Structural analysis of the GAP-related domain from neurofibromin and its implications
1998; Springer Nature; Volume: 17; Issue: 15 Linguagem: Inglês
10.1093/emboj/17.15.4313
ISSN1460-2075
Autores Tópico(s)Meningioma and schwannoma management
ResumoArticle3 August 1998free access Structural analysis of the GAP-related domain from neurofibromin and its implications Klaus Scheffzek Corresponding Author Klaus Scheffzek Max-Planck-Institut für molekulare Physiologie, Rheinlanddamm 201, 44139 Dortmund, Germany Search for more papers by this author Mohammad Reza Ahmadian Mohammad Reza Ahmadian Max-Planck-Institut für molekulare Physiologie, Rheinlanddamm 201, 44139 Dortmund, Germany Search for more papers by this author Lisa Wiesmüller Lisa Wiesmüller Present address: Heinrich Pette Institut für experimentelle Virologie an der Universität Hamburg, Martinistrasse 52, 20251 Hamburg, Germany Search for more papers by this author Wolfgang Kabsch Wolfgang Kabsch Max-Planck-Institut für medizinische Forschung, Jahnstrasse 29, 69120 Heidelberg, Germany Search for more papers by this author Patricia Stege Patricia Stege Max-Planck-Institut für molekulare Physiologie, Rheinlanddamm 201, 44139 Dortmund, Germany Search for more papers by this author Frank Schmitz Frank Schmitz Max-Planck-Institut für molekulare Physiologie, Rheinlanddamm 201, 44139 Dortmund, Germany Search for more papers by this author Alfred Wittinghofer Alfred Wittinghofer Max-Planck-Institut für molekulare Physiologie, Rheinlanddamm 201, 44139 Dortmund, Germany Search for more papers by this author Klaus Scheffzek Corresponding Author Klaus Scheffzek Max-Planck-Institut für molekulare Physiologie, Rheinlanddamm 201, 44139 Dortmund, Germany Search for more papers by this author Mohammad Reza Ahmadian Mohammad Reza Ahmadian Max-Planck-Institut für molekulare Physiologie, Rheinlanddamm 201, 44139 Dortmund, Germany Search for more papers by this author Lisa Wiesmüller Lisa Wiesmüller Present address: Heinrich Pette Institut für experimentelle Virologie an der Universität Hamburg, Martinistrasse 52, 20251 Hamburg, Germany Search for more papers by this author Wolfgang Kabsch Wolfgang Kabsch Max-Planck-Institut für medizinische Forschung, Jahnstrasse 29, 69120 Heidelberg, Germany Search for more papers by this author Patricia Stege Patricia Stege Max-Planck-Institut für molekulare Physiologie, Rheinlanddamm 201, 44139 Dortmund, Germany Search for more papers by this author Frank Schmitz Frank Schmitz Max-Planck-Institut für molekulare Physiologie, Rheinlanddamm 201, 44139 Dortmund, Germany Search for more papers by this author Alfred Wittinghofer Alfred Wittinghofer Max-Planck-Institut für molekulare Physiologie, Rheinlanddamm 201, 44139 Dortmund, Germany Search for more papers by this author Author Information Klaus Scheffzek 1, Mohammad Reza Ahmadian1, Lisa Wiesmüller2, Wolfgang Kabsch3, Patricia Stege1, Frank Schmitz1 and Alfred Wittinghofer1 1Max-Planck-Institut für molekulare Physiologie, Rheinlanddamm 201, 44139 Dortmund, Germany 2Present address: Heinrich Pette Institut für experimentelle Virologie an der Universität Hamburg, Martinistrasse 52, 20251 Hamburg, Germany 3Max-Planck-Institut für medizinische Forschung, Jahnstrasse 29, 69120 Heidelberg, Germany *Corresponding author. E-mail: [email protected] The EMBO Journal (1998)17:4313-4327https://doi.org/10.1093/emboj/17.15.4313 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Neurofibromin is the product of the NF1 gene, whose alteration is responsible for the pathogenesis of neurofibromatosis type 1 (NF1), one of the most frequent genetic disorders in man. It acts as a GTPase activating protein (GAP) on Ras; based on homology to p120GAP, a segment spanning 250-400 aa and termed GAP-related domain (NF1GRD; 25-40 kDa) has been shown to be responsible for GAP activity and represents the only functionally defined segment of neurofibromin. Missense mutations found in NF1 patients map to NF1GRD, underscoring its importance for pathogenesis. X-ray crystallographic analysis of a proteolytically treated catalytic fragment of NF1GRD comprising residues 1198-1530 (NF1-333) of human neurofibromin reveals NF1GRD as a helical protein that resembles the corresponding fragment derived from p120GAP (GAP-334). A central domain (NF1c) containing all residues conserved among RasGAPs is coupled to an extra domain (NF1ex), which despite very limited sequence homology is surprisingly similar to the corresponding part of GAP-334. Numerous point mutations found in NF1 patients or derived from genetic screening protocols can be analysed on the basis of the three-dimensional structural model, which also allows identification of the site where structural changes in a differentially spliced isoform are to be expected. Based on the structure of the complex between Ras and GAP-334 described earlier, a model of the NF1GRD-Ras complex is proposed which is used to discuss the strikingly different properties of the Ras-p120GAP and Ras-neurofibromin interactions. Introduction Neurofibromatosis type 1 (NF1), also termed von Recklinghausen neurofibromatosis, is a common autosomal dominant disease affecting ∼1 in 3500 individuals (Riccardi, 1981, 1991; Riccardi and Eichner, 1986). Multiple so-called 'café au lait' spots, Lish nodules of the iris, and neurofibromas, benign cutaneous tumours, are hallmarks of the disease with important diagnostic relevance. While several manifestations are benign in character some of them involve a significant risk of developing malignant or fatal traits, such as plexiform lesions or optic glioma (Gutmann and Collins, 1993). Therefore, NF1 has been grouped together with familial cancer syndromes (Bader, 1986). The NF1 gene has been identified to code for a large transcript that is disrupted or mutated in patients affected with NF1 (Cawthon et al., 1990; Viskochil et al., 1990; Wallace et al., 1990). Since the gene appeared to be inactivated in tumours it has been postulated to act as a tumour suppressor (Ponder, 1990; Stanbridge, 1990). In spite of conflicting genetic results, experimental evidence accumulated during the past few years indicate that loss of heterozygosity (LOH) precedes fibrosarcomas and other lesions, with a somatic mutation following an inherited mutant allele (Serra et al., 1997). NF1 codes for a protein comprising 2818 amino acids (Marchuk et al., 1991) termed neurofibromin (∼280 kDa) that is found in the particulate fraction bound to a very large protein (DeClue et al., 1991). Analysis of the predicted protein sequence revealed homology between a segment spanning 250 amino acids with high and an additional 150 amino acids with lower homology to Ras-specific GTPase activating proteins (Xu et al., 1990a) such as p120GAP (Trahey and McCormick, 1987) and its yeast homologues IRA1 and IRA2 (Tanaka et al., 1990a,b, 1991). Biochemical analyses and functional complementation tests in yeast established this segment as a functional GAP-related domain (NF1GRD) that is able to stimulate GTP-hydrolysis on normal but not oncogenic Ras (Ballester et al., 1990; Martin et al., 1990; Xu et al., 1990b), thus linking neurofibromatosis to the regulation of the Ras-MAP kinase pathway and its activation by oncogenes (Lowy and Willumsen, 1993). It has indeed been found that Ras is mostly in the GTP-bound form in cell lines established from malignant tumours without functional neurofibromin (Basu et al., 1992; DeClue et al., 1992; Guha et al., 1996), suggesting that neurofibromin is a major regulator of Ras in the parental cells due to its GAP activity. Further studies indicate that another mechanism of NF1-mediated tumour suppression is possibly independent of its GAP activity (Nakafuku et al., 1993; Johnson et al., 1994; Griesser et al., 1995). Four differentially spliced isoforms have been detected so far (Gutmann et al., 1995), one of which, the type II transcript, is characterized by a 21 amino acids insertion within the GAP-related domain (GRD; Nishi et al., 1991; Suzuki et al., 1991; Andersen et al., 1993). Somatic mutations in NF1 have been shown to map to the GRD and affect the interaction with Ras (Li et al., 1992; Purandare et al., 1994; Upadhyaya et al., 1997; Klose et al., 1998), underscoring the importance of this part of the protein for proper neurofibromin function. Neurofibromin has been shown to associate with microtubules (Gregory et al., 1993) thus linking its function to aspects of structural reorganization of the cytoskeleton. It binds tubulin and the binding region presumably overlaps with NF1GRD since tubulin inhibits GAP activity (Bollag et al., 1993). The fundamental physiological importance of neurofibromin is underscored by the observation that mice with a targeted disruption of the neurofibromin locus are embryonically lethal and show abnormalities of neural crest-derived tissues (Brannan et al., 1994; Jacks et al., 1994). We have previously determined the structure of GAP-334 (Scheffzek et al., 1996), the NF1GRD homologue in p120GAP, and of its complex with Ras (Scheffzek et al., 1997). Together with biochemical analyses (Mittal et al., 1996; Ahmadian et al., 1997b) this enabled us to characterize the Ras-RasGAP interaction and to elucidate the mechanism of GTPase activation. In order to address the specific issues underlying neurofibromin function, we report here the crystal structure of NF1GRD, determined from a proteolytically treated fragment comprising residues 1198-1530 (NF1-333). Significant but limited homology with GAP-334 suggested structural similarity but also differences that might account for the strikingly different biochemical behaviour of the two proteins: (i) on a structural level, the minimal domain with full catalytic activity obtainable by limited proteolysis or recombinant expression is 270 residues for p120GAP, compared with 230 for neurofibromin (Ahmadian et al., 1996). (ii) For neurofibromin, a fragment of 91 residues has been defined to retain some catalytic activity and yet does not contain residues thought to be critical for catalysis, and an even smaller fragment with anti-oncogenic activity from the C-terminus of the catalytic fragment has been reported (Nur-E-Kamal et al., 1993; Fridman et al., 1994). The putative catalytic activity of these fragments could not be reconciled with the structure of GAP-334 or of its complex with Ras, raising the possibility of significant deviations in the NF1GRD-Ras interaction pattern. (iii) The affinity of neurofibromin for Ras-GTP is 50- to 100-fold higher than that of p120GAP, and the kinetics of association and dissociation are much faster for the latter (Eccleston et al., 1993; Ahmadian et al., 1997a). (iv) Although the catalytic activity of both GAPs is generally sensitive to the presence of detergents, specific inhibition of neurofibromin can be obtained by applying selective compounds such as dodecyl maltoside (Bollag and McCormick, 1991). On the basis of our structural model we analyse these features together with mutations found in NF1 patients or derived from mutational studies. In addition, we discuss aspects of NF1-tubulin interaction and, on the basis of the Ras-RasGAP complex, propose a Ras-NF1GRD complex model that should be an approximation of the transition state of the GTPase reaction. Results and discussion Structure determination and model quality The structure was determined by X-ray crystallographic analysis of crystals obtained from proteolytically treated NF1-333 (described in Materials and methods). Since attempts to solve the structure by molecular replacement using the coordinates of GAP-334 (protein data bank accession code 1WER) as a search model were not successful, we used the Multiple Isomorphous Replacement (MIR) method. Using mercury and platinum derivatives we obtained a heavy atom model suitable for initial phase determination with a data set of 2.5 Å resolution, collected from six untreated crystals on the synchrotron beam line X11 at EMBL (c/o DESY; Hamburg, Germany), as native reference (Table I; Materials and methods). The initial electron density map (Figure 1A) was readily interpretable and could accommodate a Cα-model corresponding to the central domain (GAPc) of GAP-334 (Scheffzek et al., 1996), which served as a guide during subsequent model building and refinement. Remaining density within the asymmetric unit could be explained in part by N-terminal extension of this model up to residue 1206. Two further helices turned out to represent segments derived from the C-terminal region of NF1-333 (Figure 1A). Analysis of dissolved crystals or of the protein solution used for crystallization by polyacrylamide gel electrophoresis revealed two major components to be present in the crystals. N-terminal sequencing along with mass spectroscopy suggested that protease treatment of NF1-333 had nicked the polypeptide chain close to the position of Ser1474. The structural integrity of NF1-333 appears to remain basically unaffected by protease digestion. The observed crystal packing is consistent with an NF1GRD protein cleaved in the segment containing the proteinase K cleavage site, and would not be compatible with uncleaved NF1-333 in the asymmetric unit. Figure 1.Aspects of structure determination (stereo views). (A) Experimental MIR map, (contoured at 20% of the maximum) calculated with phases derived from the heavy atom model. The Cα-traces of the central (blue) and extra domain (yellow) are included. (B) Segment of the 2Fo−Fc map (contoured at 1.2 σ) covering the C-terminal half of helix α7c and the variable loop (L6c) after structure refinement with the model included. A segment of a neighbouring molecule covering residues 1373-1377 [see (C)] is shown in pink. (C) Section comparing NF1GRD (blue) and GAP-334 (red) in the region of the loop preceding α6c, showing a three residue insertion in GAP-334 (see Figure 2B). Arginine 1375 contacts the N-terminal region of the finger loop. The corresponding situation is found for Lys884 in GAP-334. Download figure Download PowerPoint Table 1. Structure determination Crystal: a = 88.2 Å; b = 58.3 Å; c = 74.8 Å; α = γ = 90°, β = 118.1°; spacegroup C2 Native CH3HgCla CH3HgCl/K2PtCl4b ICH2COOH/K2PtCl4c Data collection: resolution (Å) 2.5 3 3.5 3 No. obs. reflections 50 100 14 694 13 533 18 658 No. uniq. reflections 11 665 6191 4019 6262 completeness (%) 99.8 91.6 96.5 92.6 Rsymd (%) 7.3 5.4 6.3 4.9 RFe (%) - 33.1 38.3 26.3 MIR-analysis: resolution (Å) 15-2.5 15-3 15-3.5 15-3 No. sites - 3 5 3 RCf - 0.5 0.43 0.76 FH/Eg - 1.8 2.1 0.9 h 0.5 a 1 mM, 12 h; b 1 mM, 7 h/+0.1 mM, 15 h; c 2 mM, 24 h/0.2 mM, 23 h. d , where Ihi is the scaled intensity of the ith symmetry-related observation of reflection h, and Ih is the mean value. e , where FPH and FP are the derivative and native structure amplitudes. f g , where FH are the heavy atom scattering factors. h = mean figure of merit. A high degree of mobility appears to be characteristic of the NF1-333 in our crystals with extensive regions remaining without well defined electron density. In successive rounds of interactive model building (program 'O'; Jones et al., 1991 and refinement (program X-PLOR; Bruenger, 1991), residues showing sufficient electron density were incorporated into the model, that presently contains 260 residues of NF1-333 defining regions 1206-1304, 1331-1403, 1412-1463, 1485-1503 and 1514-1530, with interspacing segments predominantly ill-defined in the electron density map. In the present model, ∼30 residues were built as alanine or glycine because of unclear side-chain density. A segment of the final 2Fo−Fc map is shown in Figure 1B. Refinement statistics and model quality are summarized in Table II, including those for the GAP-334 model which had not been completed at the time of its publication (Scheffzek et al., 1996). The current R-factor is 27% (Rfree = 37%), which is comparatively high and at least in part reflects residual electron density that could not be explained in terms of ordered polypeptide chains. For the following reasons we are very confident that the current NF1GRD model is largely correct: (i) we used the MIR method for structure determination, thus our initial phases are unbiased by the GAP model that only served as a guide for model building; (ii) coordinates of heavy atom sites are consistent with the mercury compound binding to cysteines; (iii) a loop which by sequence comparison contains three amino acids less than the corresponding stretch in GAP-334 is correspondingly shorter in NF1-333 (Figure 1C; see below). Thus, together with the results of structure refinement, several lines of evidence indicate that our structure determination presents a valid model of NF1GRD, suitable for analysis of various mutations associated with NF1 and with the structure of the Ras-GAP-334 complex at hand to discuss effects on the interaction with Ras. Table 2. Refinement statistics Refinement NF1-333 GAP-334a Resolution (Å) 30-2.5 5-1.6 No. reflections 10 926 (F >2 s) 40 803 Rcrystb (%) 27 22.1 Rfreec (%) 37 27.1 (Å2) 46.8 26.1 r.m.s. bond length (Å) 0.008 0.007 r.m.s. bond angle (°) 1.2 0.9 a Refinement statistics for the final model as released in the PDB. b , where Foh and Fch are the observed and calculated structure factor amplitudes for reflection h. c R-factor calculated for 10% randomly chosen reflections not included in the refinement. Overall structure The purely helical protein is similar in structure to GAP-334 with a central domain (NF1c; segments 1253-1304, 1331-1403 and 1412-1463) containing the conserved residues and an extra domain (NF1ex) that comprises segments 1206-1252, 1485-1503 and 1514-1530 (Figure 2A and C). For graphic representation and clarity the regions missing from our NF1GRD model were complemented by the corresponding segments of the GAP-334 model where they are mostly well-defined. The two structures superimpose (Figure 2B) with an r.m.s.d. value of 1.83 Å when comparing the positions of 243 corresponding Cα-atoms using program 'O' (Jones et al., 1991). The largest differences are seen in the extra domain where helix α1ex appears to be shifted by ∼3 Å in comparison with the corresponding helix in GAPex. The central domain coincides with a minimal catalytic fragment of neurofibromin with full GAP activity, which was originally obtained by proteolysis and can be expressed as a recombinant fusion protein and cleaved (Ahmadian et al., 1996). Interestingly, the corresponding fragment of p120GAP could not be expressed in Escherichia coli as a soluble protein. This suggests that despite close similarities in the primary sequence, subtle differences exist in the interaction patterns between the extra and the central domain in these two GAPs, and might influence flexibility and/or exposure of hydrophobic residues. With regard to the absence of sequence homology within the extra domains of GAP-334 and NF1GRD, and to the fact that they are not involved in the interaction with Ras-GTP, their structures are surprisingly similar. This suggests a conserved function for this part of the proteins, possibly serving structural 'assistance' for the central catalytic GAP modules. A detailed analysis of this issue will have to await the structure determination of other RasGAPs, which show no or very limited sequence homology outside the central catalytic domain (Figure 2C). Figure 2.Structure of NF1-333. (A) Ribbon representation of the NF1-333 model. The central domain (NF1c) is in blue and the extra domain (NF1ex) in yellow. Regions that are not visible in our model were 'complemented' by the corresponding segments derived from the GAP-334 model and are shown in red (see Figure 2B). Helices α6c and α7c forming the bottom of the Ras-binding groove are shown in light blue. Side chains of selected residues presumably involved in polar interactions with Ras (see text) are indicated. K1436 (light grey) appears to be flexible and is modelled stereochemically. (B) Structural overlay of NF1-333 (blue) and GAP-334 (red) in stereo representation. (C) Sequence alignment of RasGAPs using the models of NF1-333 and GAP-334 as references; assignment of the secondary structure elements is according to the program DSSP (Kabsch and Sander, 1983), thin bars indicate 310 helices, dotted lines regions of high mobility. Abbreviations of species: hs, Homo sapiens; sc, Saccharomyces cerevisiae; bt, Bos taurus; ss, Sus scrofa; rn, Rattus norvegicus; mm, Mus musculus; ce, Caenorhabditis elegans; dm, Drosophila melanogaster. The assignment for GAP-334 is based on the final refined model (Table II) deposited with the Protein Data Bank (PDB; accession code 1WER). The minimum catalytic domain (NF230) and the fragment reported by Nur-E-Kamal et al. (1993) (NF91) are indicated as are the proteinase K cleavage site (PK) and the location of the type II transcript insertion (21 aa ins) (see Figure 3). The positions of patient mutations are highlighted in yellow. Invariant residues are in red, conserved residues in blue. Amino acids reportedly involved in Ras-RasGAP interaction (Scheffzek et al., 1997) are shown in light blue boxes for p120GAP. Download figure Download PowerPoint Regions of high mobility predominantly include segments connecting helices as indicated in Figure 2A and B. As extrapolated from the GAP-334 model, an extended chain linking the C-terminal end of NF1c to NF1ex contains the proteinase K cleavage site; since the corresponding region is flexible in GAP-334 as well, it is unlikely that this is due to cleavage in NF1-333: rather, flexibility makes this region more accessible to the protease. It is possible that the extensive mobility which is also reflected in the high Wilson B-factor (∼50 Å2) provides an explanation why poor diffraction quality is common in most of our previously obtained crystals containing fragments of NF1GRD. The Ras-binding groove Given the similarity of GAP-334 and NF1-333, we use corresponding structural annotations and can identify the Ras-binding site as the groove in the surface of NF1c, which is bordered mainly by the finger loop (L1c) along with part of helix α2c and by the variable loop (L6c; Figure 2A and C) (Scheffzek et al., 1997). Helix α6c is less distorted than in GAPc with only Phe1392, next to the FLR-finger print motif of RasGAPs, interrupting the ideal helix geometry. Various hydrophobic and polar residues cover the floor of the groove, comprising conserved/invariant residues and amino acids that are different from their GAP-334 counterparts. The most prominent deviation is seen in position 1419 where a lysine protrudes into the groove, corresponding to an isoleucine (Ile931) in GAP-334 (see below). p120GAP and NF1GRD are inhibited by a large number of lipids, some of which act differentially (Bollag and McCormick, 1991; Golubic et al., 1991; Serth et al., 1991; Tsai et al., 1991). Using appropriate concentrations of arachidonate, phosphatidate and phosphatidylinositol-3,4-bisphosphate, NF1GRD can be inhibited without disturbing p120GAP activity. Likewise, dodecyl maltoside has been used to distinguish between neurofibromin and p120GAP activity in crude cell extracts (Bollag and McCormick, 1991). It is probable that the amino acid composition in the groove region contributes significantly to these differential properties. The structure suggests that these properties arise from a few residues in this region. Mutational analysis would be a powerful tool to address this question in more detail. The finger loop appears to be more flexible than in GAP-334; as in GAP-334 the stretch preceding the finger arginine (Arg1276) is stabilized by a phenylalanine and a leucine participating in a hydrophobic core. The orientation of the loop is stabilized by the FLR-arginine (Arg1391), the structural and functional equivalent of Arg903 in GAP-334. The N-terminal region of the finger loop appears to be stabilized by Arg1375 which is packed against the neighbouring Phe1376 and belongs to the loop connecting helices α5c and α6c. In GAP-334 an equivalent situation can be observed, where Lys884, packed against Trp885, interacts with the N-terminal part of the finger loop. The loop carrying Lys884 contains three residues more than the NF1GRD counterpart and therefore appears as a 'bulge' in a structural overlay (Figure 1C). The variable loop is in a similar conformation to that which it adopts in GAP-334 with Ala948 as an extra amino acid in GAP-334, in support of the idea that this loop is of variable length in RasGAPs (Scheffzek et al., 1997). The minimum catalytic domain, a controversy? Conflicting evidence to localize the minimal fragment of NF1GRD able to stimulate Ras-mediated GTP-hydrolysis has been reported. Originally, a 483 residue fragment of neurofibromin was classified as a GAP-related domain (Martin et al., 1990), and was later shown to have similar enzymatic properties as full-length neurofibromin (Bollag and McCormick, 1993). In analogy to p120GAP where the 334 C-terminal amino acids were shown to be sufficient for GAP activity (Marshall et al., 1989), NF1-333, homologous in sequence to GAP-334, and similar constructs were shown to be fully active and were extensively analysed kinetically and by equilibrium methods (Wiesmüller and Wittinghofer, 1992; Brownbridge et al., 1993; Eccleston et al., 1993; Nixon et al., 1995; Ahmadian et al., 1997a). Finally, proteolysis experiments and recombinant expression studies showed a 230 residue fragment (Asp1248 to Phe1477; Figure 2C) to retain full catalytic activity (Ahmadian et al., 1996). From these experiments together with the crystal structures of GAP-334 (Scheffzek et al., 1996) and NF1GRD (this paper) we conclude that the central domains indeed represent minimum RasGAP-modules. Using a deletion cloning approach a fragment of 91 amino acids (NF91; 1441-1531; Figure 2C) was identified to reverse the malignant phenotype induced by v-H-Ras, and to carry GAP activity, although 20-fold lower than NF1GRD (Nur-E-Kamal et al., 1993). In the structure this fragment would start at the C-terminal end of the variable loop and comprise helices α8c, α4ex-α6ex (Figures 2C and 3), and thus be located completely outside the region that has been found to interact with Ras in the Ras-RasGAP complex (Scheffzek et al., 1997) and contains none of the critical residues of RasGAPs. One would have to postulate that the GAP activity reported for the NF91 fragment or its smaller relative NF78 (Fridmann et al., 1994) occurs by a mechanism that uses a protein-protein interface different from that described for the Ras-RasGAP complex. Site-directed mutagenesis of Arg1441 which is in a region reported to be critical for the function of this fragment might be a useful approach to address this issue. Figure 3.Sites of NF1GRD mutations. Ribbon drawing of NF1-333 to illustrate the location of mutations found in NF1-patients (light grey spheres, indicated in grey boxes) or derived from mutational analyses (dark grey spheres). The position where 21 amino acids are inserted in the type II transcript is indicated. The extension of the Δ53 deletion (Gutmann et al., 1993) is shown in grey, the NF91 fragment reported by Nur-E-Kamal et al. (1993) is in green. Download figure Download PowerPoint Patient mutations and structure-function studies Locations of mutations discussed in this section are summarized in the model shown in Figure 3. A variety of alterations in the NF1 gene have been found in tissues from NF1 patients, with no apparent hot-spot region (Shen et al., 1996). Very large deletions of up to 190 kb, and nonsense mutations all lead to truncation of the protein product, whereas small deletions induce frameshifts. A limited number of missense mutations has been described, 20% of which are found in the catalytic and 1% in the extra domain of NF1GRD, some of which affect the GAP-activity (Figures 2A and C, and 3) (Li et al., 1992; Purandare et al., 1994; Upadhyaya et al., 1997; Klose et al., 1998; P.Nürnberg, unpublished). Lysine 1423 appears to be the most frequently altered residue and has been found mutated to glutamate or glutamine in neurofibromas as well as in solid tumours not associated with neurofibromatosis, which reportedly inhibits the GAP activity (Li et al., 1992; Upadhyaya et al., 1997). Extensive analyses of this residue by site-directed mutagenesis have tested every natural amino acid substitution along with biochemical/biological characterization of the mutant proteins (Poullet et al., 1994). These studies showed that the original wild-type residue lysine in position 1423 is the only amino acid that results in a functional protein, and suggest decreased Ras affinity as the major effect of Lys1423 mutations (Poullet et al., 1994), in line with earlier data on Lys1423→Ser by Gutmann et al. (1993). In the structure, Lys1423 is located on helix α7c from which it protrudes into the surface groove to interact with Glu1437 (Figures 1B and 4). As an analogous interaction is weak in isolated GAP-334 (Scheffzek et al., 1996) but very prominent in complex with Ras (Scheffzek et al., 1997), it appears to be additionally stabilized during the interaction with the Ras target; it is conceivable that charge inversion in this region (as in the mutation Lys1423→Glu) not only disrupts a favourable internal interaction but might also contribute to the accumulation of negative charges in the interface region which is unfavourable for the mostly acidic effector region of Ras entering the surfac
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