Oligomerization-dependent Association of the SAM Domains from Schizosaccharomyces pombe Byr2 and Ste4

SAM (sterile alphamotif) domains are protein-protein interaction modules found in a large number of regulatory proteins. Byr2 and Ste4 are two SAM domain-containing proteins in the mating pheromone response pathway of the fission yeast, Schizosaccharomyces pombe. Byr2 is a mitogen-activated protein kinase kinase kinase that is regulated by Ste4. Tu et al. (Tu, H., Barr, M., Dong, D. L., and Wigler, M. (1997) Mol. Cell. Biol. 17, 5876–5887) showed that the isolated SAM domain of Byr2 binds a fragment of Ste4 that contains both a leucine zipper (Ste4-LZ) domain as well as a SAM domain, suggesting that Byr2-SAM and Ste4-SAM may form a hetero-oligomer. Here, we show that the individual SAM domains of Ste4 and Byr2 are monomeric at low concentrations and bind to each other in a 1:1 stoichiometry with a relatively weak dissociation constant of 56 ± 3 μm. Inclusion of the Ste4-LZ domain, which determines the oligomeric state of Ste4, has a dramatic effect on binding affinity, however. We find that the Ste4-LZ domain is trimeric and, when included with the Ste4-SAM domain, yields a 3:1 Ste4-LZ-SAM:Byr2-SAM complex with a tight dissociation constant of 19 ± 4 nm. These results suggest that the Ste4-LZ-SAM protein may recognize multiple binding sites on Byr2-SAM, indicating a new mode of oligomeric organization for SAM domains. The fact that high affinity binding occurs only with the addition of an oligomerization domain suggests that it may be necessary to include ancillary oligomerization modules when searching for binding partners of SAM domains. SAM (sterile alphamotif) domains are protein-protein interaction modules found in a large number of regulatory proteins. Byr2 and Ste4 are two SAM domain-containing proteins in the mating pheromone response pathway of the fission yeast, Schizosaccharomyces pombe. Byr2 is a mitogen-activated protein kinase kinase kinase that is regulated by Ste4. Tu et al. (Tu, H., Barr, M., Dong, D. L., and Wigler, M. (1997) Mol. Cell. Biol. 17, 5876–5887) showed that the isolated SAM domain of Byr2 binds a fragment of Ste4 that contains both a leucine zipper (Ste4-LZ) domain as well as a SAM domain, suggesting that Byr2-SAM and Ste4-SAM may form a hetero-oligomer. Here, we show that the individual SAM domains of Ste4 and Byr2 are monomeric at low concentrations and bind to each other in a 1:1 stoichiometry with a relatively weak dissociation constant of 56 ± 3 μm. Inclusion of the Ste4-LZ domain, which determines the oligomeric state of Ste4, has a dramatic effect on binding affinity, however. We find that the Ste4-LZ domain is trimeric and, when included with the Ste4-SAM domain, yields a 3:1 Ste4-LZ-SAM:Byr2-SAM complex with a tight dissociation constant of 19 ± 4 nm. These results suggest that the Ste4-LZ-SAM protein may recognize multiple binding sites on Byr2-SAM, indicating a new mode of oligomeric organization for SAM domains. The fact that high affinity binding occurs only with the addition of an oligomerization domain suggests that it may be necessary to include ancillary oligomerization modules when searching for binding partners of SAM domains. glutathioneS-transferase β-mercaptoethanol N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine Ras-associating SAM domains (also known as Pointed, SPM, and HLH domains) are frequently found in eukaryotic regulatory proteins ranging from receptor tyrosine kinases to transcription factors (1Ponting C.P. Protein Sci. 1995; 4: 1928-1930Crossref PubMed Scopus (142) Google Scholar, 2Schultz J. Ponting C.P. Hofmann K. Bork P. Protein Sci. 1997; 6: 249-253Crossref PubMed Scopus (270) Google Scholar, 3Kyba M. Brock H.W. Dev. Genet. 1998; 22: 74-84Crossref PubMed Scopus (64) Google Scholar). Structures of several SAM domains reveal a common tertiary fold but show a diverse array of oligomeric states and binding schemes (4Slupsky 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 (123) Google Scholar, 5Chi S.W. Ayed A. Arrowsmith C.H. EMBO J. 1999; 18: 4438-4445Crossref PubMed Scopus (150) Google Scholar, 6Smalla M. Schmieder P. Kelly M. Ter Laak A. Krause G. Ball L. Wahl M. Bork P. Oschkinat H. Protein Sci. 1999; 8: 1954-1961Crossref PubMed Scopus (70) Google Scholar, 7Stapleton D. Balan I. Pawson T. Sicheri F. Nat. Struct. Biol. 1999; 6: 44-49Crossref PubMed Scopus (213) Google Scholar, 8Thanos C.D. Bowie J.U. Protein Sci. 1999; 8: 1708-1710Crossref PubMed Scopus (126) Google Scholar, 9Thanos C.D. Goodwill K.E. Bowie J.U. Science. 1999; 283: 833-836Crossref PubMed Scopus (200) Google Scholar, 10Kim 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, 11Kim C.A. Gingery M. Pilpa R.M. Bowie J.U. Nat. Struct. Biol. 2002; 9: 453-457PubMed Google Scholar). Some SAM domains, such as that from the Ets family transcription factor TEL, can self-associate to form an open-ended polymeric structure (10Kim 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), whereas the closely related Ets-1, GABPα, and Erg SAM domains are monomeric (4Slupsky 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 (123) Google Scholar, 12Slupsky C.M. Gentile L.N. McIntosh L.P. Biochem. Cell Biol. 1998; 76: 379-390Crossref PubMed Scopus (15) Google Scholar, 13Mackereth C.D. Schärpf M. Gentile L.N. McIntosh L.P. J. Biomol. NMR. 2002; (in press)PubMed Google Scholar). The SAM domains from Eph receptor tyrosine kinases can either be monomeric or dimeric or may possibly form an extended oligomeric structure (7Stapleton D. Balan I. Pawson T. Sicheri F. Nat. Struct. Biol. 1999; 6: 44-49Crossref PubMed Scopus (213) Google Scholar, 9Thanos C.D. Goodwill K.E. Bowie J.U. Science. 1999; 283: 833-836Crossref PubMed Scopus (200) Google Scholar, 14Thanos 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). SAM domains have also been described in interactions with non-SAM domain-containing proteins. For example, the SAM domain of BAR (46Zhang H., Xu, Q. Krajewski S. Krajewska M. Xie Z. Fuess S. Kitada S. Godzik A. Reed J.C. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 2597-2602Crossref PubMed Scopus (161) Google Scholar), a protein involved in the regulation of apoptosis, associates with both Bcl-2 and Bcl-XL (13Mackereth C.D. Schärpf M. Gentile L.N. McIntosh L.P. J. Biomol. NMR. 2002; (in press)PubMed Google Scholar). Cdk10, a member of the Cdc2 family of kinases, binds the SAM domain of Ets-2 and thereby regulates the activity of this transcription factor (15Kasten M. Giordano A. Oncogene. 2001; 20: 1832-1838Crossref PubMed Scopus (91) Google Scholar). The mitogen-activated protein kinase Erk2 docks on the SAM domain of Ets-1, enhancing the kinetics of phosphorylation at an adjacent N-terminal target site within this transcription factor (16Seidel J.J. Graves B.J. Genes Dev. 2002; 16: 127-137Crossref PubMed Scopus (147) Google Scholar). Although several complexes between nonidentical SAM domains have been described like TEL/TEL2 (17Carroll M. Tomasson M.H. Barker G.F. Golub T.R. Gilliland D.G. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14845-14850Crossref PubMed Scopus (246) Google Scholar, 18Golub T.R. Goga A. Barker G.F. Afar D.E. McLaughlin J. Bohlander S.K. Rowley J.D. Witte O.N. Gilliland D.G. Mol. Cell. 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Wainwright S.M. Ish-Horowicz D. Dibb N.J. Nature. 2001; 411: 330-334Crossref PubMed Scopus (57) Google Scholar) Scm/ph (3Kyba M. Brock H.W. Dev. Genet. 1998; 22: 74-84Crossref PubMed Scopus (64) Google Scholar, 11Kim C.A. Gingery M. Pilpa R.M. Bowie J.U. Nat. Struct. Biol. 2002; 9: 453-457PubMed Google Scholar, 25Peterson A.J. Kyba M. Bornemann D. Morgan K. Brock H.W. Simon J. Mol. Cell. Biol. 1997; 17: 6683-6692Crossref PubMed Scopus (123) Google Scholar), their recognition mechanisms have not yet been characterized. Here we investigate one example of a hetero-SAM domain interaction that occurs between the Byr2 and Ste4 proteins in the fission yeastSchizosaccharomyces pombe. Sexual differentiation in S. pombe is controlled via a mitogen-activated protein kinase pathway that includes Ste4 and Byr2 (26Hughes D. Yamamoto M. Kuriyan J. Taylor B.L. Signal Transduction: Prokaryotic and Simple Eukaryotic Systems. Academic Press Inc., New York1993Google Scholar). Byr2 is a mitogen-activated protein kinase kinase kinase that is activated by interactions with both Ras1 and Ste4 (27Wang Y., Xu, H.P. Riggs M. Rodgers L. Wigler M. Mol. Cell. Biol. 1991; 11: 3554-3563Crossref PubMed Scopus (150) Google Scholar, 28Masuda T. Kariya K. Shinkai M. Okada T. Kataoka T. J. Biol. Chem. 1995; 270: 1979-1982Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 29Bauman P. Albright C.F. Biochimie (Paris). 1998; 80: 621-625Crossref PubMed Scopus (9) Google Scholar). The SAM domain of Byr2 has previously been shown to bind to the N-terminal 160 amino acids of Ste4, a region containing a SAM domain followed immediately by a putative leucine zipper (Ste4-LZ) domain. A speculative model of Byr2 activation has therefore emerged in which Byr2 and Ste4 interact via their SAM domains, leading to oligomerization of Byr2 by virtue of the Ste4 leucine zipper domain (30Tu H. Barr M. Dong D.L. Wigler M. Mol. Cell. Biol. 1997; 17: 5876-5887Crossref PubMed Scopus (72) Google Scholar). Here we find that although the two SAM domains do bind to each other, the role of the Ste4-LZ domain is not to oligomerize Byr2. Instead, the leucine zipper domain of Ste4 trimerizes, thereby displaying three SAM domains that together bind a single Byr2-SAM domain with high affinity. The region of the Byr2 gene encompassing its SAM domain (amino acids 1–70; SPBC1D7.05 in theS. pombe GeneDB,www.genedb.org/genedb/pombe/index.jsp) was PCR-amplified from a S. pombe cDNA library and cloned into a modified pET-3c (Novagen) expression vector containing a C-terminal six-histidine tag. The expressed protein sequence comprised amino acids 1–70 of Byr2, followed by RDHHHHHH. The DNA sequences encoding the SAM domain (amino acids 9–72) and the Ste4-LZ domain (amino acids 83–152) of Ste4 (SPAC1565.04c in the S. pombe GeneDB) were cloned similarly into the pET-3c vector with the same C-terminal His6 sequence, plus a MEKTR leader sequence. Two different Ste4 constructs were prepared containing both the Ste4-LZ domain and the SAM domain. The Ste4-LZ-SAM-A construct in the pTrcHisB (Invitrogen) vector encoded the sequence MGDSDDSY and then amino acids 1–152 of Ste4, followed by RDHHHHHH. The Ste4-LZ-SAM-B, consisting of residues 1–157, was also PCR-amplified from a S. pombecDNA library and cloned into pET28a with no added purification tags. The GST-Byr21 construct was made by subcloning amino acids comprising 1–66 of the Byr2-SAM domain into a vector The expressed protein was Byr2 amino acids followed by RDHHHHHH. Byr2-SAM, and were expressed in J. Mol. Biol. 1991; PubMed Scopus Google Scholar). The Ste4-LZ-SAM-A and the proteins were expressed in C.M. 1996; PubMed Scopus Google Scholar). The were at in containing or to of and then by the addition of for the of from a were in of and by The protein in the was to a of and in the same The protein was then with of and was in and an The protein at and was in of the expressed proteins were their C.A. J. Chem. Scopus Google Scholar). A of Byr2-SAM was with and the was to a The proteins were with and β-mercaptoethanol at a of The to the complex was and a followed by a of the of was in and of of the proteins were with the for at 4 The were with of the and then with of of the proteins in the same for at 4 The were three with of the followed by of the proteins with of and The proteins were a The binding was by concentrations of with Byr2-SAM in in Ste4-LZ-SAM:Byr2-SAM ranging from to with to the protein The proteins were in a containing and a consisting of 19 A was with of a protein or of a of the Ste4-LZ protein was to the and with the at a of were a Byr2-SAM was to by the at in containing 3 and at a of a was of Byr2-SAM in was at a of both and of the Byr2-SAM domain was on the only was with to response of Byr2-SAM on the For binding we a of the described by et D.G. M.D. Graves B.J. Biochem. 1998; PubMed Scopus Google Scholar). The was with at a of a was were for at an the Ste4-LZ-SAM-A concentrations of and in were the were an a for Ste4-LZ-SAM-A was to the the to that the response to the same as that with this of protein not of the extended which were for this we found it to the with the al. D.G. M.D. Graves B.J. Biochem. 1998; PubMed Scopus Google Scholar) the as a the modified may be in with the or the for high affinity interactions several The response at each was the binding response were then and the dissociation constant was by the to a binding the of a 1:1 is the response at of and A to the were the and as For binding with of Ste4-SAM domain at concentrations of and were at a of The response at were by and ranging between and in the of the binding for each The was three of the Ste4-SAM domain and each in of The binding were to a binding as The is an of three were at 4 the isolated SAM domains) or other in a from to 4 for the SAM domains, from to 3 for the Ste4-LZ domain, and for other proteins. The were in and at or to the was to a and the was within the of the were for for for of the SAM for for and for the were from the amino of the proteins acids and as and New Scholar) and to the in and Science. The of Scholar). were to an for a single the dissociation the was to multiple of a of for two different in at two with the of Ste4 and Byr2 are proteins by al. (30Tu H. Barr M. Dong D.L. Wigler M. Mol. Cell. Biol. 1997; 17: 5876-5887Crossref PubMed Scopus (72) Google Scholar) showed that the 160 amino acids of Ste4, which include a SAM domain, associates with the SAM domain of Byr2 SAM domains are known protein-protein interaction we the isolated SAM domains can bind to each binding were a of shown in the Ste4-SAM domain is to bind to the whereas no is with SAM domains from or on (3Kyba M. Brock H.W. Dev. Genet. 1998; 22: 74-84Crossref PubMed Scopus (64) Google Scholar, 11Kim C.A. Gingery M. Pilpa R.M. Bowie J.U. Nat. Struct. Biol. 2002; 9: 453-457PubMed Google A.J. Kyba M. Bornemann D. Morgan K. Brock H.W. Simon J. Mol. Cell. Biol. 1997; 17: 6683-6692Crossref PubMed Scopus (123) Google no binding is for with is an SAM domain from the protein A.J. Kyba M. Bornemann D. Morgan K. Brock H.W. Simon J. Mol. Cell. Biol. 1997; 17: 6683-6692Crossref PubMed Scopus (123) Google no binding is for with no binding is for with is an SAM domain from the protein (3Kyba M. Brock H.W. Dev. Genet. 1998; 22: 74-84Crossref PubMed Scopus (64) Google Scholar). no binding is for with with The of the to Ste4-LZ-SAM-A by the that the trimeric Ste4-LZ-SAM-A protein binds to with The of the to Ste4-SAM by weak binding between the two SAM domains. The in each from the whereas to the stoichiometry of we shown in Ste4-SAM as a single with a of to that of the of the and the Byr2-SAM a of when the was to a single is the for Byr2-SAM of The was found to with protein and suggesting that Byr2-SAM is a a of at different concentrations and a dissociation constant of Byr2-SAM and Ste4-SAM were together at a 1:1 we found a of at the and which to the of for the the results that Ste4-SAM is Byr2-SAM is monomeric at low concentrations but and Ste4-SAM and Byr2-SAM form a 1:1 4 that were to the binding affinity between Byr2-SAM and Ste4-SAM domains. of their relatively weak the and dissociation were to we were to the response for each of Ste4-SAM shown in the response are well by a binding with a dissociation constant of 56 ± 3 and a stoichiometry of 1:1 between Ste4-SAM and that the isolated SAM domains are at for the between the Byr2 and Ste4 proteins. Ste4 contains a leucine zipper domain C-terminal to its SAM domain that may be involved in the oligomeric state of this domain, Ste4-LZ and Ste4-LZ-SAM-A were expressed and that both the Ste4-LZ and Ste4-LZ-SAM-A proteins are For Ste4-LZ we a of which is with the of For Ste4-LZ-SAM-A we found a in with a of that Ste4-SAM is we the oligomerization of Ste4-LZ-SAM-A to the of the Ste4-LZ domain. shown in the Ste4-LZ from an at a with a high suggests that the Ste4-LZ domain is as be for an extended We to the trimeric of the Ste4 SAM domain the interaction with Ste4-LZ-SAM-A binding to Byr2-SAM was in shown in with the results previously H. L. Wigler M. Mol. Cell. Biol. 1996; 16: PubMed Scopus (57) Google Scholar), Ste4-LZ-SAM-A binds to the Byr2-SAM and not to the proteins. the stoichiometry of this we the complex by shown in a of was for the complex by is with the of for a 3:1 of Ste4-LZ-SAM-A to the 1:1 stoichiometry of binding for the isolated SAM domains, the 3:1 in the of the trimeric Ste4-LZ domain is We therefore an of the of the in the complex a binding at a Byr2-SAM well the and Byr2-SAM were in and the and of were by shown in the binding sites on were at a of to is with the stoichiometry and described the affinity of the we Byr2-SAM was and Ste4-LZ-SAM-A was in the was but it to to the binding a of the binding dissociation mechanisms are relatively We therefore to binding a model is not to concentrations of Ste4-LZ-SAM-A were a was shown in the is well described by a binding two we dissociation of and or an of 19 ± 4 for the 3:1 complex between the Ste4-LZ-SAM-A and Byr2-SAM proteins. the trimeric Ste4-LZ with the Ste4-SAM domain binding affinity for Byr2-SAM with that with the individual Ste4-SAM domain. results that the interaction between Byr2 and Ste4 is by their individual SAM domains. of the Ste4-SAM domain by the adjacent Ste4-LZ domain the affinity of this with an stoichiometry of 3 Ste4-LZ-SAM to Although we to binding from the Ste4-LZ region within the of the Ste4-LZ-SAM Ste4-LZ not bind to Byr2-SAM the of the region immediately adjacent to the Ste4-SAM domain may binding to Byr2-SAM by the structure or of the Ste4-SAM domain. the of Ste4-SAM a well with a to that for other SAM domains. we only a in the of as with the isolated Ste4-SAM domain it that the dramatic in binding affinity is a of interactions between three Ste4-SAM domains with a Byr2-SAM to this shown in the isolated Byr2-SAM and Ste4-SAM domains form a 1:1 complex with affinity 56 between binding on each affinity sites when three Ste4-SAM domains are in by the trimeric and leucine zipper domain, the of Ste4 and Byr2 in the complex has to be only results in a of the for binding to 19 of and dissociation for proteins and protein complexes in a new binding that Ste4-SAM domains recognize different sites on the Byr2-SAM domain in an Although of in interactions is not For example, a dimeric receptor binds to a monomeric different binding on the M. Science. PubMed Scopus Google Scholar). SAM domains are known to multiple binding to polymeric (10Kim 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 C.A. Gingery M. Pilpa R.M. Bowie J.U. Nat. Struct. Biol. 2002; 9: 453-457PubMed Google Scholar). is a for the interaction of Ste4 with Byr2 in the S. pombe mating The of S. pombe or other in either the Ste4 or Byr2 SAM domain that SAM domains are for activation of the pheromone response pathway P. Albright C.F. Biochimie (Paris). 1998; 80: 621-625Crossref PubMed Scopus (9) Google Scholar, H. Barr M. Dong D.L. Wigler M. Mol. Cell. Biol. 1997; 17: 5876-5887Crossref PubMed Scopus (72) Google Scholar, H. L. Wigler M. Mol. Cell. Biol. 1996; 16: PubMed Scopus (57) Google Scholar, K. K. H. 1991; 19: PubMed Scopus Google Scholar). of the Ste4-LZ region in Ste4 a in K. K. 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Biol. 1997; 17: 5876-5887Crossref PubMed Scopus (72) Google Scholar). model in of results indicating that Ste4-LZ-SAM not the oligomerization state of Byr2-SAM, although it be for the proteins in their The C-terminal region in Ste4 has also been shown to be a factor in the pheromone response of this region pombe K. K. H. 1991; 19: PubMed Scopus Google Scholar), and it is that the C-terminal region a role in the of Byr2 to of the mating sequence a to Ras-associating domains J. F. Bork P. Ponting C.P. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: PubMed Scopus Google Scholar, I. L. N.J. T. J. R. F. Ponting C.P. Bork P. 2002; PubMed Scopus Google Scholar), suggesting that Ste4 a role in binding to for two the of the complex and the structure of a complex that the binding sites on G. C. A. F. A. Nature. 1995; PubMed Scopus Google Scholar, K. P. S. H. Wigler M. A. 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Biochem. 1998; PubMed Scopus Google Scholar). suggests that Byr2 can bind to in the of the interaction with Ste4. the domain may have a different in Ste4 or may bind that has yet to be The fact that high affinity binding between the SAM domains of Byr2 and Ste4 only occurs in the of the Ste4 as a Although SAM domains are protein-protein interaction the binding partners of only a SAM domains are The of may or other of binding partners are not with isolated SAM domains. an oligomerization may to be For example, the SAM domain of the receptor has been in binding to a protein tyrosine Genes Dev. 1998; PubMed Scopus Google Scholar), but this interaction only occurs receptor interactions may in other as that the SAM domains from and form open-ended (10Kim 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, 11Kim C.A. Gingery M. Pilpa R.M. Bowie J.U. Nat. Struct. Biol. 2002; 9: 453-457PubMed Google Scholar). The interaction of Byr2 and Ste4, is the well example of a complex SAM domain in interaction the of SAM domains in protein-protein interactions in S. pombe cDNA library was by at the in We A. H. M. D. F. and for on the

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