- Email: [email protected]
The interaction of BMP-7 and ActRII implicates a new mode of receptor assembly
Update
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Vol.28 No.10 October 2003
| Research Focus
The interaction of BMP-7 and ActRII implicates a new mode of receptor assembly Walter Sebald and Thomas D. Mueller Department of Physiological Chemistry II, Biocenter, University of Wuerzburg, Am Hubland, D-97074 Wuerzburg, Germany
The recently described structure of bone morphogenetic protein 7 in complex with the extracellular domain of the activin type receptor II provides a new and important paradigm to add to the list of possible modes of receptor assembly. A new mode of a ligand-mediated cooperative receptor assembly without receptor– receptor contacts yields new and exciting insights into the molecular signal transduction mechanism in the transforming growth factor-b superfamily. An allosteric mechanism was proposed in a seminal TIBS review [1] for epidermal growth factor (EGF)-receptor signalling: a ligand-induced conformational change in the extracellular domain (ECD) of the receptor leads to dimerization by direct receptor– receptor contacts. This model has been confirmed by the crystal structure of the ECD – ligand complex [2] (Figure 1a).
In the cytokine-receptor system (e.g. the growthhormone-receptor system), ligands have multiple binding sites for receptor chains (single transmembrane receptor subunits). Recruitment of the second receptor chain is achieved by cooperation of low-affinity binding by the second epitope of the ligand and direct receptor– receptor interaction [3] (Figure 1b). Pre-assembled receptor complexes (i.e. before ligand binding), which constitute a further oligomerization mode, might play an important part in receptor-assembly dynamics [4,5] (Figure 1c). These complexes seem to be arrested in a non-productive conformation, preventing activation of the intracellular domains. The blockade is released by ligand binding and a productive complex is created. Some reports describe receptors forming preassembled complexes that have two or more transmembrane segments. The presence of multiple transmembrane
(a) Allosteric receptor oligomerization
(b) Multiple ligand epitopes and receptor–receptor interaction
(c) Pre-assembled complex
(d) Cooperative binding mode
Ti BS
Figure 1. Oligomerization modes for receptors with single membrane-spanning segments. Receptor chains (single transmembrane receptor subunits) are shown in grey, ligands in light blue. Red stars denote the transactivation of a receptor chain, which leads to activation of the intracellular signalling cascade. (a) Allosteric conformational changes of the extracellular domain upon ligand binding leads to receptor assembly and activation (e.g. epidermal growth factor-receptor paradigm [1]). (b) Homodimerization of the receptor is induced by two ligand epitopes and direct contacts between the extracellular domains of the receptor chains (e.g. growth hormone-receptor paradigm [3]). (c) Preformed receptor complexes existing in non-active form become activated upon ligand binding [4,5]. Some reports postulate transmembrane allosteric effects that are transmitted across the membrane by assemblies of multiple transmembrane segments (erythropoietin-receptor paradigm [20]; see also ‘piston-model’ [6]). (d) In the cooperative assembly mode the low-affinity receptor is not able to bind the ligand efficiently without the presence of the high-affinity receptor [9]. High-affinity chains are highlighted in yellow, low-affinity chains in green. No direct contacts occur between the receptor ectodomains (BMP –activin-receptor complex paradigm [7]). Mode (c) and (d) can be realized in the same receptor system resulting possibly in different downstream signalling [5]. Corresponding author: Walter Sebald ([email protected]). http://tibs.trends.com
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segments enables them to transmit conformational changes across the membrane, thus allowing allosteric mechanisms (‘piston’ model [6]). The bone morphogenetic protein-7 (BMP-7)–activin type II receptor (ActRII) ECD complex adds yet another paradigm for a cooperative mode of receptor assembly [7] (Figure 1d), providing new insights into how receptor chains might cooperate to accomplish affinity for their ligands. Crystal structure of the BMP-7–ActRII ligand–receptor complex Bone morphogenetic proteins (BMPs), activins, transforming growth factor-b (TGF-b), growth and differentiation factors, and anti-Mullerian hormones constitute the large TGF-b superfamily of secreted signalling molecules that control numerous crucial events in embryonal development and tissue – organ homeostasis of adult animals [8]. The BMPs fall into discrete subgroups based on aminoacid sequence similarity. The BMP-2/4 and BMP-5/6/7/8 subgroups, as well as the activin subgroup, are present in almost all multicellular animals from worms to flies to mammals [9,10]. BMP-7 is homodimeric and its structure has a twofold axis of symmetry (i.e. the monomer subunits can be transformed into each other by a rotation of 1808 around the axis of symmetry). Both the fold of the handshaped monomer with a central cystin knot and the architecture of the dimer are highly conserved throughout the TGF-b superfamily. Two distinct types of receptor chains, designated type I and II receptor based on their amino-acid homology, are required for signalling. Both types of receptor consist of a small extracellular domain that is linked to the membrane-spanning segment by a short peptide and a cytoplasmic part that contains a Ser/ Thr-kinase domain. Signalling by BMP-7 is mediated through a sequential binding mechanism; in the first step, BMP-7 is bound to its high-affinity type II receptor. In the subsequent step, the low-affinity type I receptor is recruited to the complex leading to trans-phosphorylation of the type I receptor at a glycine- and serine-rich region (GS-box) by the type II receptor kinase. This leads to activation of the type I receptor Ser/Thr-kinase and starts intracellular signalling to the nucleus via SMAD proteins. Recently, Senyon Choe and coworkers determined the structure of the complex of a BMP (BMP-7) and a type II receptor (ActRII) ECD [7]. The complex consists of the BMP-7 dimer and two ActRII receptor ECDs, and its structure exhibits a perfect twofold axis of symmetry. The dyad of the complex is likely to be oriented perpendicular to the membrane, with the C termini of the ActRII ECDs facing the membrane ˚ (Figure 2a). The ActRII ECDs and separated by , 80 A bind to the convex back-sides of either BMP-7 subunit. This ‘knuckle’ epitope for the interaction with type II receptors is absolutely conserved among BMPs and activin A, and has been identified in BMP-2 by mutational analysis [11] and in activin A by X-ray analysis of the activin A – ActRIIB complex [12]. By contrast, the epitope of TGF-b for binding to TGFreceptor type II is located at a ‘finger-tip’ epitope [13]. http://tibs.trends.com
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(a)
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Figure 2. The heterohexameric complex of bone morphogenetic protein (BMP)-7– BMP receptor IA (BMPR-IA)–activin type II receptor (ActRII) consisting of one ligand dimer bound to four receptor extracellular domains (ECDs). The components are shown in blue and yellow (BMP-7), green (ECDs of BMPR-IA) and red (ECDs of ActRII). The BMPR-IA ECDs were docked to the BMP-7 –ActRII complex [7] by superposition with the BMP-2 –BMPR-IA complex [14]. (a) The twofold axis of symmetry (broken line) is perpendicular to the membrane. The peptides connecting the ActRII ECDs (16 residues; blue and red) and the BMPR-IA ECDs (9 residues; green and red) with the membrane-spanning segments are depicted in an extended conformation. (b) Top view of the heterohexameric complex demonstrating that the receptor domains have no direct contacts. The C termini of the BMPR-IA and ActRII ECDs are ,30 A˚ apart.
How is cooperativity generated? The structure of this heterotrimeric complex represents the first step in BMP-7-mediated activin-receptor activation: the ligand dimer is bound to two high-affinity receptor chains and is prepared to recruit two low-affinity type I chains into the signalling complex. The result of this second activation step, the heterohexameric complex, can now be modelled by combining the previously determined structures of the complexes of BMP-2 and its type I receptor BMPR-IA [14], and BMP-7 and the type II receptor ActRII (Figure 2). Two type II receptor ECDs representing the high-affinity chains bind to the two convex sides of the BMP-7 dimer – the ‘knuckle’ epitope. Two type I receptor ECDs (the low-affinity chains) are then recruited into the complex by binding to the two concave sides of the symmetrical BMP-7 dimer – the ‘wrist’ epitope. The final signalling complex consists of the ligand dimer bound to four receptor chains, two type II and two type I receptors. The C termini of all four ECDs point towards the membrane. Remarkably, the extracellular parts of the receptor chains seem to have no mutual contacts and are connected only through the ligand. This
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was unexpected because in previously accepted receptor activation schemes [9,15] the recruitment of the lowaffinity type I receptor requires interaction with epitopes of both ligand and type II receptor chain [3]. Nevertheless, the high-affinity type II receptor ActRII definitely exerts a cooperative effect on the type I receptor–BMP complex assembly. How can this cooperative binding mode be explained, if the ECDs have no contacts among each other? This question leads to the central issues of the paper of Greenwald et al. [7]: by which mechanism is the lowaffinity chain recruited into the complex? Which roles do the ligand and the high-affinity receptor play in a cooperative binding mechanism? The term low-affinity chain is based on the observation that low-affinity receptors cannot be crosslinked with BMP-7 or activin on whole cells in the absence of high-affinity type II receptors. This is true for the complete chain as well as for the chain with its cytoplasmic domain deleted [7]. The cooperative binding shown in the crosslinking experiments were also demonstrated for the interaction of truncated receptor chains ActRI or ActRIB and ActRII that lacked their cytoplasmic domains. Thus, the cytoplasmic domains do not contribute to dimerization. Another possibility, that the transmembrane domains of type I and II receptors associate and, therefore, promote complex formation, was not excluded. In the model of the complex, the visible C termini of the ECDs of BMPR-IA ˚ apart. However, in reality, the and ActRII are , 30 A chains of BMPR-IA ECD and ActRII ECD are longer by 9 and 16 residues, respectively. These peptides are not visible in the electron density of the structures (probably owing to disorder in the crystal) and can easily be modelled to join at the point where they connect to the membrane segments. Is ligand allostery a new aspect of cooperativity? A low, but measurable, affinity (KD , 100 mM) was determined between the ActRI ECD and BMP-7 in biosensor-based interaction analysis employing immobilized BMP-7. Remarkably, a fivefold increase (KD , 20 mM) in the binding affinity of the type I ActRI ECD in the presence of the type II ActRII ECD was observed [7]. Free BMP-7 shows conformational differences compared with BMP-7 in complex with ActRII ECD [7,16]. These differences are in the ‘wrist’ and ‘knuckle’ epitopes and, therefore, might be indicative of allosteric effects. However, the small increase in binding affinity of approximately fivefold argues for a limited role of allosteric conformational changes in cooperative binding and does not really represent a transition from low-affinity to high-affinity binding. Two-dimensional interactions in the membrane The membrane could transform an interaction that is low affinity in solution into a high-affinity one [17,18]. The ligand is accumulated on the membrane by binding to the high-affinity receptor chain and, thereby, a high local concentration of the ligand is achieved. In addition, in the first step of complex formation, the ligand diffusing in free space (3-dimensional) is bound to the high-affinity receptor that is diffusing in the cell membrane (2-dimensional, one http://tibs.trends.com
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less degree of freedom). In the subsequent step, this intermediate complex recruits the low-affinity receptor, and the diffusional freedom of both components is restricted in two dimensions. Consequently, more productive encounters are expected for the binding to the second receptor than for a situation in which all components are freely diffusing in solution. Therefore, association rates are probably adequate for oligomer formation and signal induction. The half-life of the heterohexameric complex was not specified in the Greenwald paper, but it can be estimated to be in the 10 – 100 ms range from the kinetic constants given in the paper. Similar half-lives have been observed for many enzyme – substrate complexes [19]. Thus, an affinity of micromolar KD might lead to receptor oligomerization in the membrane for a period of time that is sufficient for the transphosphorylation reaction. Concluding remarks Heteromeric receptor complexes with high-affinity and low-affinity chains and with a cooperative mode of assembly have been reported not only for BMP-7 and the TGF-b superfamily [9], but also for numerous other receptor systems including cytokine receptors [18]. The analysis and modelling of the BMP-7 receptor has now clearly demonstrated that cooperative binding in this system is not entirely generated by ECD –ECD contacts and not by contacts of the cytosolic domains. Possible interactions between transmembrane segments still need to be established experimentally. However, low affinities increased by allosteric effects as well as by 2D-interaction dynamics in the membrane should account for the observed biological activity of the receptors, that is, ligand-dependent trans-phosphorylation of the type I chains and initiation of cytosolic signalling. References 1 Schlessinger, J. (1988) Signal transduction by allosteric receptor oligomerization. Trends Biochem. Sci. 13, 443– 447 2 Schlessinger, J. (2002) Ligand-induced, receptor-mediated dimerization and activation of EGF receptor. Cell 110, 669– 672 3 Bernat, B. et al. (2003) Determination of the energetics governing the regulatory step in growth hormone-induced receptor homodimerization. Proc. Natl. Acad. Sci. U. S. A. 100, 952– 957 4 Krause, C.D. et al. (2002) Seeing the light: preassembly and ligandinduced changes of the interferon g receptor complex in cells. Mol. Cell. Proteomics 1, 805– 815 5 Nohe, A. et al. (2002) The mode of bone morphogenetic protein (BMP) receptor oligomerization determines different BMP-2 signaling pathways. J. Biol. Chem. 277, 5330 – 5338 6 Ottemann, K.M. et al. (1999) A piston model for transmembrane signaling of the aspartate receptor. Science 285, 1751– 1754 7 Greenwald, J. et al. (2003) The BMP7/ActRII extracellular domain complex provides new insights into the cooperative nature of receptor assembly. Mol. Cell 11, 605 – 617 8 Hogan, B.L. (1996) Bone morphogenetic proteins in development. Curr. Opin. Genet. Dev. 6, 432– 438 9 Massague, J. (1998) TGF-b signal transduction. Annu. Rev. Biochem. 67, 753 – 791 10 Souchelnytskyi, S. et al. (2002) TGF-b signaling from a threedimensional perspective: insight into selection of partners. Trends Cell Biol. 12, 304– 307 11 Kirsch, T. et al. (2000) BMP-2 antagonists emerge from alterations in the low-affinity binding epitope for receptor BMPR-II. EMBO J. 19, 3314– 3324 12 Thompson, T.B. et al. (2003) Structures of an ActRIIB:activin A
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17 Grasberger, B. et al. (1986) Interaction between proteins localized in membranes. Proc. Natl. Acad. Sci. U. S. A. 83, 6258 – 6262 18 Letzelter, F. et al. (1998) The interleukin-4 site-2 epitope determining binding of the common receptor g chain. Eur. J. Biochem. 257, 11 – 20 19 Schomburg, D. et al. (2002) Springer Handbook of Enzymes, 2nd edn, Springer Verlag 20 Livnah, O. et al. (1999) Crystallographic evidence for preformed dimers of erythropoietin receptor before ligand activation. Science 283, 987– 990
complex reveal a novel binding mode for TGF-b ligand:receptor interactions. EMBO J. 22, 1555– 1566 Hart, P.J. et al. (2002) Crystal structure of the human TbR2 ectodomain – TGF-b3 complex. Nat. Struct. Biol. 9, 203 – 208 Kirsch, T. et al. (2000) Crystal structure of the BMP-2 – BRIA ectodomain complex. Nat. Struct. Biol. 7, 492 – 496 Wrana, J.L. et al. (1994) Mechanism of activation of the TGF-b receptor. Nature 370, 341 – 347 riffith, D.L. et al. (1996) Three-dimensional structure of recombinant human osteogenic protein 1: structural paradigm for the transforming growth factor b superfamily. Proc. Natl. Acad. Sci. U. S. A. 93, 878 – 883
0968-0004/$ - see front matter q 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.tibs.2003.08.001
Genes within genes within bacteria J. Christopher Ellis and James W. Brown Department of Microbiology, North Carolina State University, Campus Box 7615, Raleigh, NC 27695-7615, USA
the ribosomal protein L34 (rpmH; Figure 1), and the two are located near the origin of replication [1 – 3]. This co-localization of genes in a wide range of bacterial genomes implies an important linkage in their regulation of expression, but the mechanism of this regulation has not been investigated. These genes in Escherichia coli have been demonstrated to be part of the same operon, with two major and one minor promoter upstream of rpmH, and two putative transcription-termination signals downstream of rpmH [3– 5]. The levels of expression of the encoded proteins are quite different; the ribosomal protein
Recently, an unusual gene structure has been described in species of the genus Thermus, in which the rpmH (ribosomal protein L34) coding sequence was found to be entirely overlapped by the unusually large rnpA (RNase P protein subunit) sequence. Gene overlap is common in viruses, but has not been seen to this extent in any bacterium. Generally, in bacteria, the gene encoding the protein subunit of RNase P (rnpA) is located immediately downstream of and in the same orientation as the gene encoding (a) Escherichia coli
yidD rpmH
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V
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P T
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RBS ...GGGCCATGGCCCCAGGGAGGGAGATGGATGAAAAGGACGTGGCAACCCAACC... Ti BS
Figure 1. Comparison of rpmH with rnpA gene structure, which are common in bacteria and Thermus. The usual gene structure in bacteria is exemplified by that of Escherichia coli (a) [3 –5], the overlapping gene structure of Thermus species is exemplified by Thermus thermophilus (b) [8]. Promoters, putative transcription terminators and ribosome-binding sequences (RBS) for rpmH (which encodes the ribosomal protein L34) and rnpA (which encodes the protein subunit of RNase P) expression are indicated by P (large for major promoters; small for minor promoters), T and RBS, respectively. Coding sequences are indicated by large arrows; homologous sequences in rpmH and rnpA are blue and red, respectively. The region of translational initiation in T. thermophilus is expanded; the start codons for rpmH and rnpA are underlined.
Corresponding author: James W. Brown ([email protected]). http://tibs.trends.com
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Vol.28 No.10 October 2003
| Research Focus
The interaction of BMP-7 and ActRII implicates a new mode of receptor assembly Walter Sebald and Thomas D. Mueller Department of Physiological Chemistry II, Biocenter, University of Wuerzburg, Am Hubland, D-97074 Wuerzburg, Germany
The recently described structure of bone morphogenetic protein 7 in complex with the extracellular domain of the activin type receptor II provides a new and important paradigm to add to the list of possible modes of receptor assembly. A new mode of a ligand-mediated cooperative receptor assembly without receptor– receptor contacts yields new and exciting insights into the molecular signal transduction mechanism in the transforming growth factor-b superfamily. An allosteric mechanism was proposed in a seminal TIBS review [1] for epidermal growth factor (EGF)-receptor signalling: a ligand-induced conformational change in the extracellular domain (ECD) of the receptor leads to dimerization by direct receptor– receptor contacts. This model has been confirmed by the crystal structure of the ECD – ligand complex [2] (Figure 1a).
In the cytokine-receptor system (e.g. the growthhormone-receptor system), ligands have multiple binding sites for receptor chains (single transmembrane receptor subunits). Recruitment of the second receptor chain is achieved by cooperation of low-affinity binding by the second epitope of the ligand and direct receptor– receptor interaction [3] (Figure 1b). Pre-assembled receptor complexes (i.e. before ligand binding), which constitute a further oligomerization mode, might play an important part in receptor-assembly dynamics [4,5] (Figure 1c). These complexes seem to be arrested in a non-productive conformation, preventing activation of the intracellular domains. The blockade is released by ligand binding and a productive complex is created. Some reports describe receptors forming preassembled complexes that have two or more transmembrane segments. The presence of multiple transmembrane
(a) Allosteric receptor oligomerization
(b) Multiple ligand epitopes and receptor–receptor interaction
(c) Pre-assembled complex
(d) Cooperative binding mode
Ti BS
Figure 1. Oligomerization modes for receptors with single membrane-spanning segments. Receptor chains (single transmembrane receptor subunits) are shown in grey, ligands in light blue. Red stars denote the transactivation of a receptor chain, which leads to activation of the intracellular signalling cascade. (a) Allosteric conformational changes of the extracellular domain upon ligand binding leads to receptor assembly and activation (e.g. epidermal growth factor-receptor paradigm [1]). (b) Homodimerization of the receptor is induced by two ligand epitopes and direct contacts between the extracellular domains of the receptor chains (e.g. growth hormone-receptor paradigm [3]). (c) Preformed receptor complexes existing in non-active form become activated upon ligand binding [4,5]. Some reports postulate transmembrane allosteric effects that are transmitted across the membrane by assemblies of multiple transmembrane segments (erythropoietin-receptor paradigm [20]; see also ‘piston-model’ [6]). (d) In the cooperative assembly mode the low-affinity receptor is not able to bind the ligand efficiently without the presence of the high-affinity receptor [9]. High-affinity chains are highlighted in yellow, low-affinity chains in green. No direct contacts occur between the receptor ectodomains (BMP –activin-receptor complex paradigm [7]). Mode (c) and (d) can be realized in the same receptor system resulting possibly in different downstream signalling [5]. Corresponding author: Walter Sebald ([email protected]). http://tibs.trends.com
Update
TRENDS in Biochemical Sciences
segments enables them to transmit conformational changes across the membrane, thus allowing allosteric mechanisms (‘piston’ model [6]). The bone morphogenetic protein-7 (BMP-7)–activin type II receptor (ActRII) ECD complex adds yet another paradigm for a cooperative mode of receptor assembly [7] (Figure 1d), providing new insights into how receptor chains might cooperate to accomplish affinity for their ligands. Crystal structure of the BMP-7–ActRII ligand–receptor complex Bone morphogenetic proteins (BMPs), activins, transforming growth factor-b (TGF-b), growth and differentiation factors, and anti-Mullerian hormones constitute the large TGF-b superfamily of secreted signalling molecules that control numerous crucial events in embryonal development and tissue – organ homeostasis of adult animals [8]. The BMPs fall into discrete subgroups based on aminoacid sequence similarity. The BMP-2/4 and BMP-5/6/7/8 subgroups, as well as the activin subgroup, are present in almost all multicellular animals from worms to flies to mammals [9,10]. BMP-7 is homodimeric and its structure has a twofold axis of symmetry (i.e. the monomer subunits can be transformed into each other by a rotation of 1808 around the axis of symmetry). Both the fold of the handshaped monomer with a central cystin knot and the architecture of the dimer are highly conserved throughout the TGF-b superfamily. Two distinct types of receptor chains, designated type I and II receptor based on their amino-acid homology, are required for signalling. Both types of receptor consist of a small extracellular domain that is linked to the membrane-spanning segment by a short peptide and a cytoplasmic part that contains a Ser/ Thr-kinase domain. Signalling by BMP-7 is mediated through a sequential binding mechanism; in the first step, BMP-7 is bound to its high-affinity type II receptor. In the subsequent step, the low-affinity type I receptor is recruited to the complex leading to trans-phosphorylation of the type I receptor at a glycine- and serine-rich region (GS-box) by the type II receptor kinase. This leads to activation of the type I receptor Ser/Thr-kinase and starts intracellular signalling to the nucleus via SMAD proteins. Recently, Senyon Choe and coworkers determined the structure of the complex of a BMP (BMP-7) and a type II receptor (ActRII) ECD [7]. The complex consists of the BMP-7 dimer and two ActRII receptor ECDs, and its structure exhibits a perfect twofold axis of symmetry. The dyad of the complex is likely to be oriented perpendicular to the membrane, with the C termini of the ActRII ECDs facing the membrane ˚ (Figure 2a). The ActRII ECDs and separated by , 80 A bind to the convex back-sides of either BMP-7 subunit. This ‘knuckle’ epitope for the interaction with type II receptors is absolutely conserved among BMPs and activin A, and has been identified in BMP-2 by mutational analysis [11] and in activin A by X-ray analysis of the activin A – ActRIIB complex [12]. By contrast, the epitope of TGF-b for binding to TGFreceptor type II is located at a ‘finger-tip’ epitope [13]. http://tibs.trends.com
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Figure 2. The heterohexameric complex of bone morphogenetic protein (BMP)-7– BMP receptor IA (BMPR-IA)–activin type II receptor (ActRII) consisting of one ligand dimer bound to four receptor extracellular domains (ECDs). The components are shown in blue and yellow (BMP-7), green (ECDs of BMPR-IA) and red (ECDs of ActRII). The BMPR-IA ECDs were docked to the BMP-7 –ActRII complex [7] by superposition with the BMP-2 –BMPR-IA complex [14]. (a) The twofold axis of symmetry (broken line) is perpendicular to the membrane. The peptides connecting the ActRII ECDs (16 residues; blue and red) and the BMPR-IA ECDs (9 residues; green and red) with the membrane-spanning segments are depicted in an extended conformation. (b) Top view of the heterohexameric complex demonstrating that the receptor domains have no direct contacts. The C termini of the BMPR-IA and ActRII ECDs are ,30 A˚ apart.
How is cooperativity generated? The structure of this heterotrimeric complex represents the first step in BMP-7-mediated activin-receptor activation: the ligand dimer is bound to two high-affinity receptor chains and is prepared to recruit two low-affinity type I chains into the signalling complex. The result of this second activation step, the heterohexameric complex, can now be modelled by combining the previously determined structures of the complexes of BMP-2 and its type I receptor BMPR-IA [14], and BMP-7 and the type II receptor ActRII (Figure 2). Two type II receptor ECDs representing the high-affinity chains bind to the two convex sides of the BMP-7 dimer – the ‘knuckle’ epitope. Two type I receptor ECDs (the low-affinity chains) are then recruited into the complex by binding to the two concave sides of the symmetrical BMP-7 dimer – the ‘wrist’ epitope. The final signalling complex consists of the ligand dimer bound to four receptor chains, two type II and two type I receptors. The C termini of all four ECDs point towards the membrane. Remarkably, the extracellular parts of the receptor chains seem to have no mutual contacts and are connected only through the ligand. This
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was unexpected because in previously accepted receptor activation schemes [9,15] the recruitment of the lowaffinity type I receptor requires interaction with epitopes of both ligand and type II receptor chain [3]. Nevertheless, the high-affinity type II receptor ActRII definitely exerts a cooperative effect on the type I receptor–BMP complex assembly. How can this cooperative binding mode be explained, if the ECDs have no contacts among each other? This question leads to the central issues of the paper of Greenwald et al. [7]: by which mechanism is the lowaffinity chain recruited into the complex? Which roles do the ligand and the high-affinity receptor play in a cooperative binding mechanism? The term low-affinity chain is based on the observation that low-affinity receptors cannot be crosslinked with BMP-7 or activin on whole cells in the absence of high-affinity type II receptors. This is true for the complete chain as well as for the chain with its cytoplasmic domain deleted [7]. The cooperative binding shown in the crosslinking experiments were also demonstrated for the interaction of truncated receptor chains ActRI or ActRIB and ActRII that lacked their cytoplasmic domains. Thus, the cytoplasmic domains do not contribute to dimerization. Another possibility, that the transmembrane domains of type I and II receptors associate and, therefore, promote complex formation, was not excluded. In the model of the complex, the visible C termini of the ECDs of BMPR-IA ˚ apart. However, in reality, the and ActRII are , 30 A chains of BMPR-IA ECD and ActRII ECD are longer by 9 and 16 residues, respectively. These peptides are not visible in the electron density of the structures (probably owing to disorder in the crystal) and can easily be modelled to join at the point where they connect to the membrane segments. Is ligand allostery a new aspect of cooperativity? A low, but measurable, affinity (KD , 100 mM) was determined between the ActRI ECD and BMP-7 in biosensor-based interaction analysis employing immobilized BMP-7. Remarkably, a fivefold increase (KD , 20 mM) in the binding affinity of the type I ActRI ECD in the presence of the type II ActRII ECD was observed [7]. Free BMP-7 shows conformational differences compared with BMP-7 in complex with ActRII ECD [7,16]. These differences are in the ‘wrist’ and ‘knuckle’ epitopes and, therefore, might be indicative of allosteric effects. However, the small increase in binding affinity of approximately fivefold argues for a limited role of allosteric conformational changes in cooperative binding and does not really represent a transition from low-affinity to high-affinity binding. Two-dimensional interactions in the membrane The membrane could transform an interaction that is low affinity in solution into a high-affinity one [17,18]. The ligand is accumulated on the membrane by binding to the high-affinity receptor chain and, thereby, a high local concentration of the ligand is achieved. In addition, in the first step of complex formation, the ligand diffusing in free space (3-dimensional) is bound to the high-affinity receptor that is diffusing in the cell membrane (2-dimensional, one http://tibs.trends.com
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less degree of freedom). In the subsequent step, this intermediate complex recruits the low-affinity receptor, and the diffusional freedom of both components is restricted in two dimensions. Consequently, more productive encounters are expected for the binding to the second receptor than for a situation in which all components are freely diffusing in solution. Therefore, association rates are probably adequate for oligomer formation and signal induction. The half-life of the heterohexameric complex was not specified in the Greenwald paper, but it can be estimated to be in the 10 – 100 ms range from the kinetic constants given in the paper. Similar half-lives have been observed for many enzyme – substrate complexes [19]. Thus, an affinity of micromolar KD might lead to receptor oligomerization in the membrane for a period of time that is sufficient for the transphosphorylation reaction. Concluding remarks Heteromeric receptor complexes with high-affinity and low-affinity chains and with a cooperative mode of assembly have been reported not only for BMP-7 and the TGF-b superfamily [9], but also for numerous other receptor systems including cytokine receptors [18]. The analysis and modelling of the BMP-7 receptor has now clearly demonstrated that cooperative binding in this system is not entirely generated by ECD –ECD contacts and not by contacts of the cytosolic domains. Possible interactions between transmembrane segments still need to be established experimentally. However, low affinities increased by allosteric effects as well as by 2D-interaction dynamics in the membrane should account for the observed biological activity of the receptors, that is, ligand-dependent trans-phosphorylation of the type I chains and initiation of cytosolic signalling. References 1 Schlessinger, J. (1988) Signal transduction by allosteric receptor oligomerization. Trends Biochem. Sci. 13, 443– 447 2 Schlessinger, J. (2002) Ligand-induced, receptor-mediated dimerization and activation of EGF receptor. Cell 110, 669– 672 3 Bernat, B. et al. (2003) Determination of the energetics governing the regulatory step in growth hormone-induced receptor homodimerization. Proc. Natl. Acad. Sci. U. S. A. 100, 952– 957 4 Krause, C.D. et al. (2002) Seeing the light: preassembly and ligandinduced changes of the interferon g receptor complex in cells. Mol. Cell. Proteomics 1, 805– 815 5 Nohe, A. et al. (2002) The mode of bone morphogenetic protein (BMP) receptor oligomerization determines different BMP-2 signaling pathways. J. Biol. Chem. 277, 5330 – 5338 6 Ottemann, K.M. et al. (1999) A piston model for transmembrane signaling of the aspartate receptor. Science 285, 1751– 1754 7 Greenwald, J. et al. (2003) The BMP7/ActRII extracellular domain complex provides new insights into the cooperative nature of receptor assembly. Mol. Cell 11, 605 – 617 8 Hogan, B.L. (1996) Bone morphogenetic proteins in development. Curr. Opin. Genet. Dev. 6, 432– 438 9 Massague, J. (1998) TGF-b signal transduction. Annu. Rev. Biochem. 67, 753 – 791 10 Souchelnytskyi, S. et al. (2002) TGF-b signaling from a threedimensional perspective: insight into selection of partners. Trends Cell Biol. 12, 304– 307 11 Kirsch, T. et al. (2000) BMP-2 antagonists emerge from alterations in the low-affinity binding epitope for receptor BMPR-II. EMBO J. 19, 3314– 3324 12 Thompson, T.B. et al. (2003) Structures of an ActRIIB:activin A
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17 Grasberger, B. et al. (1986) Interaction between proteins localized in membranes. Proc. Natl. Acad. Sci. U. S. A. 83, 6258 – 6262 18 Letzelter, F. et al. (1998) The interleukin-4 site-2 epitope determining binding of the common receptor g chain. Eur. J. Biochem. 257, 11 – 20 19 Schomburg, D. et al. (2002) Springer Handbook of Enzymes, 2nd edn, Springer Verlag 20 Livnah, O. et al. (1999) Crystallographic evidence for preformed dimers of erythropoietin receptor before ligand activation. Science 283, 987– 990
complex reveal a novel binding mode for TGF-b ligand:receptor interactions. EMBO J. 22, 1555– 1566 Hart, P.J. et al. (2002) Crystal structure of the human TbR2 ectodomain – TGF-b3 complex. Nat. Struct. Biol. 9, 203 – 208 Kirsch, T. et al. (2000) Crystal structure of the BMP-2 – BRIA ectodomain complex. Nat. Struct. Biol. 7, 492 – 496 Wrana, J.L. et al. (1994) Mechanism of activation of the TGF-b receptor. Nature 370, 341 – 347 riffith, D.L. et al. (1996) Three-dimensional structure of recombinant human osteogenic protein 1: structural paradigm for the transforming growth factor b superfamily. Proc. Natl. Acad. Sci. U. S. A. 93, 878 – 883
0968-0004/$ - see front matter q 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.tibs.2003.08.001
Genes within genes within bacteria J. Christopher Ellis and James W. Brown Department of Microbiology, North Carolina State University, Campus Box 7615, Raleigh, NC 27695-7615, USA
the ribosomal protein L34 (rpmH; Figure 1), and the two are located near the origin of replication [1 – 3]. This co-localization of genes in a wide range of bacterial genomes implies an important linkage in their regulation of expression, but the mechanism of this regulation has not been investigated. These genes in Escherichia coli have been demonstrated to be part of the same operon, with two major and one minor promoter upstream of rpmH, and two putative transcription-termination signals downstream of rpmH [3– 5]. The levels of expression of the encoded proteins are quite different; the ribosomal protein
Recently, an unusual gene structure has been described in species of the genus Thermus, in which the rpmH (ribosomal protein L34) coding sequence was found to be entirely overlapped by the unusually large rnpA (RNase P protein subunit) sequence. Gene overlap is common in viruses, but has not been seen to this extent in any bacterium. Generally, in bacteria, the gene encoding the protein subunit of RNase P (rnpA) is located immediately downstream of and in the same orientation as the gene encoding (a) Escherichia coli
yidD rpmH
RBS P
rnpA
RBS
>P>P>
TT
dnaA (b) Thermus thermophilus
rpmH
RBS
rnpA P>P>
yidD
T
livG rpmH M M
D
K E
R K
T W D
V
Q A
P T
T ...
rnpA
Q ...
RBS ...GGGCCATGGCCCCAGGGAGGGAGATGGATGAAAAGGACGTGGCAACCCAACC... Ti BS
Figure 1. Comparison of rpmH with rnpA gene structure, which are common in bacteria and Thermus. The usual gene structure in bacteria is exemplified by that of Escherichia coli (a) [3 –5], the overlapping gene structure of Thermus species is exemplified by Thermus thermophilus (b) [8]. Promoters, putative transcription terminators and ribosome-binding sequences (RBS) for rpmH (which encodes the ribosomal protein L34) and rnpA (which encodes the protein subunit of RNase P) expression are indicated by P (large for major promoters; small for minor promoters), T and RBS, respectively. Coding sequences are indicated by large arrows; homologous sequences in rpmH and rnpA are blue and red, respectively. The region of translational initiation in T. thermophilus is expanded; the start codons for rpmH and rnpA are underlined.
Corresponding author: James W. Brown ([email protected]). http://tibs.trends.com