) and Ras association (RalGDS/AF6 Ras associating) (RA) domains. The name implies that they bind to Ras (or Ras-like) GTP-binding proteins, and a few of them have been documented to qualify as true Ras effectors, defined as binding only to the activated GTP-bound form of Ras. Here we have expressed a large number of these domains and investigated their interaction with Ras, Rap and M-Ras. While their (albeit weak) sequence homology suggest that the domains adopt a common fold, not all of them bind to Ras proteins, irrespective of whether they are called RB or RA domains. We used fluorescence spectroscopy and isothermal titration calorimetry to show that the binding affinities vary over a large range, and are usually specific for either Ras or Rap. Moreover, the specificity is dictated by a set of key residues in the interface. Stopped-flow kinetic analysis showed that the association rate constants determine the different affinities of effector binding, while the dissociation rate constants are in a similar range. Manual sequence analysis allowed us to define positively charged sequence epitopes in certain secondary structure elements of the ubiquitin fold (b1, b2 and a1) which are located at similar positions and comprise the hot spots of the binding interface. These residues are important to qualify an RA/RB domain as a true candidate Ras or Rap effector. q 2005 Elsevier Ltd. All rights reserved.

*Corresponding author

Keywords: RA domains; Ras proteins; protein interactions; kinetics; thermodynamics

Introduction Ras is a GTP binding protein involved in many signal transduction processes such as control of growth, differentiation and apoptosis. It acts as a † S.W. and C.K. contributed equally to this work. Present addresses: A. Kra¨mer, School for Biomedical Science and Institute for Molecular Bioscience, The University of Queensland, St Lucia, Brisbane, Qld 4072, Australia; C. Herrmann, Physikalische Chemie 1, RuhrUniversita¨t Bochum, 44780 Bochum, Germany. Abbreviations used: RalGDS, Ral guanine dissociation stimulator; RBD, Ras binding domain; RA, RalGDS/AF6 or Ras association; PLC3, phospholipase C epsilon; ITC, isothermal titration calorimetry; mant, Nmethylanthraniloyl, a fluorophor attached to the 2 0 or 3 0 position of the nucleotide; DTE, dithioerythritol; AF6, ALL fused protein 6; RIN1, Ras interacting 1; Nore1, novel Ras effector 1. E-mail address of the corresponding author: [email protected]

molecular switch that cycles between an inactive GDP- and an active GTP-bound state.1,2 In the resting state it contains tightly bound GDP, whose dissociation is increased by many orders of magnitude by the action of guanine nucleotide exchange factors. This leads to the activation and GTP loading of Ras. In the GTP-bound form it interacts with effector proteins that are defined as proteins having a high affinity to the GTP- but not the GDP-bound form of Ras. Signal transduction to the effectors is terminated by the GTPase reaction that is intrinsically very slow, and is stimulated by GTPaseactivating proteins (GAPs) which increase the reaction by many orders of magnitude.3 The first effector of Ras to be identified was the Ser/Thr protein kinase c-Raf1 which initiates a cascade of protein kinases, the MAP kinase module, to activate MEK1, which in turn activates the MAPKs ERK1 and ERK2.4,5 c-Raf-1 contains a Ras binding domain (RBD): a stable 81-residue protein domain, sufficient to bind to Ras in a

0022-2836/$ - see front matter q 2005 Elsevier Ltd. All rights reserved.

742

RA Domain Characterisation

Figure 1. Proteins containing RA, RB and UBQ domains. (a) Domain structure of selected proteins containing RA (Ras association) and RB (Ras-binding) domains. The Figure has been prepared following SMART nomenclature and domain assignment.36,37 (b) Superposition of the structures, as ribbon diagrams, of RalGDS21 (PDB entry 1LFD; green), Raf8 (PDB

743

RA Domain Characterisation

GTP-dependent manner. The structural analysis showed that it has an ubiquitin fold which interacts with Ras (and Ras-like proteins) by forming an inter-protein b-sheet, involving the outer strands of the two proteins.6–8 The interface of that complex was shown to be mostly determined by complementary charge interactions, the surface of Ras being negatively charged and the surface of RafRBD being positive. The structure of the RBD of the Schizosaccharomyces pombe protein Byr2, genetically and hierarchically a homologue of the mammalian MEK1 or Raf, was also solved both alone9,10 and in complex with Ras.11 While the overall fold is similar, the details in the intermolecular interactions forming the interface with Ras are different. RalGDS (Ral guanine dissociation stimulator) and the isoforms Rgl, Rgr Rlf, and the a,b,g PI(3) kinases have also been identified as effectors of Ras.12–15 They contain an RA domain (RalGDS/AF6 or Ras association domain), which in spite of low sequence homology has a similar structure. The structures of the RA/RB domains, and of their complexes with Ras proteins, show that they share the same fold and also bind to the effector switch I region of Ras via a similar binding mode.16–20 This interaction, however, involves a different set of residues.21–23 In the case of the complex between Ras and PI(3)Kg, which is the first structure with a complete effector, rather than the RA/RB domain alone, the interaction also involves switch II, and this interaction is thought to regulate the activity of the enzyme allosterically. Using yeast two-hybrid analysis and pull-down techniques, a large number of additional putative effectors have been identified, such as adenylate cyclase in Saccharomyces cerevisiae,24 AF6,25,26 phospholipase C3,27 Nore1 (novel Ras effector 1)28,29 and other isoforms of the tumour suppressor RASSF1.30 In a recent publication, Rodriguez-Viciana and coworkers have performed an extensive study on the interaction of RA and RB domains, in complex with various members of the Ras sub-family, using pulldown experiments.31 The RA (RalGDS/AF6) domain has been defined by Ponting & Benjamin, based on database searches and sequence analysis.32 This domain is present in members of the RalGDS family, RIN1 and many proteins not previously recognized as downstream effectors of Ras such as AF6 and the Drosophila homologue canoe, Myr-5, DAG kinase, Ste50 and Ste4. Surprisingly, Raf-RBD scored very low in the sequence comparison and was not considered to belong to this domain family although the authors assumed, as was later verified by structural

analysis, that the RA and RB domain have the same fold. Since ubiquitin also has the same fold, we will define from now the overall ubiquitin-fold family, comprising RA/RBD and ubiquitin, as UB domains. A further complication in the search for effectors for Ras arises from the fact that the superfamily of Ras-like proteins includes a Ras subfamily containing, among others, the proteins Rap (five isoforms) and R-Ras (three isoforms). These are believed to have functions at least partially overlapping with Ras and also seem to interact with RA and RB domain containing proteins.33,34 Thus, R-Ras and Rap1 have been shown to bind to various RA or RB domains in vitro, in a GTP-dependent manner, and in the case of RalGDS the binding is tighter than to Ras.35 Furthermore, numerous functional studies have shown interactions between Ras-like proteins and effectors containing RA or RB domains to be functionally meaningful. At present, 108 RA and 20 RB domains, from human proteins, are listed in the SMART database.36,37 The role of many of these in signal transduction, via Ras/Rap/R-Ras, is not obvious. Not counting possible redundancies, it seems unlikely that all of them are true Ras effectors. Using model building, it has in fact been shown that Myr-5 is unlikely to form a productive interface with Ras.38 In order to define the requirements for productive binding of predicted RA and RB domains to Ras-GTP, or possibly other Ras-like proteins, we have measured the interaction of a large number or RA and RB domains, using different methodologies. Based on these experiments, we have derived common denominators for efficient complex formation. Using this information in the accompanying paper (Kiel et al.)39 and through the use of homology modelling and prediction of binding energy, we develop a methodology to do genome-wide prediction for this type of interaction.

Results Domain structure and homology Figure 1(a) shows the domain organization of a number of proteins containing an RA/RB domain which we have selected for our study. The proteins were selected from databases and have in most cases been shown to bind to Ras or a Ras-like protein, usually by non-equilibrium pull-down and/or two-hybrid analysis. The RA/RB domain

entry 1GUA; blue), and Ubiquitin87 (PDB entry 1AAR; red). (c) Sequence alignment of RA, RB and UBQ domains. The alignment of the first three sequences was based on an overlay of their respective structures (mRalGDS, 1LFD; cRaf; 1CLY; Ubiquitin, 1AAR) as shown in (b). In yellow, we marked the regions of secondary structure as indicated in the boxes above. The stars indicate hydrophobic contacts stabilizing the hydrophobic core in the respective UB domain protein. All sequences are grouped either to the RA, the RBD and the UB domain families as described by SMART36,37 and the consensus sequence is given using the abbreviations given in SMART.36,37 We coloured conserved positively charged amino acid side-chain residues in blue, hydrophobic residues in red and aromatic side-chains in green.

744 is present in different locations within the corresponding proteins, including both the N and C-terminal ends; no pattern of domain organisation is recognizable, arguing that the domain is a structural module that appears to have been “shuffled around” in the course of evolution. In order to align the sequences, one could use standard sequence alignments procedures. However, when the sequence identity is low, misalignments of certain secondary structure elements can take place. To improve the quality of the alignment, we first took all the high-resolution 3D structures of UB domains and superimposed them by pairs using the Swiss-PdbViewer40 software and the “fragment superimpose” option (Figure 1(b)). Based on this superimposition we did a sequence alignment of these domains, within and between members of the subfamilies, and we used it to align manually the rest of the sequences (Figure 1(c), top). While members of the UBQ family do not bind to Ras proteins, the classification into RA and RB domain families is not necessarily a reflection of their binding properties, but rather of sequence variation. The alignment in Figure 1(c) shows the pattern of conserved residues which as described by the threedimensional structures are located in the hydrophobic core. Therefore, this excludes the possibility that the conserved residues are involved in a conserved interactive function, but suggests that they are rather involved in stabilizing the fold of the protein. Notably, sequence conservation is rather poor in the secondary structure elements (b1, b2, a1, b3, b4, a2 and b5). Based on our alignment, we classify nearly all domains that bind to Ras proteins into the RA domain family, including many of the verified effector proteins, like RalGDS, AF6 and Nore1, which have been described previously as RBDs, on the basis of functional studies. By contrast, the number of domains in the actual RBD family, which contains Raf, Tiam1 and RGS 12/14, is rather small. Byr2, the S. pombe homologue of Raf could be aligned better with the consensus of the RA domain family than that of the RBD family. Overall, the sequence alignments suggest that the classification in RBD and RA domains is rather artificial, at least from a structural point of view. Domain characterization The physical isolation of domains from a multidomain protein can potentially result in partial or global denaturation of the isolated domain. As a result, binding to a target protein can sometimes be detected with the intact protein, while analysis of the isolated domain simultaneously produces negative results. Therefore, to determine the structural integrity of all the RA and RB domains we have analysed them by circular dichroism (CD). Figure 2 shows the Far–UV CD spectra for all the domains analysed here, at a protein concentration of 20 mM. Most of them show a similar spectrum, with a minimum around 208 nm and negative ellipticity

RA Domain Characterisation

around 222 nm, as expected for a mixture of alpha and beta structure. An exception is mTiam1 that shows a CD spectrum typical of a coil–coil helical structure with a minimum at 200 nm (typical of random-coil). In addition, CD spectra at concentrations between 130 mM and 200 mM have been recorded (see Figures in Supplementary Data). We find that the shapes of the spectra are very similar, indicating that no aggregation occurs within this concentration range. Since the RA domain of Rain was very unstable and tended to aggregate, we have used the GST-Rain fusion protein. In these cases, binding Kd values are only upper values. Equilibrium binding studies using fluorescence spectroscopy Fluorescence spectroscopy to measure binding has several advantages over other methods, the most important being the low concentration required. Using low concentration is important to avoid aggregation problems which could hamper Kd measurements. However, in order to obtain affinity data from equilibrium titration experiments via fluorescence, a sufficiently large signal change is required upon binding. Fluorescent mant-nucleotides (mGDP or mGTP) bound to Ras-like proteins, have been shown to be very sensitive to changes in their environment and have been used to study the interaction between Ras proteins and GAPs, GEFs and effectors.41 While binding between Ras and GAPs can easily be monitored by mant-nucleotides, Ras$mGTP or mGppNHp has failed to show a sufficiently large change in fluorescence on binding to the Raf-RBD,42 or to the RA domains of RIN1 and RalGDS, thus making equilibrium titrations impossible. The signal is, however, sufficiently large to be used for stopped-flow kinetic experiments43 (see also below). We have previously shown that fluorescence labelling of Ras proteins with Aedans is a valuable tool to study the mechanism of GAP-stimulated Ras, Ran and Rap GTPase reactions.44–46 To do this, the Aedans fluorophore was coupled to cysteine at position 32 or 86, of Ras (Ras32, Ras86) or Rap, which from structural considerations7 is sufficiently close to the effector binding site. The change in fluorescence was measured after adding saturating amounts (approximately 60 mM) RA/RB domains to Ras32, Ras86 or Rap86, bound to the nonhydrolysable GTP-analogue GppNHp. The strongest change in fluorescence (of around 45%) was obtained for Raf and RASSF1, and they were used to determine the affinity for Ras32$GppNHp (Figure 3). Plotting the fluorescence increase against the RA/RB domain concentration, and fitting the data using equation (1) (see Materials and Methods), the affinities for Raf and RASSF1 were determined to be 22.3 nM and 19.2 mM, respectively. The affinity for Raf-RBD is close to that described before, using the more time-consuming GDI assay under low salt conditions.42 In general, the magnitude of change in fluorescence using different RA/RB domain

RA Domain Characterisation

745

Figure 2. Circular dicroism spectra for UB domains in the far–UV region. (a) Spectra for cRaf, RalGDS, spByr2, AF6_RA1 and AF6_RA2. (b) Spectra for RIN1, RIN2, PL3_RA1, PLC3_RA2, and PLC3_RA1RA2. (c) Spectra for mNore1, RASSF1, scCYR1, Krit1, GST-Rain, PDZGEF, and mTiam. All spectra were obtained at 20 mM protein concentration in phosphate buffer at pH 7.0. Twenty accumulations were averaged to obtain each spectrum. The values for q were normalized to the concentration and to the cuvette length, as well as to the number of amino acid residues in each domain.

proteins is extremely small or not recognizable for some of the domains (data not shown). Fluorescence could therefore not be used for a more global analysis of binding affinities. Isothermal titration calorimetry The small magnitude of the fluorescent change could be due to lack of binding, or could reflect the structural variability in the interaction between RA/RB domains and Ras proteins. To rule out the latter, we used isothermal titration calorimetry

(ITC), which does not require any labelling of proteins. ITC has the additional advantage of giving information not only on the affinity but also on other thermodynamic properties, like the enthalpy and entropy changes of the interaction. For this, we used H-Ras, Rap1B and M-Ras bound to the nonhydrolyzable GTP analogue GppNHp and titrated with increasing amounts of the recombinant minimal RA/RBD domain, as described.47 A typical experiment for an affinity in the lower micromolar range (Figure 4(a)) shows the interaction between Ras$GppNHp and mNore1 (KdZ0.20 mM at 25 8C).

746

Figure 3. Dissociation equilibrium constants determined by fluorescence measurements. A solution of fluorescently labelled Ras32$GppNHp (200 nM) was titrated with (a) increasing concentrations of the RBD from Raf or (b) the RA domain from RASSF1, as indicated. The relative fluorescence was plotted against the effector concentrations. A fit based on equation (1) leads to dissociation constants (Kd) of 22.3 nM (Ras32-Raf) and 19.2 mM (Ras32-RASSF1).

The interaction between Ras$GppNHp and scCYR in Figure 4(b) (KdZ18.9 mM) is an example of low affinity complex formation, that is close to the limit of accuracy for this method. ITC experiments were repeated, in some cases two or three times, sometimes using different protein batches. From repeating the experiments, the errors were estimated to be 0.4 kcal/mol for the DG8 values, 0.5 kcal/mol for the DH8, and 0.3 for the n-values (stoichiometry of the interaction). Errors could be larger because of concentration errors or partial unfolding of the domains. The thermodynamic parameters obtained from ITC measurements are summarized in Table 1, together with previous data for the interaction of Raf, RalGDS and spByr2 with H-Ras and Rap1B.47 Using the standard ITC assay, we do not find any measurable affinity between Ras and the RA/RB domains of Rain, Tiam1, and the first RA domain of

RA Domain Characterisation

PLC3 (phospholipase C epsilon), Rap1 and the second RA domain of AF6, Rain, mTiam1 and PLC3_RA1. This indicates that either the interaction is too low to be measurable by ITC (KdO50 mM) or that the enthalpy of the interaction is too low to be measurable under our standard conditions. It is possible that the enthalpy change is zero at standard temperature and binding is driven exclusively by favourable entropy changes. Therefore, we have also performed measurements at temperatures different from 25 8C and find no measurable enthalpy in such cases. Since it is unlikely that the change in heat capacity (as a function of temperature dependence of the enthalpy) is also zero, we can conclude that there is no binding between these isolated domains and the corresponding Ras proteins. Affinities between Ras proteins and the real or putative effector domains vary between 80 nM for the Ras–Raf interaction, and 33.3 mM for Ras-Krit and Ras-PDZGEF. The weak interaction with adenylylcyclase from S. cerevisiae is most likely due to the use of mammalian Ras rather than the correct binding partner (Ras2p) from S. cerevisiae. The affinities between Rap and the same set of effectors are less divergent and vary between 0.37 mM for the AF6 interaction, and 8.4 mM for the PLC3 interaction. As for the specificity of the interactions, we find that several of the RA/RB domains show a pronounced specificity towards either Ras or Rap, whereas M-Ras (also called R-Ras3) shows a pattern of affinities more similar to that of Ras, except for PLC3, where its affinity is between that of Ras and Rap. We find a large difference for the RA1 domain of AF6/Canoe, where Rap has a 7-fold higher affinity, whereas RIN1 shows a tenfold higher affinity for Ras. PDZGEF and Krit1 bind Rap with a relatively low affinity of 5.2 mM and 4.7 mM and show a weak interaction with Ras, that is probably insignificant. The second domain of PLC3 binds with micromolar affinity to Ras, M-Ras and Rap, with an eightfold higher affinity for Ras than for Rap. Various effector proteins in the database contain two putative RA/RBD domains in tandem, such as AF6/Canoe and PLC3. Our binding studies show that the first RA domain of AF6 binds with high affinity whereas the second site has a, probably insignificant, affinity of 23.3 mM for Ras and no measurable affinity for Rap. In the case of PLC3, we find that the first RA domain has no measurable affinity. To exclude the possibility that the first RA domain binds Ras proteins only in the presence of the second, or whether one is required for proper folding of the other, a construct of PLC3 containing both domains (PLC3_RA1RA2) was expressed. It binds with similar affinity as the second domain alone and shows a stoichiometry of binding close to 1 : 1. Concerning Krit1, we could only obtain measurable binding data using a much larger construct with residues 266–736, which contains ankyrin repeats and an RA in the context of a FERM

747

RA Domain Characterisation

Figure 4. Calorimetric characterization of Ras/effector interactions by isothermal titration calorimetry. (a) Calorimetric titration of 1 mM Ras$GppNHp, out of the syringe, into 100 mM mNore1 in the sample cell. (b) Titration of 1 mM Ras$GppNHp into 100 mM scCYR1. (c) Thermodynamic parameters for binding of AF6_RA1, RIN1, mNore1, PLC3_RA1RA2 and Krit1 to H-Ras, Rap1B (and M-Ras). Green bars show changes in free energy (DG8), blue bars show enthalpy changes (DH8) and yellow bars show entropy changes (TDS8) upon binding, as derived from ITC experiments (Table 2).

domain.48 FERM domains consist of three tightly linked sub-domains, one of which shows a ubiquitin fold. ITC experiments for the Krit1 construct 416-524 (RA domain alone) and construct 416-736 (complete FERM) did not give any analysable results. Thus it is possible that some of the domains for which we have not detected binding, could indeed interact with Ras in the context of the full protein. This could be due either to a conformational change in the domain, or to partial unfolding in the absence of the rest of the protein (see accompanying paper39).

Thermodynamic analysis We have observed previously that for a few RA/RB domain interactions the enthalpy and entropy contributions to the binding energy vary considerably.47 By extending our measurements to a larger number of effectors, we observed a wide range of thermodynamic properties of the binding reaction. Leaving out the low affinity interactions, which are probably insignificant, the entropy contribution (as TDS) is especially variable: it ranges from a positive, favourable value of

748

RA Domain Characterisation

Table 1. Thermodynamic parameters for binding of RA/RB domain proteins to H-Ras, Rap1B and M-Ras bound to GppNHp GTP-binding protein H-Ras Rap1B M-Ras H-Ras Rap1B M-Ras H-Ras Rap1B H-Ras Rap1B M-Ras H-Ras Rap1B H-Ras Rap1B M-Ras H-Ras Rap1B M-Ras H-Ras Rap1B Ras Rap1B H-Ras Rap1B M-Ras H-Ras Rap1B H-Ras Rap1B M-Ras H-Ras Rap1B H-Ras Rap1B M-Ras H-Ras Rap1B H-Ras Rap1B M-Ras H-Ras Rap1B

Effector

n

K da (mM)

DH8 (kcal/mol)

DG8b (kcal/mol)

TDS8c (kcal/mol)

AF6_RA1 AF6_RA1 AF6_RA1 AF6_RA2 AF6_RA2 AF6_RA2 Rain Rain RalGDS RalGDS RalGDS RASSF1C RASSF1C mNore1 mNore1 mNore1 RIN1 RIN1 RIN1 RIN2 RIN2 PDZGEF PDZGEF mTiam1 mTiam1 mTiam1 Craf Craf PLC3_RA1 PLC3_RA1 PLC3_RA1 PLC3_RA2 PLC3_RA2 PLC3_RA1RA2 PLC3_RA1RA2 PLC3_RA1RA2 scCYR1 scCYR1 SpByr2 SpByr2 SpByr2 Krit1 Krit1

0.9 0.8 1.1 0.9

2.2 0.24 2.8 35

K7.7 K9.1 K7.6 K6.1

1.9 8.3 6.5 5.0

1.3 0.7

1.0c 0.077c 3.7 39

K8.3c K9.8c K7.4 K6.3

K6.2c K4.7c 4.0 1.2

1.0 0.8 1.0 0.9 0.9 0.9 0.8 0.6 1.0 0.8

0.32 2.8 0.31 0.88 4.3 0.59 11 5.7 33 4.5

K5.8 K0.8 K1.1 K1.1 w0 w0 w0 w0 K14.5c K14.5c K3.5 K5.0 w0 K9.0 K5.7 K5.4 K12.9 K8.1 K10.8 K13.3 K4.7 K3.4 K4.3 w0 w0 w0 K5.2c K3.7c w0 w0 w0 K6.5 K8.1 K8.3 K7.5 K2.3 K4.2

K8.9 K7.6 K8.9 K8.3 K7.3 K8.5 K6.8 K7.2 K6.1 K7.3

K0.1 1.9 3.5 K4.6 K0.9 K2.3 K6.5 2.5 3.0 2.8

K9.7c K8.5c

4.5c 4.8c

K8.3 K7.1 K8.2 K6.7 K7.0 K6.5

1.9 K1.1 K0.1 K0.9 4.7 2.3

K9.2c K9.8c K10.3 K6.1 K7.3

0.2c 0.8c 6.9 5.3 1.2

0.080c 0.67c

0.7 0.5 0.8 0.7 1.1 1.0

1.0 0.7 0.8

0.82 8.4 0.98 13 7.5 16.9 O100 0.18c 0.067c 0.030 33 4.7

K9.0c K9.0c K3.4 K0.8 K6.1

Measurements were done in 50 mM Tris-buffer (pH 7.4) containing 5 mM MgCl2 and 2 mM DTE using ITC at 25 8C. Data were taken from Rudolph et al.47 (ITC data, 50 mM Tris-buffer (pH 7.4) containing 5 mM MgCl2, 100 mM NaCl and 2 mM DTE at 25 8C). a KdZ1/Ka. b DG8ZKRTlnKa. c DGZDHKTDS.

8.3 kcal/mol, for the Rap–AF6_RA1 interaction, to a negative and thus unfavourable K6.5 kcal/mol, for the Ras–RIN2 interaction. The latter case is compensated by the very strong favourable enthalpy contribution of K13.3 kcal/mol. We find a purely enthalpic contribution in the case of mNore1 and Ras, a mostly favourable enthalpic contribution in the case of mNore1 and Rap1, while the interaction between Krit1 and Ras is mainly driven by the favourable entropy. The thermodynamics of the Ras, as compared to the Rap and M-Ras interactions, are sometimes similar and sometimes different (Figure 4(c)). For the interaction of Rap1 with Krit1, which has been considered to be an effector of Rap, there is a more favourable entropy

contribution for the Rap versus the Ras complex, which accounts for the higher affinity. Likewise, the AF6 interaction with Rap has a highly favourable entropy and a negligible enthalpy contribution, which is totally reversed as compared to Ras.47 For other RA/RB domains, the principle thermodynamic profiles are quite similar. Thus, binding of AF6_RA1 to H-Ras, Rap and M-Ras is always driven by favourable enthalpy and entropy changes, while binding of RIN1 to the three Ras proteins is enthalpically driven, and counteracted by unfavourable entropy changes. In the case of Nore1 and PLC3_RA1RA2, binding is in all cases driven by favourable enthalpy changes, while entropy changes are close to zero or small and positive.

749

RA Domain Characterisation

Kinetics of interaction between RA/RB domains and Ras proteins Kinetic parameters of the interaction of Ras with the established effectors Raf-RBD, RalGDS and AF6_RA1 have been measured before and show a two-step binding reaction.43,49,50 The kinetics of RA/RB domain proteins binding to Ras were measured using stopped-flow experiments and the fluorescent mant group attached to GppNHp. Here, rapid mixing of the effector proteins with Ras$mGppNHp produces observable fluorescence changes for AF6_RA2, RASSF1C, mNore1, RIN1, PLC3_RA2, PLC3_RA1RA2 and spByr2, while fluorescence changes obtained for, mTiam1, Krit1, Rain and scCYR were too small to be useful. Using pseudo first-order conditions (concentration of the effector being at least tenfold higher), the observed time-dependent change in fluorescence can be fitted to a single exponential to

Figure 5. Kinetics of binding of Ras$mGppNHp to effectors, measured by stopped-flow. (a) Plot of observed rate constants kobs against the RIN1 concentration, for binding of RIN1 to Ras$mGppNHp. A fit based on a two-step binding equation leads to K1Z27 mM and k2Z 534 sK1, yielding konZ20 mMK1 sK1. (b) Binding of PLC3_RA2 to Ras$mGppNHp. The association rate constant was obtained from the concentration dependence of kobs, as the slope of a linear fit (konZ1.9 mMK1 sK1). All measurements were done in 50 mM Tris-buffer (pH 7.4) containing 5 mM MgCl2 and 2 mM DTE at 10 8C.

give kobs (data not shown). These observed rate constants increase with higher effector concentration, as shown for RIN1 in Figure 5(a). The increase is linear within low concentrations of effector, but becomes saturated at high concentrations, indicating a two-step binding process. The first step represents the formation of an initial encounter complex, followed by an isomerization reaction to the final complex, occurring at several hundreds per second. The data were analysed to obtain the affinity of the encounter complex K1, the isomerization rate k2 and the overall association rate constant konZk2/K1. (Table 2). For the two PLC3 constructs, no saturation was reached. (As the association rate constant is slow, we might come to saturation at much higher concentration.) Hence K1 and k2 could not be determined here and the association rate constant was obtained from the linear fit (Figure 5(b)). Dissociation rate constants were determined by displacing Ras$mGppNHp complexed with the RA/RB domains, using excess unlabelled Ras$GppNHp in the stopped-flow apparatus. Single exponential fits to the fluorescent transients gave dissociation rate constants (koff; not shown) which are in the range of 4–16 sK1. The errors for determination of kinetic parameters are in the order of 10%, mainly due to errors in concentration determination. The error is expected to be lower for the dissociation rate constants (koff values), because the dissociation process is independent of the RA or RB domain concentration. The kinetic parameters are summarized in Table 2, together with data obtained previously. The K1 and k2 values for RIN1 and mNore1 are in a similar range to those obtained earlier for AF6, RalGDS and Raf, considering the different buffer conditions used. For the two PLC3 constructs the kon values are much lower, indicating that electrostatic interactions may not be as pronounced in PLC3. While the equilibrium binding affinities obtained from the kinetic constants are usually very similar to those obtained from ITC measurements, the mNore1/Ras$mGppNHp complex affinity, calculated from KdZkoff/kon, is tenfold higher than that determined by isothermal titration calorimetry. It is due to an unusually slow dissociation of Ras$mGppNHp and this, in turn, could be due to the mant-group contributing to complex formation. Apart from an experimental artefact, the only other explanation could be that the fluorescent change of Ras upon dissociation from mNore is delayed due to an induced conformational change in the complex. However, we do not have any experimental evidence for this. Functional epitopes in the interfaces The three-dimensional structures of the Rap-Raf, Raps-Raf, Ras-RalGDS and Ras-Byr2 effector complexes have been solved. 7,8,11,21,22 These studies have identified a number of residues involved in forming the interface. Here, after having established a large data set about binding

750

RA Domain Characterisation

Table 2. Kinetic parameters of RA and RB domains binding to Ras$mGppNHp Linear range Effector AF6_RA1 RalGEF mNore1 RIN1 RafRBD PLCe_RA2 PLCe_RA1RA2

Saturation

kon (mMK1 sK1)

koff (sK1)

Kd (mM)

K2 (sK1)

K1 (mM)

konZk2/K1 (mMK1 sK1)

6.4a 8.8b 8.9 12.0 66.1 1.89 1.71

15.3a 11.0b 0.18 16.0 4.34 5.49 5.94

2.4a 1.3b 0.020 1.33 0.066 2.9 3.5

310a 300b 451 534 415c

40a 61b 36.7 27.1 11.7c

7.75a 4.9b 12.3 19.7 35.5c

Measurements were done in 50 mM Tris-buffer (pH 7.4) containing 5 mM MgCl2 and 2 mM DTE using stopped flow at 10 8C. a Data were taken from Linnemann et al.49 (20 mM Tris-buffer (pH 7.4) containing 5 mM MgCl2, 100 mM NaCl and 2 mM DTE at 10 8C). b Data were taken from Linnemann et al.50 (20 mM Tris-buffer (pH 7.4) containing 5 mM MgCl2, 100 mM NaCl and 2 mM DTE at 10 8C). c Data were taken from Spoerner et al.63 (40 mM Hepes-buffer (pH 7.4) containing 10 mM MgCl2, 150 mM NaCl and 2 mM DTE at 10 8C).

and non-binding RA/RB domains (Tables 1 and 2), we re-examined the sequence alignment of the RA/RB domains in order to identify structural epitopes required for productive complex formation (see accompanying paper39). From this it appears that all RA/RB domains which are experimentally verified as true Ras/Rap binding proteins, present some positively charged, solventexposed Arg/Lys residues in similar positions in b-strands 1 and 2 and in a-helix 1, all located in the interior of the interface (Figure 1(b)). To verify the influence of these residues on complex formation, alanine mutants of these positions in different RA/RB domains were generated and their affinities measured using ITC. Our results are summarized in Table 3 and a representative graphical view of some of the data are shown in Figure 6(a). Mutation of Arg59 (b1), Arg67 (b2) and Lys84 (a1) (to Ala) have variable, but rather drastic effects on the complex formation of Raf with Ras (between 12 and 31-fold), while the effect of the R89L mutation

(a1) is too drastic to be measured accurately using ITC or fluorescence. For Arg89, a contribution to the binding energy of at least 5 kcal/mol was estimated.51 The interaction between Ras and RalGDS is decreased by the R16A (b1) and K28A (b2) mutations. The importance of a positively charged epitope in helix a1 of RalGDS is highlighted by the effect of the D47K and K48A mutations, which increase or decrease affinity, respectively.22,52 For the AF6_RA1 domain, we find that the mutations R44A (b1), K58A (b2) and K87A (a1) reduce binding affinity between 4.5 and 22-fold, for the interaction with both Ras and Rap, such that no binding to Ras could be detected for the R44A mutant (Table 3). Likewise, residues Arg74, Arg83 and Lys101 of spByr2 are required for binding, as are Arg629, Lys642 and Lys662 of RIN1. The K236A, K283A and K303A mutations in mNore1 reduce binding to Ras between 20 and 50-fold, while the already weak binding to Rap is reduced between one- and threefold. This is all consistent with a requirement for

Table 3. Influence of K/R mutations in b1, b2 and a1 on binding affinity to H-Ras and Rap1B bound to GppNHp Relative Kd (Mut/WT) GTP-binding protein H-Ras Rap1B H-Ras H-Ras H-Ras Rap1B H-Ras Rap1B H-Ras Rap1B H-Ras

Effector

Kd (mM)

b1

b2

a1

AF6_RA1 AF6_RA1 RalGDS RafRBD mNore1 mNore1 RASSF1C RASSF1C RIN1 RIN1 spByr2

2.2 0.3 3.5a 0.08b 0.2 2.8 20.2 O 100 1.2 4.3 0.03c

R44A: O 45 R44A: 14.0 R16A: 5.2a R59A: 31.3b K236A: 11.7 K236A: 0.95

K58A: 7.1 K58A: 17.3 K28A: 4.0a R67A: 12.5b K283A: 50.0 K283A: 2.5

R629A: 3.5 R629A: 6.2 R74A: 1333

K642A: O 83 K642A: 2.3 R83A: 1000c

E77K: 0.055/K78A: 6.2 E77K: 0.37/K78A: 4.7 D47K: 0.071a/K48A: 6.7a K84A: 31.3b K302D: 3.75/K303A: 20.9 K302D: 0.36/ K303A: 1.55 R161D: O 5.0 R161D: ! 0.22 T661D: 1.3/ K662A: 4.9 T661D: 0.83/ K662A: 2.3 K101A: 1000c

Measurements were done in 50 mM Tris-buffer (pH 7.4) containing 5 mM MgCl2 and 2 mM DTE at 25 8C using ITC. ITC experiments for AF6 mutants were done at 30 8C. a Data were taken from Kiel et al.52 (ITC-data, 15 mM Hepes-buffer (pH 7.4), 5 mM MgCl2 at 25 8C). b Data were taken from Rudolph et al.47 (ITC data, 50 mM Tris-buffer (pH 7.4) containing 5 mM MgCl2, 100 mM NaCl). c Data were taken from Scheffzek et al.11 (GDI data, 50 mM Tris-buffer (pH 7.4) containing 5 mM MgCl2, 100 mM NaCl and 2 mM DTE at 37 8C).

RA Domain Characterisation

751

Figure 6. Epitopes defining binding affinity and specificity. (a) Changes in free energy of binding for RA/RB domains, with Lys/Arg mutations in b1, b2 and a1 in complex with RasGppNHp. Blue, green and yellow bars show changes in DG8 for mutation of Lys/Arg residues to Ala in b1, b2 and a1, respectively. (b) Specificity of RA/RB domain interactions with Ras and Rap. Influence of charge reversal in epitope a1 on change in dissociation constant (Kd). (c) Surface representation of the RalGDS,21 the Byr211 and the Raf structure,8 taken from the respective complexes. Highlighted are epitopes in b1 (blue), b2 (green) and a1 (yellow) important for affinity between RA/RB domains and Ras proteins. The red spot in a1 indicates the amino acid position that defines the specificity of RA domains towards Rap and Ras: RA domains with a positive charge at this position bind with higher affinity to Ras, while a negative charge in this position leads to preferential binding in complex with Rap.

positively charged residues on b1, b2 and a1, which form the interface in the previously determined structures (Figure 6(c)). While a positive charge in the interface is generally required, it is not sufficient. Thus, the affinity of the second RA domain of AF6 is much lower than the first, although the distribution of positive charges in b1, b2 and a1 are identical.

Obviously, other residues in the epitope modify the affinities, and this is borne out by the threedimensional structures which show a number of non-conserved pair-wise interactions between RB/RA domains and Ras/Rap.7,8,11,21–23 Since structures for the relevant complexes are not available, we investigate the influence of these modifying residues in the accompanying paper,

752 using homology modelling and energy calculations for RA and RB domains in complex with Ras and Rap.39 Specificity of Ras versus Rap binding While Ras and Rap1 have apparently no overlapping biological function, they both bind to the same set of effectors as shown by in vitro and double-hybrid studies. Although the binding affinities in vitro of Ras and Rap, to c-Raf and RalGDS, can be fairly different,8,35 the biological significance of different affinities is unclear. Specificity of binding between proteins of the Ras subfamily and RB/RA domains is borne out and extended by data presented here (Table 1). For AF6_RA1, RalGDS, mNore1, PDZGEF, spByr2 and Krit1, the affinity for Rap is much better than for Ras, while RIN2 and the S. cerevisiae CYR have similar affinities. For mNore1, RIN1, Raf and PLC3_RA2, affinity is highest for Ras. It has been found previously, that residues 30 and 31 (which are Asp30/Glu31 in Ras and Glu30/Lys31 in Rap) and the charge of the residue that is N-terminal to the crucial Lys residue in a1 of the RB/RA domain, are both important for the specificity for Ras and Rap.8 The importance of this epitope to determine the specificity has also been described in two double mutant cycle analysis studies of RalGDS and RGL.51,53 Here, we find that the E77K-mutation in AF6_RA1 leads to a 22-fold increased affinity in complex with Ras, while the affinity in complex with Rap is approximately the same (Table 3; Figure 6(b)). The RalGDS D47K-mutation shows an increased affinity to Ras and no significant change in affinity to Rap. Consistent with this, the K303A mutation eliminates the specificity of mNore1 for Ras/Rap. While the charge reversal mutant K302D also decreases specificity, the effect is not strong enough to reverse it. A dramatic effect is observed for the charge reversal in a1 of RASSF1C. Whereas, no binding to Rap could be detected, the R161D mutant now has an affinity to Rap similar to that of wild-type for Ras, but does no longer bind to Ras.

Discussion Biophysical characterization of Ras-RA/RB interactions We have analysed the interaction of a number of RA/RB domains with H-Ras, Rap and M-Ras using various biophysical solution methods, not all of which are suitable to detect a signal change upon binding. In the case of mant-labelled guanine nucleotides, the change in fluorescence was too small to be used for equilibrium titration studies, but was sufficiently large for kinetic studies, as was observed previously for the Ras/Raf interaction.35,43 Using Ras, labelled with the Aedeans fluorophore on position 32 or 64 (Ras32$GppNHp and

RA Domain Characterisation

Ras64$GppNHp), only some signal changes were sufficiently large to measure equilibrium binding of RA/RB domains. Thus, the absence of a fluorescence signal does not indicate absence of binding. ITC has been shown to be a valuable tool for investigating protein–protein interactions as it does not require any label and directly measures the enthalpy change upon complex formation.54,55 This method has been shown to be well suited to study in detail the thermodynamics of Ras–effector interactions.47,51,52 Using ITC, we were able to determine the affinities and thermodynamic parameters of nearly all expressed domains in complex with H-Ras, Rap and M-Ras. We could not determine any enthalpy change for the interaction of Rain and mTiam1. In order to exclude the possibility that the enthalpy change is zero at standard conditions, we tested binding also at temperatures different from 25 8C, with the same results. Thus, we can rule out zero enthalpy change as the reason for non-binding. However, we cannot discard the possibility that we do not observe any binding due to partial unfolding or incorrect folding of the isolated domains. Binding for Rain and Tiam has been observed by pull-down experiments with the full length proteins and the RA/RB domain which is in contrast to our findings.56,57 The CD spectrum of the RA domain mTiam1 indicates that it might be not folded properly. Surprisingly, Arthur et al. have shown that the DH/PH domain combination is required for the binding of Rap to Tiam, confirming that other parts of the protein can contribute to binding.58 Similarly, Rain could only be purified as GST-fusion protein and the RA domain alone was insoluble after cleavage, indicating folding problems. We find that the relative amount of enthalpy and entropy contributions to the free energy of complex formation vary considerably between the complexes analysed, and often compensate each other. This phenomenon of enthalpy–entropy compensation was found to be a general finding in many protein–protein complexes, especially in water.59–61 We might speculate whether favourable entropy contributions are an indication of a more hydrophobic contact area. Indeed, we find a high correlation between the hydrophobic and polar solvation energy and the entropy (with correlation coefficients of K0.65 and 0.67, respectively) in the modelled complexes (see accompanying paper39). Based on this, a strong entropy contribution might indeed reveal a predominantly hydrophobic contact area. The stopped-flow experiments show saturation kinetics indicative of a two-step binding mechanism.43,49,50 This is clearly in-line with the expectation that an electrostatic complementary surface, as seen in previously published structures of such complexes, enhances the rate constant of association by favouring the formation of a low affinity encounter complex. The rate-limiting isomerization step has a rate constant of between 160 sK1 and 534 sK1 for the various complexes. It is

RA Domain Characterisation

postulated to correspond to the isomerization of Ras (or Rap) between a binding and non-binding conformation, which has been observed by P-NMR studies of various complexes,49,62,63 with a rate constant of O1000 Hz at 25 8C calculated by line shape analysis.62 It is a general mechanism in Ras–effector interactions to achieve moderate to high affinities by a combination of fast association and fast dissociation. This is physiologically relevant for signal transduction since recruitment of effctors is visible within minutes after stimulation of the cells with growth factor, with a peak of Ras activity at the plasma membrane observed after several minutes.64,65 A fast dissociation of the effector complex is also necessary to explain the deactivation of Ras. Since Ras deactivation kinetics cannot be correlated with the intrinsic GTP hydrolysis, RasGAP has to be recruited. This in turn would require the prior release of the effector, since GAP binding and effector interactions are mutually exclusive. Domain specificity For many RA/RB domains we observe a certain specificity of binding towards Ras and Rap, whereas M-Ras behaves more or less similar to Ras. It is arguable whether or not the relative affinities of the isolated domains for Ras and Rap, measured in vitro, reflect the specificity of that interaction in vivo. This question has become even more important since it has become evident that the biological roles of Ras and Rap are different, but that Ras and Rap consistently interact with the same set of effectors, at least in vitro. On the other hand, the downstream effectors and their modes of action are not well understood for many signal transduction pathways activated by Rap, such as integrin and T-cell activation. In the case of AF6/Canoe, consistent with our affinity measurements, there is strong genetic evidence from Drosophila that it acts together with Rap rather than Ras in the same pathway, leading to dorsal closure, Also, although both Ras and Rap interact with full length AF6 as shown by co-precipitation studies, Rap co-localizes with AF6 at the membrane.66 Together with the finding that AF6 associates with the actin cytoskeleton regulator profilin, this indicates a biological function in actin modelling and adhesion complex formation. In addition, AF6 has been found to control integrinmediated cell adhesion by regulating Rap1 activation through a specific recruitment of Rap1GTP and SPA-1.67 In the case of PLC3, it has been demonstrated both in vivo27 and in vitro,68 that binding to Ras and Rap is mediated through its RA2 domain. This is in agreement with our data demonstrating binding of Ras to PLC3_RA2 and PLC3_RA1RA2 but no binding using the first RA domain alone (PLC3_RA1). Concerning the specificity for various Ras proteins, it has been found that Rap1A and Rap2B were able to activate PLC3 significantly (by

753 measuring the increase in level of production of inositol phosphates) but the effect was not so strong as for H-Ras and no activation was observed for RIN and M-Ras.69 Again, this basically agrees with our ITC results showing a higher affinity in complex with H-Ras than with Rap1, and weak binding for M-Ras. PDZGEF has been shown in vitro to bind specifically to Rap1A and Rap2B,70 which agrees with our quantitative analysis. PDZGEF exerts RapGEF function in perinuclear compartments including the Golgi apparatus,71,72 but the function of the RA domain in this RapGEF is unclear. However, since Ras has been shown to bind and to stimulate its own RasGEF Sos in a positive feedback loop,73 PDZ-GEF could be regulated in a similar manner. The tumour suppressor RASSF1 has the potential to serve as a Ras effector and thereby mediate Rasdependent apoptosis.30,74 However, we could only detect weak binding in complex with Ras, and none with Rap, and it is not entirely obvious how the affinity in vitro is correlated to biological activity in vivo. However, mNore1, a homologue of RASSF1 with 48% identity, has a high affinity for Ras which is also much higher than for Rap, in-line with its proposed biological function; it was shown to be a Ras-regulated tumour suppressor in the lung, that mediates Ras-dependent apoptosis.75,76 Conversely, it has recently been shown that an analogue of RasSF1, RapL or RasSF5, is a specific downstream effector of Rap involved in integrin activation.77 RIN1, which was cloned as one of the earliest putative Ras effectors with unknown function,78 has now been identified as a GEF for Rab5. It stimulates receptor mediated endocytosis and thereby links Ras signalling and endocytosis via RIN1,79 which is in agreement with our results, which show higher affinity for Ras (and M-Ras) than for Rap. The function of RIN2 appears to be different,80 and its affinity to Ras is very low, consistent with those data. Nothing is known about the function of Krit1, and its involvement in cerebral cavernous malformations,81,82 but our affinity measurements showing preferred binding to Rap1 are clearly in-line with two-hybrid data.83 There is a certain possibility that additional domains in Ras effectors modify the affinities measured for the isolated RA/RB domains in a positive or negative manner. For example, in the study of Rodriguez-Viciana and co-workers, a much stronger interaction for the full length AF6 has been found with Ras than with Rap,31 while all studies of the single (first) RA domain show a preferential binding to Rap rather than to Ras. In addition, since Ras proteins are inserted into membranes via C-terminal lipophilic modifications, and interactions with effectors occur on the membranes, it is also possible that affinities are modified by the membranes themselves; even weak affinities might lead to productive signalling due to twodimensional enrichment of the interaction partners, on the surface of the membranes. Further studies

754 with full-length Ras effectors are required to answer such questions. The RA/RB family and their interaction with Rasproteins There is a growing list of RA/RB domains in the SMART 36,37 and similar domain databases. Previous structural analysis of a number of these domains, and the conservation of residues comprising the hydrophobic core, indicate that these domains adopt the ubiquitin fold. As described by sequence alignments, these proteins have been subgrouped as Ras association (RA), Ras binding (RB) domains. We show that there are no functional or structural reasons for these categories, since some proteins from the RA domain subfamily are truly Ras (or Rap) effectors and some are not, while the Tiam1-RBD, which better aligns with Raf, does not bind to either Ras or Rap (at least in vitro). Previously, it has been shown that the RA domain from Myr5 is unlikely to bind to Ras or Rap, based on surface exposed residues.38 This is in-line with our results on the requirement for some positively charged epitopes on any of the following secondary structure elements: b1, b2 and a1 of the ubiquitin fold. It would thus be more appropriate to combine all ubiquitin fold proteins into one family and to devise sequence analysis tools that can select true Ras binding proteins from the ubiquitin superfamily. Obviously, it is not the ubiquitin fold per se that is recognized by Ras proteins but rather epitopes on its surface. The specific recognition of a partner protein out of several members of a protein sub-family, having the same fold, is a central property in a lot of biological processes.84 Based on the studies presented here, would it now be possible to predict whether a newly identified RA/RB domain would bind to the (or any) Ras protein? We have shown that positively charged residues on certain epitopes of the fold are more or less essential, such that their presence or absence would be a strong indicator for efficient binding. However, we have also shown that the presence of positively charged residues is usually required, but not sufficient, for binding, and that in rare cases (PLC3) a positive charge can be replaced by a Gln residue. Thus, simple sequence rules cannot be applied to predict interaction between RA/RB domains and Ras proteins. In order to explore systematically the sequence space that leads to productive binding, and to decipher the rules that render an ubiquitin fold protein into a true Rasbinding protein, we have employed model building and energy calculation studies (see accompanying paper39).

Materials and Methods Cloning of RA and RB domains AF6-RA2 (residues 244–351), RIN1 (residues 619–745),

RA Domain Characterisation

RIN2 (residues 782–875) Krit1 (residues 416–524), Krit1 (residues 266–736) and Krit1 (residues 416–736) were amplified from a human WI38 cDNA library (provided by M. Hanzal-Bayer, Dortmund) and scCYR1 (residues 640–780) was amplified from genomic DNA of S. cerivisiae and cloned into the pGEX expression vector. Rain (residues 120–233) (template given by N.Y. Mitin, West Lafayette), RASSF1C (residues 58–218) (template given by R. Dammann, DuarteCA) and mNore1 (residues 199–358) (template kindly provided by J. Avruch, Boston), mTiam1 (residues 736–841) (template kindly provided by J. Collard, Amsterdam), as well as PDZGEF (residues 577–728), PLC3_RA1 (residues 2132–2131), PLC3_RA2 (residues 2001–2243), PLC3_RA1RA2 (residues 2001–2243) (all templates kindly provided by J.L. Bos, Utrecht) were also cloned into pGEX expression vectors. Mutants were generated using the QuikChangee Sitedirected Mutagenesis Kit (Qiagen). All mutants were verified by sequencing. Protein expression and purification The proteins AF6_RA1 (residues 1–142), CRaf (residues 53–131), RalGEF (residues 1–97, corresponding to residues 789–885 of the full length protein) and Byr2 (residues 65–180) were expressed as GST-fusion proteins as described.9,35,49 AF6_RA2, Rain, RASSF1C, mNore1, RIN1, RIN2, PDZGEF, mTiam, PLC3 and Krit1 were expressed as GST-fusion proteins in Escherichia coli BL21 cells in TB medium (containing 50 mg/ml ampicillin). Protein expression was induced by adding 0.1 mM IPTG and incubating overnight at 20–25 8C. Cell lysis was performed in 50 mM Tris-buffer (pH 7.4) containing 5 mM MgCl2, 500 mM NaCl, 5 mM dithioerythritol, 1 mM phenyl-methansulfonyl-fluoride and 1% Triton-X100, using ultrasonication. Protein purification was done using glutathione-Sepharose (Pharmacia) in 50 mM Trisbuffer (pH 7.4) containing 5 mM MgCl2, 500 mM NaCl and 2 mM dithioerythritol. After cleavage with thrombin, overnight on the column, the proteins were eluted. The last purification step was done using gel filtration chromatography (Superdex 75; Pharmacia) in 50 mM Tris-buffer (pH 7.4) and 2 mM dithioerythritol containing 5 mM MgCl2 (for purification of CRaf, Krit 1 and RIN2 100 mM NaCl was added to the buffer). Protein concentration was determined using the Bradford assay using bovine serum albumin as a standard.85 H-Ras (residues 1–189) and Rap1B (residues 1–167) were prepared from E. coli strain CK600K using the ptac-expression system, purification on a Q-Sepharose column and subsequent gel filtration, as described previously.35 M-Ras (1–208) was expressed as a GST fusion protein. Labeling and nucleotide exchange Generation and preparation of Ras32, Ras86 and Rap86 was performed as described.44 Nucleotide exchange to GppNHp, as well as to N-methylanthraniloyl-GppNHp was done as described previously86 and free nucleotides and phosphates were removed by gel filtration. The amount of protein-bound nucleotide was analysed by C18 reverse phase HPLC and quantified with a calibrator detector (Beckman Coulter) and integrator (Shimadzu). Circular dicroism Circular dicroism (CD) spectra in the far-UV region were obtained by using a Jasco-710 spectropolarimeter. Spectra were recorded in buffer containing 25 mM

755

RA Domain Characterisation

phosphate (pH 7.0) at two different protein concentrations (typically 20 mM and 200 mM). 20 scans were acquired in the range 190–250 nm at a temperature of 25 8C by taking points every 0.2 nm, with 100 nm minK1 scan-rate, an integration time of one second and a 1 nm band-width. Cells with path-lengths of 0.1 cm and 0.2 cm were used for the analysis of protein concentrations of 200 mM and 20 mM, respectively. Twenty accumulations were averaged to obtain each spectrum.

We thank Mark Isalan for critical reading of the manuscript.

Supplementary Data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j. jmb.2005.02.048.

Equilibrium titration measurements Equilibrium titration measurements were carried out in a fluorimeter (Perkin–Elmer) at 25 8C in 50 mM Tris-buffer (pH 7.4) containing 5 mM MgCl2 and 2 mM dithioerythritol. Titration was done by adding increasing amount of effectors to 200 nM Ras32$GppNHp, Ras86$GppNHp or Rap86$GppNHp, respectively. The excitation wavelength was in all cases 350 nm, the emission wavelength was 480 nm (for Ras32$GppNHp and Ras86$GppNHp) or 490 nm (for Rap86$GppNHp). The fluorescence signal was monitored until a stable signal was detected, usually after five minutes. Data were fitted according to equation (1): Fmin K ðE C L C Kd Þ K

Acknowledgements

pffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi ðE C L C Kd Þ2 K 4 !E !L !ðFmax K Fmin Þ 2 !E

(1) where Fmin, minimal fluorescence; Fmax, maximal fluorescence; E, concentration of fluorophore (Ras or Rap); L, concentration of ligand (effector).

Isothermal titration calorimetry The thermodynamic parameters are determined using an isothermal titration calorimeter (MSC-ITC, MicroCal, Inc.) as described previously,54,55 at 25 8C, in 50 mM Trisbuffer (pH 7.4) containing 5 mM MgCl2. For binding of GTP-binding proteins to effectors, usually 1 mM H-Ras, Rap1B, or M-Ras (bound to GppNHp) were titrated from the syringe into the sample cell, containing 100 mM effector protein. Titration was carried out by injecting volumes of 8 ml into the sample cell, where the time between injections was four minutes. Further data evaluation was done using the MicroCal Origin program. Several experiments were repeated two or three times and here the average values are shown.

Stopped-flow measurements Stopped-flow measurements were done using an SM17 apparatus (Applied Photophysics) at 10 8C, in 50 mM Tris-buffer (pH 7.4), containing 5 mM MgCl2 and 2 mM dithioerythritol. Association kinetics were measured by rapid mixing of 0.6 mM Ras bound to mant (m) GppNHp or 0.8 mM Ras32$GppNHp, with increasing concentrations of effector proteins, starting from 10 mM, in order to have pseudo first-order conditions. Ras$mGppNHp was excited at 370 nm, Ras32$GppNHp at 350 nm and the fluorescence was recorded through a 408 nm cut-off filter. The observed rate constants were fitted using a single exponential equation to give kobs. Association and dissociation rate constants (kon, koff) were determined as described.43

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Edited by R. Huber (Received 7 September 2004; received in revised form 10 February 2005; accepted 24 February 2005)