Detection of RBLSte11 domains in fungal MAPKKKs by structural bioinformatics
The domain recognition methods based on primary sequence information have been only partially successful in detecting the ubiquitin fold of Ras-binding domains (RBDs) and Ras association (RA) domains. Although we were able to detect the RA domain in the S. cerevisiae
Ste50 protein by application of simple homology tools (Ponting and Benjamin, 1996
; Ekiel et al., 2009
), initial database surveys (Ponting and Benjamin, 1996
) suggested an absence of the RBD or RA motifs within the Ste11 protein, a kinase that serves as the MAPKKK for a variety of signaling pathways in yeast. To identify structural motifs that could be responsible for interaction of Ste11 with Ste5 scaffold, we reanalyzed the sequence of Ste11, applying structural bioinformatics approaches based on the state-of-the-art fold recognition methods assembled within the 3D-Jury meta-predictor (Ginalski and Rychlewski, 2003
). Our analysis detected with statistical significance a ubiquitin fold–based RBD encompassing residues 117–240 within the previously identified Ste5-interacting region of the yeast Ste11 protein kinase. We termed this an RBD-like (RBL) motif, which shares all secondary structural elements characteristic of the archetypal ubiquitin β-grasp fold (Supplemental Figure S2(a)), with the highest 3D-JURY consensus fold recognition score to the RBD structure of the Ste11 homologue Byr2 of S. pombe
(Scheffzek et al., 2001
). This RBLSte11
motif is common to the Ste11MAPKKK homologues of a large number of fungi (Supplemental Figure S2(b)).
The RBL domain of Ste11 MAPKKK is essential for the pheromone response pathway
To directly assess the role of the RBL domain in signal transduction within the MAP kinase pathway required for pheromone response, we created a Ste11 mutant (Ste11ΔRBL) with an internal in-frame deletion of the region corresponding to the predicted RBL domain (residues 117–240). The resulting mutant was assayed for its ability to mate with wild-type cells of opposite mating type. Cells expressing Ste11ΔRBL are severely defective in mating and also in pheromone-induced cell cycle arrest, as well as in pheromone-induced transcriptional expression of a mating-specific reporter gene. Therefore cells containing Ste11 lacking the RBL have an essentially sterile phenotype (). To ensure that Ste11ΔRBL is a functional kinase, we used the fact that Ste11 MAPKKK is shared among several MAP kinase pathways and essential also for high-osmolarity glycerol (HOG) synthesis. In the HOG pathway, the Ste11-SAM (for “sterile alpha mating”) domain, which is N-terminal and precedes the RBL domain, is necessary for the interaction of Ste11 with Ste50 adaptor for proper localization and activation of the Ste11 kinase. We therefore analyzed the function of the Ste11ΔRBL mutant in the activation of the HOG pathway and found that, in contrast to its behavior in the pheromone response pathway, it is fully capable of activating the HOG pathway, indicating that the Ste11ΔRBL retains kinase activity and suggesting that the RBL domain is uniquely crucial for Ste11 function in the activation of the MAP kinase pathway for pheromone response ().
FIGURE 1: The RBLSte11 domain is essential for pheromone response signaling. (A) Schematic diagram of Ste11 MAPKKK with functional domains indicated: KD, kinase domain; RBL, RBD-like; SAM, sterile alpha mating. (B) The RBLSte11 domain is essential for mating. Yeast (more ...)
Solution structure of the RBLSte11 domain
To confirm the structural bioinformatics prediction for the RBLSte11
domain, we determined its structure in solution by nuclear magnetic resonance (NMR) spectroscopy. The RBLSte11
domain (amino acids [aa] 116–236) was bacterially expressed in 15
N- and 13
C-enriched medium and purified. Using NMR experiments, we obtained chemical shift assignments for the protein backbone, as well as for aliphatic and aromatic side chains. Backbone assignments were complete, with exceptions for Cys-138 and Asp-142, for which signals were missing in 1
N heteronuclear single quantum coherence (HSQC) spectra. An ensemble of 20 low-energy structures that satisfy NMR constraints was obtained using Cyana 2.1 (Güntert, 2004
; Supplemental Figure S3).
The NMR structure reveals that the predicted RBL domain indeed adopts a ubiquitin fold, comprising a mixed five-stranded β-sheet flanked by three α-helices (). The secondary structure assignments are as follows: strands β1 (aa 120–125) and β2 (aa 128–134), helix α1 (aa 142–151), strands β3 (aa 172–178) and β4 (aa 182–186), helix α2 (aa 189–196), and strand β5 (aa 206–211) are elements typical of the ubiquitin fold. The final, long C-terminal helix α3 (aa 218–233) is rarely present in this class of domains. In addition, we observed a short, unique insertion between helix α1 and strand β3 in RBLSte11 domain, which we call a β-finger, composed of two short strands βF1 (aa 158–160) and βF2 (aa 163–166). The ensemble of NMR structures shows significant protein backbone flexibility in the N-terminus preceding the ubiquitin fold, in the C-terminal end of helix α3, and also to a moderate degree in the β-finger insert (Supplemental Figure S3).
FIGURE 2: Overall structures of the RBLSte11 and PHSte5 domains. (A) Solution NMR structure of the RBLSte11 domain. Canonical ubiquitin-like fold is in cyan, the inserted β-hairpin in red, and the helical C-terminal extension in blue. (B) Modeled PHSte5 (more ...)
The structure of the RBLSte11
domain is most similar to the RBD domain of S. pombe
Byr2 (Protein Data Bank [PDB] code 1K8R; Scheffzek et al., 2001
), as indicated by various structural similarity metrics such as a Z
-score of 8.0 and a Q
-score of 0.38 (http://pdbe.org/fold
). However, there are two major differences between these structures. First, the Byr2-RBD does not have the β-finger insertion, and its C-terminal helix α3 is much shorter than the corresponding helix in the RBLSte11
The RBLSte11 domain has no detectable association with small GTPases but interacts with the PH domain of Ste5 scaffold
To understand the role of this RBLSte11
domain in pheromone response signaling, we first searched for its interaction partner(s). Because of the similarity of the RBLSte11
domain to the Byr2-RBD of S. pombe
, which has been shown to interact with Ras1 (Gronwald et al., 2001
; Scheffzek et al., 2001
), we tested the possibility of the RBLSte11
domain interacting with any of the S. cerevisiae
small GTPases. To this end we performed in vitro resin-binding assays with small GTPases of the Ras and Rho family members (a total of 11, including Ras1, Ras2, Cdc42, Rho1, Rho2, Rho3, Rho4, Rho5, Rhb1, Rsr1, and Yhr022c) as glutathione S
-transferase (GST) fusions from the yeast open reading frame library (Martzen et al., 1999
) expressed in yeast and purified as previously described (Annan et al., 2008
) and the RBLSte11
domain (aa 116–236) expressed as histidine (His)-tagged fusion protein in bacteria. For these assays the small GTPases were preloaded with the nonhydrolyzable GTP analogue GTPγS or with GTPβS (Truckses et al., 2006
). No detectable binding of RBLSte11
to these GTPases was observed (unpublished data).
The presence of PH domains in some family members of Ste5-like and Far1-like proteins was predicted (Wiget et al., 2004
; Garrenton et al., 2006
; Cote et al., 2011
). This prediction was derived from the application of fold-recognition methods (Supplemental Figure S1(a)), which detected the boundaries of the PH domain in a large number of Ste5 and Far1 fungal proteins (Supplemental Figure S1(b)). Simple application of homology tools was unable to detect this cryptic structural relationship due to the low sequence conservation characteristic of PH domains. Ste11 had been shown to interact with Ste5, and the Ste11-binding region on Ste5 was first mapped to residues 336–586 through deletion (Choi et al., 1994
). This region was further delineated through mutagenesis to residues 463–514 (Inouye et al., 1997
). It overlaps with the recently proposed lipid-binding PHSte5
domain (Garrenton et al., 2006
), which maps to residues 400–512.
To further delineate the domain boundary for the region of Ste5 that is specifically required for the interaction with the RBLSte11
domain, we used a modified cytoplasmic yeast, two-hybrid system (Wu, Jansen, and Yerko, unpublished data) developed based on our finding that the interaction of Ste50 and Ste11 through their SAM domains, in the HOG pathway activation, can be replaced with other protein–protein interacting modules (Wu et al., 2006
We replaced the Ste50-SAM domain with the Ste11-interacting region of Ste5, so that the activity of the modified HOG pathway in cells (ste50Δ ssk2Δ ssk22Δ
) depended on the ability of the Ste5 fragment in the Ste50 chimera to interact with Ste11 (). Deletions from both the N- and C-termini of the Ste5 fragment delineated a Ste5 fragment composed of residues 373–537 that was able to activate the HOG pathway when fused to Ste50ΔSAM (). Further deletion analysis indicated that a Ste5 fragment of aa 373–523 was still functional, albeit with somewhat reduced activity compared with the larger fragment. However, the fragment consisting of aa 373–515 of Ste5 was unable to activate HOG pathway (unpublished data). These results show that the Ste5 region interacting with Ste11 largely overlaps with the PH domain of Ste5. Because we showed previously that the Ste11 regulatory region encompassing the RBL domain is required for interactions with Ste5 (Wu et al., 1999
), we concluded that the Ste5–Ste11 association is mediated through PH–RBL domain interactions. This interaction of the PHSte5
domain with the RBLSte11
domain is specific, as other versions of PH domains show no interaction with the RBL domain ().
FIGURE 3: Ste11 interacts with the PHSte5 domain. (A) Schematic representation of the interaction of Ste11 with Ste50 through their respective SAM domains in the natural HOG pathway (left) and through the RBL and grafted PHSte5 domain in the altered HOG pathway. (more ...)
To test whether the RBLSte11 domain and PHSte5 domain can interact directly in vitro, we performed pull-down resin-binding assays with independently expressed and differentially tagged fragments: a Ste11 fragment consisting of aa 116–236 with two Ste5 constructs encompassing aa 373–537 and aa 373–523, respectively. When bacterially expressed GST fusions of the Ste5 fragments were mixed with a bacterial extract containing the His-tagged RBLSte11, both Ste5 fragments were able to pull down the RBLSte11 domain, although the smaller Ste5 (373–523) fragment showed somewhat lower efficiency. The GST protein alone was used as a negative control, and no Ste11 fragment was retained on the column. These results established a direct interaction between Ste5 and Ste11 through a PH domain and an RBD-like domain. Further analysis indicated that the longer form of the PHSte5 domain behaved better in solution, and it was chosen along with the RBLSte11 domain for subsequent studies.
The bacterially expressed and purified RBLSte11 domain and the PHSte5 domain appeared to be predominantly monomers and were able to form a complex at a 1:1 ratio as judged by size exclusion chromatography. The complex appeared to be more stable in solution than either partner separately. The apparent affinity of the interaction of the complex was determined to be ~200 nM using surface plasmon resonance with the immobilized PHSte5 domain on the surface and the RBLSte11 domain in the flowing phase (Supplemental Figure S4(a)). The physical interaction of these two domains was also demonstrated by NMR analysis using an 15N-labeled RBLSte11 domain titrated with an unlabeled PHSte5 domain. A set of amino acid residues showed specific chemical shifts upon the addition of PHSte5 domain, indicating their involvement in the interaction (Supplemental Figure S4(b)).
Mutational analysis of the PHSte5 domain and structural mapping of the Ste11-interacting site
The RBL domain–PH domain interaction represents a new type of RBD interaction complex, as Ras-binding domain modules typically associate with small GTPases. To probe the structural basis of this interaction and the role of this interaction in signal transduction in the pheromone response MAP kinase pathway, we identified functionally important residues of the PHSte5
domain by both random and site-directed mutagenesis. We first screened for mutations that disrupted the function of the PH domain in the context of the PHSte5
-Ste50 chimera shown in . Approximately 150 clones that satisfied this criterion were selected after sequencing and classification according to the nature of the substitutions, and 10 representative mutants from random mutagenesis were chosen along with 6 mutants of site-directed mutagenesis, including I504T mutant based on previous work (Inouye et al., 1997
), for further functional analysis ().
These mutants were transferred into STE5 under the control of its own promoter using in vivo recombination in yeast cells deleted for the endogenous STE5. The resulting yeast strains bearing different single point mutations in the PHSte5 domain were assayed for their ability to direct pheromone signaling. All the mutants showed severe defects in pheromone response, with some exhibiting a totally sterile phenotype ( and ).
FIGURE 4: Mutational analysis of the essential role of the PHSte5 domain in pheromone response. (A) β-Galactosidase assay (top) and Halo assay (bottom) of yeast cells (ste5Δ) transformed with STE5 alleles carrying mutations in the PH domain for (more ...)
To confirm that these PHSte5-domain mutants have altered interactions with the RBLSte11, were cloned Ste5 mutants corresponding to residues 373–537, expressed them in bacteria, and used them for the in vitro binding assay with a bacterially expressed RBLSte11 domain (116–236). PHSte5-domain mutants showed severely decreased or no binding to the RBLSte11 domain, and the extent of the decrease in the interaction correlated well with that of the decease in the pheromone response (). These results indicate that the interaction of the PHSte5 domain with the RBLSte11 domain is essential for the pheromone response signal transduction pathway. Further analysis indicated that this interaction played a critical role in Ste11 MAPKKK activation, as an activated allele of STE11 largely bypassed the signaling defects of the Ste5 mutants ().
Modeled structure of the PHSte5 domain
To analyze the spatial relationship between the loss-of-binding mutants and to gain insight into the residues forming the interface between the RBL domain and the PH domain, we applied structural bioinformatics and homology modeling to construct a three-dimensional (3D) model of the PHSte5 domain encompassing residues 374–537. This model was refined by a 20-ns molecular dynamics (MD) simulation, at which point it attained structural convergence, with only a solvent-exposed loop region in the C-terminal end showing significant fluctuations at room temperature over the last 5 ns of MD simulation (Supplemental Figure S5). The average minimized structured over the last 1 ns of MD simulation is of good quality as validated by several methods (Supplemental Figure S6).
The modeled structure of the PHSte5
domain () was based on the PH domain of the guanine nucleotide exchange factor collybistin (PDB code 2DFK; Xiang et al., 2006
). It comprises the canonical PH fold (residues Leu-406–Asp-511) consisting of an antiparallel seven-stranded β-barrel (β4-β3-β2-β1-β7-β6-β5) capped by a C-terminal α-helix (αC). The axis of αC has a noticeable curvature. The canonical PH fold is flanked by N- and C-terminal extensions (Thr-374–Leu-405 and Phe-512–Gly-537) that interact with each other via helical regions present within these extensions and contact the outside of the β-barrel on strands β1-β2-β3. The N-terminal helix αN (Leu-375–Asn-389) is well formed and sandwiched between the β-barrel and the short C-terminal helix αC1 (residues Ile-529–Phe-532). Terminal helical extensions are predicted also in other PH domains from fungal Ste5 and Far1 homologues, and these are linked to the canonical PH fold by structurally varying linkers (Supplemental Figure S1(a)). The longer linker connecting the αC1 helix appears particularly flexible (i.e., unstructured) in our 3D model of the yeast PHSte5
domain (Supplemental Figure S5).
The mutagenized residues of the PHSte5 domain that affected mating and that modified Ste11 binding ability were projected on this 3D model ( and ). Mutations that significantly affected the mating activity (<1%) and had undetectable or severely decreased Ste11 binding define a contiguous surface patch on one face of the PHSte5-domain structure. They are located in the β5-β6-β7-αC region: T465A (β5–β6 loop), K472E (β6), S484P (β6–β7 loop), N491I (β7-–αC loop), and S494P, Q501R, K502R, and I504, in the αC helix. The exceptions are R379G in the αN helix in the N-terminal extension and F514L at the beginning of the loop following the αC helix. Two mutations affecting mating to a lesser extent (~10%) and having decreased Ste11 binding relative to wild-type Ste5 are R407K (at the beginning of the β1 strand) and Y487H (β7) map also to the same surface area. Overall the Ste11-interacting surface of the PHSte5 domain is centered on the αC helix.
FIGURE 5: Structural mapping of functional data. (A) Mutagenesis data () mapped on the modeled PHSte5 domain. Mutated residues are shown as CPK models color coded by activity change upon mutation relative to wild-type Ste5: red, <<0.1% mating (more ...)
PHSte5-domain mutants are defective only in the interaction with Ste11 and show normal cellular localization
Some known PH domains are capable of binding inositol phosphates and phosphatidylinositides (PIs) and may be functionally involved in plasma membrane targeting and association or subcellular localization (Lemmon, 2004
). The PHSte5
domain has recently been shown to bind phospholipids and to be required for plasma membrane localization of the protein. This property has been proposed to be required for the function of Ste5 in the activation of the pheromone response pathway (Garrenton et al., 2006
). A detailed analysis of the putative PI-binding sites of the PHSte5
domain is given in the Supplemental Figure S7. Briefly, the Ste11-interacting surface of the PHSte5
domain mapped by mutagenesis is distant from the canonical PI-binding site but partially overlaps with the general location of the noncanonical PI-binding site.
To demonstrate that the defect of our PHSte5
-domain mutants for pheromone response is due only to their inability to interact with Ste11 and not in membrane association, we constructed Ste5 mutants with a protein–protein interaction module that restores interaction with Ste11 and tested their ability to mate. To this end, we made an in-frame fusion of the SAM domain of Ste50 to the C-terminal region of Ste5. The fusion proteins are expected to restore the ability to interact with the Ste11 by taking advantage of the fact that Ste50 and Ste11 interact through their respective SAM domains (Wu et al. 1999
). Two Ste5 mutants chosen to be modified and assayed were Q501R and I504T, as these mutants showed a nearly sterile phenotype. These sterile Ste5 mutants became mating competent when they were fused with the SAM domain of Ste50 (), demonstrating that reestablishing the association between Ste5 mutants and Ste11 is critical for the signal transduction of the pheromone response pathway and that the mating incompetence of the Ste5 mutants is due to their inability to interact with Ste11.
FIGURE 6: PHSte5-domain mutants defective primarily in the binding of Ste11 MAPKKK. (A) Yeast cells (ste5Δ) carrying PHSte5-domain mutants that are defective in binding to RBLSte11 are defective in mating. (B) Reestablishing the interaction of Ste5 mutants (more ...)
We also constructed N-terminally green fluorescent protein (GFP)–tagged PHSte5 domain mutants to examine the subcellular localization of PHSte5-domain mutants using fluorescence microscopy. We chose three mutants with the most severe mating defects (Q501R, I504T, and F514L) to make the GFP-tagged constructs. These constructs, along with GFP-tagged wild-type Ste5, were transformed into wild-type yeast cells that are capable of forming pheromone-induced shmoos. All the mutants showed a subcellular localization similar to the wild-type Ste5: general cytoplasmic and nuclear distribution both in the absence and presence of pheromone and a sharp crescent-shaped localization to the shmoo tip in the presence of pheromone (). These results demonstrated that the Ste5 mutants are competent in plasma membrane recruitment, suggesting that the observed mating defect is likely due to the defect in the ability of these PH-domain mutants to interact with the RBL domain of Ste11.
Mutational analysis of the RBLSte11 domain and structural mapping of residues essential for pheromone response
We were interested in defining the face of the RBL module that interacts with this PHSte5
domain. To identify those residues in the RBLSte11
domain critical for the binding of the PHSte5
region, we carried out a random mutagenesis analysis. We showed previously that deleting a region encompassing the RBL domain of Ste11 permits a reduced response to mating pheromone in an otherwise wild-type background but causes sterility in a ste50Δ
yeast strain (Wu et al., 1999
). We used this observation and performed the mutagenesis analysis in a ste50Δ
strain so that the interaction of the RBLSte11
domains is the only driver of the pheromone response. Mutants that were pheromone response negative were selected and then further analyzed for their ability to activate the HOG pathway in the presence of Ste50 to eliminate nonfunctional nonsense mutations, as Ste11 lacking the RBL motif is fully capable of activating the HOG pathway (Wu et al., 1999
). Approximately 100 clones were selected for sequencing analysis and, after classification, eight representative clones with either single or double mutation were chosen for further studies. Two additional mutants generated by site-directed mutagenesis (D178R and D173R/F175A) were used as controls to show that RBLSte11
can tolerate radical mutations, as Ste11 with these mutations has a nearly normal signaling function ().
These Ste11 mutants were assayed for their function in the pheromone response pathway through their ability to allow formation of diploids, to permit pheromone-dependent cell cycle arrest, and to facilitate mating-specific gene transcription; all the mutants selected from the screening showed severe defects up to an essentially sterile phenotype. All these mutants were, however, fully able to activate the HOG pathway. This indicates that the mutational effects are specific for the function that requires the RBLSte11 domain and not due to a general loss of function of Ste11. The residues defined by the defective mutants were mapped onto the RBLSte11 structure ().
NMR-based mapping of the PHSte5 domain-binding interface on the RBLSte11-domain surface
The residues of the RBLSte11 domain involved in binding to the PHSte5 domain were also interrogated by NMR spectroscopy. To this end, we used a chemical shift perturbation approach through analysis of 1H15N HSQC spectra of the RBLSte11 domain upon addition of increasing amounts of an unlabeled PHSte5 domain. The majority of NMR signals experience some line broadening due to the large size of the complex. This indicates that the RBL domain does not undergo substantial conformational changes upon complex formation. However, a subset of amide signals undergoes a chemical shift change or extensive line broadening (Supplemental Figure S3B), allowing identification of the interface. The results are summarized in , where the affected residues are highlighted in magenta. The regions most affected by the binding of the PH domain include the N-terminal end of the strand β1, the β1/β2 loop, one residue from strand β3, the helix α2, and the following loop leading to strand β5 (). All of these regions are located close to each other in space, forming a continuous patch on the surface of the RBL domain and identifying the interface. This interface is in excellent agreement with the functional results from mutagenesis (); of eight mutants with drastically reduced mating (), seven were also picked up by NMR experiments, either identifying the same amino acid or its direct neighbor in the sequence, suggesting that the mutational analysis picked up critical residues involved in RBLSte11- and PHSte5-domain interaction. The number of identified residues that functionally impair mating is lower than the number of amino acids identified on the interface by NMR, indicating incomplete coverage by mutagenesis. However, the NMR data did not identify participation of the loop βF1/βF2 in binding the PH domain ( and ). Hence, this β-finger insert does not appear to be involved in the interaction with the Ste5 scaffold, although it may play a different functional role. The NMR data also help to interpret the three cases of impaired double mutants identified by random mutagenesis (), suggesting that most likely Asp-189 (rather than Thr-166), Asn-126 (rather than Lys-225), and Tyr-188 (rather than Ser-241) are responsible for the reduced mating. The only region located outside of this contiguous interface is the loop β1/β2, which was picked up by both mutagenesis and NMR (Asn-126 and Gly-128; ) or only by NMR experiments (the neighboring amino acid Leu-125). Most likely, some small conformational changes in this region are responsible for the observed effect.