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• W2020207222 abstract "The sterile α motif (SAM) is a 65-70-amino acid domain found in over 300 proteins that are involved in either signal transduction or transcriptional activation and repression. SAM domains have been shown to mediate both homodimerization and heterodimerization and in some cases oligomerization. Here, we present the solution structure of the SAM domain of the Saccharomyces cerevisiae protein, Ste50p. Ste50p functions as a modulator of the mitogen-activated protein kinase (MAPK) cascades in S. cerevisiae, which control mating, pseudohyphal growth, and osmo-tolerance. This is the first example of the structure of a SAM domain from a MAPK module protein. We have studied the associative behavior of Ste50p SAM in solution and shown that it is monomeric. We have examined the SAM domain from Ste11p, the MAPK kinase kinase that associates with Ste50p in vivo, and shown that it forms dimers with a self-association Kd of ∼0.5 mm. We have also analyzed the interaction of Ste50p SAM with Ste11p SAM and the effects of mutations at Val-37, Asp-38, Pro-71, Leu-73, Leu-75, and Met-99 of STE50 on the heterodimerization properties of Ste50p SAM. We have found that L73A and L75A abrogate the Ste50p interaction with Ste11p, and we compare these data with the known interaction sites defined for other SAM domain interactions. The sterile α motif (SAM) is a 65-70-amino acid domain found in over 300 proteins that are involved in either signal transduction or transcriptional activation and repression. SAM domains have been shown to mediate both homodimerization and heterodimerization and in some cases oligomerization. Here, we present the solution structure of the SAM domain of the Saccharomyces cerevisiae protein, Ste50p. Ste50p functions as a modulator of the mitogen-activated protein kinase (MAPK) cascades in S. cerevisiae, which control mating, pseudohyphal growth, and osmo-tolerance. This is the first example of the structure of a SAM domain from a MAPK module protein. We have studied the associative behavior of Ste50p SAM in solution and shown that it is monomeric. We have examined the SAM domain from Ste11p, the MAPK kinase kinase that associates with Ste50p in vivo, and shown that it forms dimers with a self-association Kd of ∼0.5 mm. We have also analyzed the interaction of Ste50p SAM with Ste11p SAM and the effects of mutations at Val-37, Asp-38, Pro-71, Leu-73, Leu-75, and Met-99 of STE50 on the heterodimerization properties of Ste50p SAM. We have found that L73A and L75A abrogate the Ste50p interaction with Ste11p, and we compare these data with the known interaction sites defined for other SAM domain interactions. Mitogen-activated protein kinase (MAPK) 1The abbreviations used are: MAPK, mitogen-activated protein kinase; MAPKK, MAPK kinase; MAPKKK, MAPK kinase kinase; SAM, sterile α motif; MOPS, 3-(N-morpholino)propanesulfonic acid; HSQC, heteronuclear single quantum correlation; NOESY, nuclear Overhauser effect spectroscopy; GST, glutathione S-transferase; ph, polyhomeotic; ML, mid-loop; EH, end helix; wt, wild type; RMS, root mean square; TROSY, transverse relaxation optimized spectroscopy.1The abbreviations used are: MAPK, mitogen-activated protein kinase; MAPKK, MAPK kinase; MAPKKK, MAPK kinase kinase; SAM, sterile α motif; MOPS, 3-(N-morpholino)propanesulfonic acid; HSQC, heteronuclear single quantum correlation; NOESY, nuclear Overhauser effect spectroscopy; GST, glutathione S-transferase; ph, polyhomeotic; ML, mid-loop; EH, end helix; wt, wild type; RMS, root mean square; TROSY, transverse relaxation optimized spectroscopy. cascades are highly conserved signaling modules found across the eukaryota. A typical MAPK module consists of three levels of protein kinases, a MAPK, a MAPK kinase (MAPKK), and a MAPKK kinase (MAPKKK) that transduce signals via sequential activation of these kinases by phosphorylation. Saccharomyces cerevisiae has five discrete MAPK modules that transduce extracellular stimuli, e.g. mating pheromone, nitrogen starvation, hyperosmolarity, perturbations in cell wall integrity, and signals that control the synthesis of spore walls (1.Banuett F. Microbiol. Mol. Biol. Rev. 1998; 62: 249-274Crossref PubMed Google Scholar). The mating pheromone response controls conjugation and mating. The pheromone binds to a receptor (Ste2p or Ste3p) and activates a heterotrimeric G protein (Gα, Gpa1p, Gβ, Ste4p, and Gγ, Ste18p). The Gβγ dimer is responsible for transducing the signal to the MAPKKK Ste11p via a path that involves Ste50p, Ste20p (a PKA-related kinase), and Ste5p (a scaffold protein). The MAPKKK Ste11p then phosphorylates the MAPKK Ste7p, which goes on to activate the MAPK, Fus3p (2.Marsh L. Neiman A.M. Herskowitz I. Annu. Rev. Cell Biol. 1991; 7: 699-728Crossref PubMed Scopus (155) Google Scholar). Activation of the transcription factor Ste12p then leads to the transcription of pheromone inducible genes, e.g. FUS1. Ste50p is thought to be a modulating protein that is found constitutively bound to Ste11p and that functions to sustain and prolong the pheromone-induced signaling response (3.Xu G. Jansen G. Thomas D.Y. Hollenberg C.P. Rad M.R. Mol. Microbiol. 1996; 20: 773-783Crossref PubMed Scopus (47) Google Scholar). The formation of pseudohyphae and invasive growth in response to nitrogen starvation also involves a MAPK cascade involving Ste11p and Ste50p (4.Rad M.R. Jansen G. Buhring F. Hollenberg C.P. Mol. Gen. Genet. 1998; 259: 29-38Crossref PubMed Scopus (40) Google Scholar). Starvation signals are routed through Ras2p, which, acting via Cdc42p (a Rho family small G protein), Ste20p and Ste50p, activates Ste11p. In this cascade the MAPKK is Ste7p, and the MAPK is Kss1p. Kss1p then goes on to activate the Tec1p-Ste12p transcription complex, which in conjunction with Flo8p co-transactivates genes, e.g. FLO11, that are essential for pseudohypha formation. The response to osmotic stress involves two MAPK cascades. The osmosensor Sln1p/Ypdp/Ssk1p activates the MAPKKKs Ssk2p and Ssk22p that activate the MAPKK Pbs2p and finally the MAPK Hog1p. The Sho1p osmosensor on the other hand activates Ste11p with the cooperation of Ste50p, which then activates Pbs2p and Hog1p. Thus, Pbs2p/Hog1p can be activated independently by two separate osmotic sensors. Although Ste50p was originally identified as a modulator of the pheromone response pathway in S. cerevisiae (5.Rad M.R. Xu G. Hollenberg C.P. Mol. Gen. Genet. 1992; 236: 145-154Crossref PubMed Scopus (41) Google Scholar), it has since been shown to have only a moderate effect on mating. It has, however, been demonstrated that Ste50p is absolutely required for the Sho1p/Ste11p control of the HOG (high osmolarity glycerol) pathway and that Ste50p may be an integral part of the Ste11p MAPKKK (6.Posas F. Witten E.A. Saito H. Mol. Cell. Biol. 1998; 18: 5788-5796Crossref PubMed Scopus (123) Google Scholar). The interaction between Ste50p and Ste11p is mediated by the N-terminal regions of these proteins (6.Posas F. Witten E.A. Saito H. Mol. Cell. Biol. 1998; 18: 5788-5796Crossref PubMed Scopus (123) Google Scholar), each of which includes a sterile α motif (SAM) domain. The SAM domain was first identified as a 65-70-residue domain found in 14 proteins including Ste50p, Ste11p, and their Schizosaccharomyces pombe homologues, Ste4 and Byr2 (7.Ponting C.P. Protein Sci. 1995; 4: 1928-1930Crossref PubMed Scopus (142) Google Scholar). Refined searches identified 60 further SAM domain-containing proteins (8.Schultz J. Ponting C.P. Hofmann K. Bork P. Protein Sci. 1997; 6: 249-253Crossref PubMed Scopus (270) Google Scholar) including the Eph family of receptor tyrosine kinases. SAM domains are now known to exist in over 300 proteins. The protein set that contains SAM domains suggested that the SAM domain is a conserved protein-protein interaction domain found in proteins involved in development and signal transduction. Extensive work on many SAM-containing proteins has shown that the SAM domains do indeed mediate homodimerization and heterodimerization. Proteins known to dimerize through SAM domain interactions now include not only receptors such as the Eph family but also many transcription factors, e.g. the Ets family (9.Slupsky C.M. Gentile L.N. Donaldson L.W. Mackereth C.D. Seidel J.J. Graves B.J. McIntosh L.P. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 12129-12134Crossref PubMed Scopus (122) Google Scholar), where the domain is known as the pointed (Pnt) domain, the polyhomeotic proteins, and the p63/p73 members of the p53 family of tumor suppressor proteins (10.Thanos C.D. Bowie J.U. Protein Sci. 1999; 8: 1708-1710Crossref PubMed Scopus (126) Google Scholar). SAM domains have also been shown to bind other non-SAM proteins and protein motifs, e.g. protein-tyrosine phosphatases (11.Stein E. Lane A.A. Cerretti D.P. Schoecklmann H.O. Schroff A.D. Van Etten R.L. Daniel T.O. Genes Dev. 1998; 12: 667-678Crossref PubMed Scopus (367) Google Scholar, 12.Serra-Pages C. Kedersha N.L. Fazikas L. Medley Q. Debant A. Streuli M. EMBO J. 1995; 14: 2827-2838Crossref PubMed Scopus (293) Google Scholar), PDZ domains (13.Hock B. Bohme B. Karn T. Yamamoto T. Kaibuchi K. Holtrich U. Holland S. Pawson T. Rubsamen-Waigmann H. Strebhardt K. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9779-9784Crossref PubMed Scopus (174) Google Scholar), and SH2 domains (14.Stein E. Cerretti D.P. Daniel T.O. J. Biol. Chem. 1996; 271: 23588-23593Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). Ste11p appears to be under complex regulatory control. The protein has an N-terminal SAM domain (residues 14-86) that binds to the Ste50p SAM domain (residues 27-109). Residues 133-335 of Ste11p have two different functions: to negatively regulate the C-terminal kinase domain and to mediate the interaction between Ste11p and Ste5p (15.Wu C.L. Leberer E. Thomas D.Y. Whiteway M. Mol. Biol. Cell. 1999; 10: 2425-2440Crossref PubMed Scopus (67) Google Scholar). Binding of Ste50p SAM to the Ste11p SAM appears to alleviate the negative intramolecular regulation of residues 133-335 on the Ste11p kinase domain. It is tempting to speculate that binding of the SAM domain induces structural changes in the regulatory domain of Ste11p allowing activation of the kinase domain. However, it appears that Ste11p and Ste50p are constitutively bound in vivo, so binding per se, although necessary for activation of Ste11p, is not sufficient (15.Wu C.L. Leberer E. Thomas D.Y. Whiteway M. Mol. Biol. Cell. 1999; 10: 2425-2440Crossref PubMed Scopus (67) Google Scholar). In fact, it seems that a C-terminal region of Ste50p is also required for a productive interaction with Ste11p. Although this C-terminal region has no identifiable domains, it is notable that it has a high degree of homology to the C-terminal region of its homologue in S. pombe Ste4. This region in Ste4 is also required for correct function (16.Tu H. Barr M. Dong D.L. Wigler M. Mol. Cell. Biol. 1997; 17: 5876-5887Crossref PubMed Scopus (71) Google Scholar). It seems that Ste11p undergoes different levels of regulation in the distinct pathways in which it is involved. In the HOG pathway Ste50p is absolutely required and is sufficient to activate Ste11p. However, in the mating response where Ste50p is important but not essential, activation of Ste11p requires interactions with both Ste50p and Ste5p (15.Wu C.L. Leberer E. Thomas D.Y. Whiteway M. Mol. Biol. Cell. 1999; 10: 2425-2440Crossref PubMed Scopus (67) Google Scholar). Here we present the structure of the SAM domain from the STE50 protein, the first SAM domain structure to be solved for a protein functionally involved in a MAPK cascade. Despite low sequence similarity to the other SAM domains whose structures have been solved (9.Slupsky C.M. Gentile L.N. Donaldson L.W. Mackereth C.D. Seidel J.J. Graves B.J. McIntosh L.P. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 12129-12134Crossref PubMed Scopus (122) Google Scholar, 17.Stapleton D. Balan I. Pawson T. Sicheri F. Nat. Struct. Biol. 1999; 6: 44-49Crossref PubMed Scopus (213) Google Scholar, 18.Thanos C.D. Goodwill K.E. Bowie J.U. Science. 1999; 283: 833-836Crossref PubMed Scopus (200) Google Scholar, 19.Thanos C.D. Faham S. Goodwill K.E. Cascio D. Phillips M. Bowie J.U. J. Biol. Chem. 1999; 274: 37301-37306Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, 20.Chi S.W. Ayed A. Arrowsmith C.H. EMBO J. 1999; 18: 4438-4445Crossref PubMed Scopus (150) Google Scholar, 21.Kim C.A. Phillips M.L. Kim W. Gingery M. Tran H.H. Robinson M.A. Faham S. Bowie J.U. EMBO J. 2001; 20: 4173-4182Crossref PubMed Scopus (199) Google Scholar, 22.Wang W.K. Bycroft M. Foster N.W. Buckle A.M. Fersht A.R. Chen Y.W. Acta Crystallogr. Sect. D Biol. Crystallogr. 2001; 57: 545-551Crossref PubMed Scopus (33) Google Scholar, 23.Kim C.A. Gingery M. Pilpa R.M. Bowie J.U. Nat. Struct. Biol. 2002; 9: 453-457PubMed Google Scholar), the structure of Ste50p SAM shows a similar fold to these proteins. The self-association of the Ste50p SAM domain has been investigated using analytical ultracentrifugation and gel filtration, which show that the domain from Ste50p is monomeric; this is supported by the NMR data. The SAM domain from the S. cerevisiae MAPKKK Ste11p by contrast is shown to be dimeric. The Ste50p SAM domain is shown to bind to the Ste11p SAM domain in an in vitro assay, and two separate mutations in the Ste50p SAM domain were found that inhibit this interaction. We examine these results in light of the proposed function of the Ste50p SAM domain as a hetero-oligomerization motif. Protein Expression and Purification—The SAM domains from Ste50p (residues 27-108) and Ste11p (residues 14-86) were amplified by PCR from a preparation of genomic yeast DNA. Restriction enzyme sites were added into the PCR primers used to facilitate cloning into expression vectors. Primer sequences used were: Ste11p SAM, 5′-CCGGGATCCCATATGGGTGACGAAAAGACC-3′ (forward) and 5′-CCGGAATTCGGATCCTCACCGTTTATCTCTCTG-3′ (reverse); and Ste50p SAM, 5′-CCGGGATCCCATATGAATAATGAAGAC-3′ (forward) and 5′-CCGGAATTCGGATCCTCAGTCCTTCCACTCCAA-3′ (reverse). Bold type indicates restriction endonuclease sites. Ste50p SAM and STE11p SAM were expressed in Escherichia coli (BL21) as GST fusion proteins using the pGEX-2T vector (Amersham Biosciences) and purified using glutathione agarose (Sigma) by standard protocols (Amersham Biosciences). The proteins were either used directly for pull-down assays or cleaved with thrombin restriction protease (Merck). Unlabeled and labeled SAM domain samples were prepared by growing the E. coli cells in 2TY or MOPS medium supplemented with 5% Celtone (Spectra Stable Isotopes), respectively. The SAM domains were then purified by size exclusion chromatography (16/60 S75; Amersham Biosciences). The protein samples for NMR spectroscopy consisted of ∼1.0 mm protein in 16 mm Na2HPO4, 4 mm NaH2PO4, 50 mm NaCl, 5 mm dithiothreitol, 10% D2O, 0.05% NaN3, pH 6.0. A mixed labeled sample of Ste50p SAM was generated by mixing a 1:1 ratio of unlabeled: 13C,15N-labeled protein and adding 8 m guanidine hydrochloride to a final concentration of 6 m. The protein was then refolded by rapid dilution (50-fold) into NMR buffer and subjected to size exclusion chromatography to remove the residual guanidine hydrochloride. The sample was then concentrated to 500 μl (∼1 mm protein) for NMR. NMR Spectroscopy—NMR experiments were recorded at 35 °C on Bruker DRX spectrometers operating at 500 and 800 MHz, equipped with H/C/N triple resonance probes with actively shielded z-gradients. 15N HSQC along with three-dimensional 15N-separated NOESY, HNCA, HN(CO)CA, CBCA(CO)NH, HNCACB, H(CC)(CO)NH, (H)CC(CO)NH, and HCCH total correlation spectroscopy were recorded at 500 MHz; a 13C-separated NOESY was recorded at 800 MHz. The data were processed and analyzed using the programs AZARA (v2.7, Wayne Boucher and Department of Biochemistry, University of Cambridge) and ANSIG (24.Kraulis P.J. Domaille P.J. Campbellburk S.L. Vanaken T. Laue E.D. Biochemistry. 1994; 33: 3515-3531Crossref PubMed Scopus (289) Google Scholar). A three-dimensional HNHA experiment was recorded to measure 3JHN-Hα coupling constants. Structure Calculation—The Ste50p SAM domain structures were calculated iteratively using the programs ARIA 1.2 (25.Linge J.P. O'Donoghue S.I. Nilges M. Methods Enzymol. 2001; 339: 71-90Crossref PubMed Scopus (333) Google Scholar) and CNS 1.0 (26.Brunger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. Sect. D Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16957) Google Scholar). Eight iterations were calculated, with 20 structures/iteration, of which seven were used for analysis of the ambiguous restraints. Finally, 100 structures were calculated, and the 25 with the lowest energy were selected for further analysis. Backbone ϕ angle restraints were obtained from an HNHA experiment. Backbone torsion angles were calculated from the backbone chemical shifts using the program TALOS (27.Cornilescu G. Delaglio F. Bax A. J. Biomol. NMR. 1999; 13: 289-302Crossref PubMed Scopus (2738) Google Scholar) and included as loose restraints with errors of ±30° or twice the standard deviation, whichever was greater. Ste11p SAM-Ste50p SAM Titration—A titration was performed by adding aliquots of unlabeled Ste50p SAM domain to 500 μl of 0.65 mm15N-labeled Ste11p SAM and then concentrating the sample back to 500 μl. Ste50p SAM was added in 0.25, 0.5, 0.75, and 1.0 molar equivalents. An 15N HSQC and size exclusion chromatography were run at each titration point. Analytical gel filtration was performed on a SMART system using a Superdex 75 PC 3.2/30 column (Amersham Biosciences). A similar titration was performed by adding aliquots of unlabeled Ste11p SAM to 550 μl of 0.3 mm15N-labeled Ste50p SAM and then concentrating the sample back to 550 μl. Unlabeled Ste11p SAM was added in 0.5, 1.0, 1.5, 2.0, and ≈11 molar equivalents. 15N HSQC and TROSY (28.Pervushin K. Riek R. Wider G. Wuthrich K. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12366-12371Crossref PubMed Scopus (2074) Google Scholar) experiments were run at each titration point. Analytical Ultracentrifugation—Sedimentation equilibrium analysis was performed using a Beckman Optima XL-I analytical ultracentrifuge. For both Ste11p SAM and Ste50p SAM, three samples were run simultaneously, with protein concentrations of 1, 2, and 3 mg/ml, respectively. The protein samples were 90 μl with 100 μl of reference buffer in the second sector of the two sector cells. The buffer used was identical to that used for the NMR experiments. All of the experiments were run at 4 °C. The data were collected at equilibrium for three different angular velocities: 25,000, 30,000, and 35,000 rpm. Detection was performed both by measuring absorbance at 280 nm and by using the Rayleigh interference optics. Because of problems arising from the differential oxidation of dithiothreitol in the sample and reference cells, all of the data analysis reported uses interference data. Data analysis was performed using WinNonlin (29.Johnson M.L. Correia J.J. Yphantis D.A. Halvorson H.R. Biophys. J. 1981; 36: 575-588Abstract Full Text PDF PubMed Scopus (778) Google Scholar). Mutagenesis—Site-directed mutagenesis of the pGEX-2T Ste50p SAM expression construct encoding residues 27-108 was performed using the QuikChange site-directed mutagenesis kit (Stratagene). The sequence of the Ste50p SAM coding region of all mutants was verified using an automated DNA sequencer (Applied Biosystems Inc.). GST Pull-down Assays—To ensure that the concentration of the GST-SAM fusion was sufficiently high on the beads, 3 ml of 50% glutathione agarose bead slurry was used to clear the fusion protein from 1 liter of culture cell extract. The fusion proteins were not digested with thrombin; instead the beads were washed a further three times with phosphate-buffered saline, 0.1% Triton. The concentration of the free STE11 and STE50 SAM domains was adjusted to 0.4 mm. The concentration of the immobilized GST-SAM domains was estimated visually from the strength of the bands on the SDS-PAGE gels to be at least equal to that of protein free in solution. 100 μl of 50% slurry of glutathione-agarose beads with a GST-SAM fusion protein bound was added to a microcentrifuge tube. The beads were then pelleted by centrifugation at 1,300 × g for 10 min, and the supernatant was removed. 50 μl of free SAM domain solution was added to the bead pellet, and the bead slurry was then incubated with rotation at 4 °C for 2 h. After incubation, the beads were pelleted by centrifugation as above. The supernatant was removed, and the beads were washed three times with 1 ml of cold phosphate-buffered saline. The samples of the beads, and the supernatant were then analyzed by SDS-PAGE (Invitrogen). Structure Determination—Backbone resonances were assigned using standard three-dimensional HNCA, HN(CO)CA, HNCACB, and CBCA(CO)NH experiments. The spectra recorded were of sufficient quality to achieve a complete backbone assignment for all residues (27-108), with the exception of Val-66 and Leu-75, whose backbone amide resonances were missing. Side chain assignments were made using three-dimensional H(CC)(CO)NNH and (H)CC(CO)NNH experiments, along with a three-dimensional HCCH total correlation spectroscopy. The side chain assignment was completed for the same range of residues. Analysis of the 15N- and 13C-separated NOESY spectra generated a total of 1,498 unambiguous and 791 ambiguous distance restraints. These were translated by ARIA into 1,013 unambiguous and 566 ambiguous, nondegenerate restraints. The final number of restraints, after eight ARIA iterations, was 1,354 unambiguous and 304 ambiguous (Table I). Assignment of the HNHA spectrum provided 35 torsion angle restraints, whereas analysis of the chemical shifts using the TALOS program gave a further 108 torsion angle restraints. The latter restraints were used loosely and discarded if in conflict with restraints provided by the HNHA. In the case of the HNHA, the torsion angles were set to ϕ = -120 ± 20° for 3JNH-Hα > 8.0 Hz and ϕ = -57 ± 20° for 3JNH-Hα < 6.0 Hz.Table IExperimental restraints and structural statisticsNumber of experimental restraintsUnambiguous1354Ambiguous304Dihedral restraints137〈SA〉a〈SA〉cbCoordinate precisionRMS deviations of backbone atoms (31-100) (Å)0.53 ± 0.110.36RMS deviations of all heavy atoms (31-100) (Å)0.98 ± 0.080.83RMS deviationsFrom the experimental restraintsNuclear Overhauser effect distances (Å)0.009 ± 0.0020.007Talos dihedral angles (°)0.572 ± 0.1110.585From idealized geometryBonds (Å)0.0013 ± 0.000070.0016Angles (°)0.312 ± 0.0080.306Impropers (°)0.180 ± 0.0130.176Final energyElj (kJ/mol)aThe Lennard-Jones potential was not used at any stage in the refinement.−734.321 ± 9.39−722.799Ramachandran analysis (%)Residues in most favored regions74.377.2Residues in additionally allowed regions23.822.8Residues in generously allowed regions1.40.0Residues in disallowed regions0.50.0a The Lennard-Jones potential was not used at any stage in the refinement. Open table in a new tab No long range nuclear Overhauser effects were observed for residues 27-31 and 100-108, and the line widths for these resonances were considerably narrower than those in the remainder of the domain. These residues are therefore not structured and have been omitted from all figures and the structural statistics. The structure of residues 31-100 is well ordered and has good covalent geometry (Fig. 1 and Table I). There were no distance restraints violated by more than 0.1 Å and no dihedral restraints violated by greater than 5°. Ste50p SAM Domain Structure—The Ste50p SAM consists of six helices, which form a compact, globular fold (Figs. 1 and 2). The N terminus forms a single turn of 310 helix from residues 32-34 (helix A′). This leads into two short α-helices, residues 37-46 (helix A) and 55-62 (helix B), followed by another 310 helix between residues 67 and 72 (helix C). Helix C is followed by another short α-helix at residues 75-81 (helix D), and the domain ends with a long C-terminal α-helix comprising residues 86-101 (helix E). Helices A and B form an anti-parallel helical hairpin on one side of a helical bundle. The other side of the bundle is made up of helix E, which packs against helices C and D in an anti-parallel manner, with one half of the coil being made up of helices C and D, interrupted by the loop. The two sides of the bundle are approximately orthogonal. The first helix, helix A′, is at the bottom of the bundle and packs in an anti-parallel fashion against the N-terminal half of helix C. The Ste50p SAM Domain Is a Monomer in Solution—Because other SAM domains are known to form homodimers or higher order oligomers, we examined the Ste50p SAM domain for any evidence of homotypic interaction. NMR can be used to solve the structures of homodimers if a sample can be produced that contains a 1:1 mixture of monomer with different labeling patterns. We produced such a sample of Ste50p SAM, in which 6 m guanidine hydrochloride was used as a denaturant prior to mixing unlabeled and 13C,15N-labeled proteins. The mixture was refolded by rapid dilution, and two NMR experiments were recorded to look for intermolecular interactions: a 13C-separated, 13C-rejected NOESY experiment and a 13C-separated NOESY recorded without carbon decoupling in the indirect proton dimension. In neither experiment was there any evidence for intermolecular nuclear Overhauser effects, suggesting that the Ste50p SAM domain is monomeric. To exclude the possibility that the absence of intermolecular contacts was due to low sensitivity in the NMR experiments, we then sought to confirm this result by other means. Given that the Kd values published for other SAM domain homo-oligomers were relatively high (in the region of 0.5-5 mm), we performed analytical ultracentrifugation using samples of the Ste50p SAM domain at concentrations of 1.0, 2.0, and 3.0 mg/ml (∼0.1, 0.2, and 0.3 mm, respectively). The sedimentation equilibrium data for the Ste50p SAM domain are shown in Fig. 3b, and the goodness of fit to a monomeric model for the data sets is shown in Fig. 3a. As can be seen the majority of the residuals are randomly distributed at approximately 0, but there is a slight upward trend at higher values of the radius, r. This is diagnostic of an aggregating protein, although given that this upward trend is restricted to the faster rotor speeds with the highest protein concentrations, it is likely to be a very small effect. The data are consistent with a molecular mass of 11.0 kDa for the Ste50p SAM domain. The Ste50p SAM domain is calculated to have a mass of 10.0 kDa. The highest concentration of the Ste50p SAM used was 0.3 mm; thus the dissociation constant for dimer formation would be above 4.9 mm. These data substantiate the monomer structure solved by NMR. The SAM Domain of Ste11p Self-associates in Solution—Because Ste50p and Ste11p interact via their SAM domains in vivo, we investigated whether the Ste11p SAM domain could self-associate. Three samples of the protein were prepared at concentrations of 1.0, 2.0, and 3.0 mg/ml (∼0.1, 0.2, and 0.3 mm, respectively) for analytical ultracentrifugation. The data obtained (Fig. 4b) were fitted to an equation describing the behavior of a single ideal species. The calculated molecular mass of the domain is 8687 Da. The molecular mass obtained from the calculation was 12.75 kDa, which exceeds the actual mass by more than 40%. In addition, the model only described the data poorly as judged by the pattern of the residuals. Fitting to a monomer to dimer equilibrium still produced a poor fit. However, simultaneous fitting of the Ka and the second virial co-efficient gave residuals that distribute well at approximately 0 (Fig. 4a). The values obtained in fringe units were lnKa = -2.00 and B = -6.4 × 10-3. Using an approximate conversion factor of 3.3 fringes (g/l) this gives an estimated Kd of 0.5 mm. The Ste50p SAM Domain Interacts with the Ste11p SAM Domain in Vitro—GST pull-down experiments were used to test the ability of Ste11p and Ste50p SAM domains to interact homotypically and heterotypically in vitro (Fig. 5). Ste50p SAM domain does not self-associate, in agreement with all the previous data (lanes 11 and 12). Addition of free Ste11p SAM to immobilized Ste50p SAM (lanes 9 and 10) shows that these two SAM domains can interact with each other. The reciprocal experiment, where Ste11p is immobilized on beads, was also performed. When free Ste50p SAM was added, an interaction between the two SAM domains was again visible (lanes 5 and 6). In addition, free Ste11p SAM was added to immobilized Ste11p SAM (lanes 7 and 8). Despite the apparent decrease in the amount of input Ste11p, it can still be seen that free Ste11p SAM can self-associate with the immobilized Ste11p, thus confirming the dimerization of Ste11p observed by analytical ultracentrifugation. It should be noted that although GST fusion proteins are known to be dimers, using the SAM domains as GST fusion proteins in these assays does not appear to hamper their abilities to homo- or heterodimerize. Analysis of the Oligomerization State of the Ste50p SAM-Ste11p SAM Complex—In an attempt to analyze the Ste11p SAM-Ste50p SAM interaction by NMR, unlabeled Ste11p SAM was titrated into 15N-labeled Ste50p SAM, and the changes in the 15N HSQC and 15N TROSY spectra were monitored (data not shown). The peaks became very broad during the titration in the HSQC spectra, with most disappearing completely during the course of the titration. A few new peaks appeared that were also very broad and weak. Interestingly, the broadening was mirrored in the TROSY spectra, indicating that it was not due to an in" @default.
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• W2020207222 date "2004-01-01" @default.
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