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Elevating the alternating-access model Renae M Ryan & Robert J Vandenberg
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© 2016 Nature America, Inc. All rights reserved.
50 years ago, Jardetzky proposed the alternating-access model, which has shaped the theoretical understanding of how substrates are carried across cell membranes. Two studies now demonstrate that transporters from distinct families undergo unexpectedly large elevator-like movements and also suggest that an ‘elevate and twist’ mechanism is a common means of achieving alternating access across the membrane. Membrane transport proteins control the movement of ions, solutes, chemical messengers, nutrients and waste across the cell membrane. They make up ~10% of all encoded proteins in humans and are important not only as drug targets but also in facilitating drug transport across membranes. In 1966, Jardetzky proposed an alternatingaccess mechanism describing the movement of substrates across the membrane1. It has been proposed that a carrier (transport protein) alternates between an extracellular- and an intracellular-facing state in which the centrally located substrate-binding site is accessible to only one side of the membrane at a time. Until recently, it had been thought that most membrane transporters use the rocker-switch model of alternating access, in which substrate binds to a site deep within the membrane, and the protein rearranges to allow access of the substrate-binding site to the inside of the cell (Fig. 1a). This type of mechanism is used widely in the major facilitator superfamily of transporters, such as the archetypal lactose permease, LacY2. In 2009, Boudker and colleagues revealed that the glutamate transporter homolog GltPh uses a twisting elevator-like mechanism of transport in which the ‘transport domain’ containing all of the cargo to be transported undergoes a 16-Å vertical movement and a 37° rotation to deliver substrate to the inside of the cell, while the ‘scaffold domain’ that anchors the protein in the membrane remains relatively static3. It had previously been thought that this elevator Renae M. Ryan and Robert J. Vandenberg are at the Discipline of Pharmacology, Sydney Medical School, University of Sydney, Sydney, New South Wales, Australia. e-mail: [email protected]
mechanism (Fig. 1b) may have been unique to the glutamate transporter family, but two unrelated proteins have now been shown to use an elevator-like mechanism of transport. In this issue, Mindell and colleagues4 and Drew and colleagues5 demonstrate that the transport domains of the dicarboxylate transporter from Vibrio cholerae (VcINDY) and an N+/H+ antiporter from Thermus thermophilus (NapA) undergo vertical translocation and rotation relative to their respective scaffold domains during substrate transport.
a
b
The only crystal structure available for the divalent anion sodium symporter (DASS) family is the inward-facing inhibitor-bound structure of VcINDY, which is a dimer of two identical protomers6. Each protomer has a similar overall architecture to a protomer of GltPh3,7 and is divided into two domains: a transport domain that contains the binding sites for substrate and coupled Na+ ions and a scaffold domain that contains the interprotomer contacts along the dimer interface (Fig. 2a,b). In this issue, Mindell and colleagues4
Rocker-switch mechanism
Elevator mechanism Gate
Transport domain
Scaffold domain
Gate
Figure 1 Alternating-access mechanisms of substrate transport across the membrane. (a) The rockerswitch mechanism involves protein rearrangements around a central substrate-binding site (orange circle) that results in the substrate being alternately exposed to either side of the membrane. (b) The elevator mechanism of transport describes a process in which the substrate is picked up on one side of the membrane, and a ‘gate’ subsequently closes. Next, part of the protein (termed the transport or core domain) undergoes a vertical translation relative to the membrane plane to carry the cargo across the membrane. For the cargo to be delivered into the cell, another gate must open to release the substrate, and the elevator then returns to the outside of the cell and is ready to pick up the next passenger. The elevator can be empty, as is the case with symporters such as VcINDY, or it may carry another ion or substrate across the membrane if the transporter operates via an antiport mechanism, as does NapA. Adapted with permission from ref. 19, Nature Publishing Group.
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VcINDY Inward facing
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Figure 2 The two domains of elevator transporters. (a–c) The aspartate transporter GltPh (PDB 2WNX) (a), the succinate transporter VcINDY (PDB 4F35) (b) and the Na+/H+ exchanger NapA (PDB 4BWZ) (c), viewed from the extracellular side of the membrane. GltPh exists as a trimer (of three identical protomers), whereas VcINDY and NapA are dimers (of two identical protomers). Although these proteins are unrelated in sequence, the tertiary structure of all three transporters reveals two distinct domains: a transporter domain that contains all of the cargo to be transported across the membrane (purple helices) and a scaffold domain that anchors the protein in the membrane and contains the interprotomer contacts (also known as the oligomerization domain; blue helices). GltPh is shown with bound aspartate and two Na+ ions, and VcINDY is shown with bound citrate and one Na+ ion. Aspartate and citrate are shown as spheres, and Na+ ions are indicated in yellow. In NapA, the ion-binding residue (Asp157) is shown in orange spheres, and the ‘linker’ helix (transmembrane domain 6) is shown in gray. (d) VcINDY viewed in the plane of the membrane in the inward-facing conformation (left; PDB 4F35) and the outward-facing conformation (right; Protein Model Database PM0080216 (ref. 4)). (e) NapA in the inward-facing conformation (left; PDB 5BZ2 (ref. 5)) and outward-facing (right; PDB 4BWZ) conformation. In d and e, the scaffold domain (blue helices) and the transport domain (purple helices) are indicated, and the elevator movement of the transport domain is indicated by the movement of the transported species (VcINDY, Na+/citrate; NapA, Asp157). The linker helix (TM6) of NapA is shown in gray ribbon. Structures were prepared with PyMOL (http://www.pymol.org/).
use a repeat-swap method to predict the outward-facing structure of VcINDY. This method has successfully been used to predict the inward-facing structure of GltPh8,9. The outward-facing model of VcINDY reveals an ~15-Å vertical translation and an ~43° rotation of the transport domain relative to the scaffold domain (Fig. 2d). This twisting elevator movement has been verified by Mindell and colleagues4 through a series of cross-links between pairs of introduced cysteine residues, thus confirming that the transport domain indeed undergoes this large movement. When this elevator movement is restricted by cross-linking of the transport domain to the scaffold domain, the transport 188
process is inhibited, thus demonstrating that large movements of the transport domain are required for transport. In addition, crosslinks designed to bridge the scaffold domain at the dimer interface do not affect the ability of VcINDY to transport substrate, thus revealing that large-scale movements of the two protomers relative to each other are not compulsory for substrate translocation. There are several available crystal structures of Na+/H+ antiporters from different species and in different states of the transport cycle10–13. Although there is general agreement that these antiporters can be divided into two domains consisting of a transport (or core) domain and a scaffold (or dimerization) domain (Fig. 2c),
interpretation of the movements required for Na+/H+ antiport has been controversial. These antiporters have been proposed to operate via a traditional rocker-switch mechanism with small rearrangements of the protein around a central binding site13 and also via an elevatorlike mechanism similar to that of GltPh11. To clarify this issue, Drew and colleagues5 have solved the structure of NapA in the outward- and inward-facing states, revealing substantial movements of the core ion-binding domain relative to the dimer or scaffold domain in the same protein. In this study, with the aim of trapping the transporter in an inward-facing state, the authors introduced two cysteine residues. These two residues form a disulfide bond, and when they are cross-linked, the function of the transporter is inhibited. Drew and colleagues5 have crystallized this inward-facing disulfide-trapped protein and have compared the structure to the outward-facing structure determined in detergent11 and also in lipidic mesophase5. Using a combination of molecular dynamics and biochemical analysis, the authors conclude that the transport domain undergoes a rigid-body vertical translation (7–10 Å) relative to the scaffold domain, but with minimal twist (Fig. 2e). Nevertheless, it appears that NapA uses an elevator-like mechanism rather than a rocker-switch mechanism. There are several transporters with a domain architecture similar to that of both VcINDY14,15 and NapA16,17 that have also been predicted to use a twisting elevator mechanism, and it will be interesting to determine how many other transport proteins use this mechanism of transport. Given the important role of tertiary protein structure in determining transporter mechanism, it is likely that other members of the VcINDY and NapA families, and even transporters from unrelated families, will be found to use a similar mechanism for substrate transport. Indeed, a recent study has reported two structures of a citrate transporter (CitS): one in an inward-facing conformation and one in an outward-facing conformation18. Similarly to VcINDY and NapA, this unrelated protein also exists as a dimer with each protomer divided into a transport and scaffold domain. During the transport process, the transport domain undergoes a 16-Å translation and a 31° rotation and also appears to use a twisting elevator mechanism of transport. Although there is variability in the extent of the translation and rotation of the transport domains among these elevator transporters3–5,18, all of these examples reveal that the substrate-binding site undergoes a vertical translation during substrate translocation, and transport is not achieved solely through rearrangement of the protein around the
volume 23 number 3 March 2016 nature structural & molecular biology
news and views substrate-binding site, as occurs in the rockerswitch mechanism. On this 50th anniversary of Jardetzky’s alternating-access proposal, a wealth of available structural and biophysical information on many transport proteins is allowing researchers to visualize the physical reality of his theoretical proposal. Subtle variations in the complex process of membrane transport can now begin to be teased apart, and the alternating-access model can be refined to include translation and rotation of the protein—two properties that Jardetzky excluded from the ‘allosteric pump’ model1. In summary, these studies have provided two new examples of transporters that use an elevator mechanism. It is now clear
that this mechanism is not unique to the glutamate transporter family, and there may be many more transport proteins—unrelated in sequence and varied in biological function— that also use a twisting elevator to move their substrates across membranes. COMPETING FINANCIAL INTERESTS The authors declare no competing financial interests. 1. Jardetzky, O. Nature 211, 969–970 (1966). 2. Yan, N. Annu. Rev. Biophys. 44, 257–283 (2015). 3. Reyes, N., Ginter, C. & Boudker, O. Nature 462, 880–885 (2009). 4. Mulligan, C. et al. Nat. Struct. Mol. Biol. 23, 256–263 (2016). 5. Coincon, M. et al. Nat. Struct. Mol. Biol. 23, 248–255 (2016).
6. Mancusso, R., Gregorio, G.G., Liu, Q. & Wang, D.N. Nature 491, 622–626 (2012). 7. Boudker, O., Ryan, R.M., Yernool, D., Shimamoto, K. & Gouaux, E. Nature 445, 387–393 (2007). 8. Crisman, T.J., Qu, S., Kanner, B.I. & Forrest, L.R. Proc. Natl. Acad. Sci. USA 106, 20752–20757 (2009). 9. Vergara-Jaque, A., Fenollar-Ferrer, C., Kaufmann, D. & Forrest, L.R. Front. Pharmacol. 6, 183 (2015). 10. Hunte, C. et al. Nature 435, 1197–1202 (2005). 11. Lee, C. et al. Nature 501, 573–577 (2013). 12. Lee, C. et al. J. Gen. Physiol. 144, 529–544 (2014). 13. Paulino, C., Wöhlert, D., Kapotova, E., Yildiz, Ö. & Kühlbrandt, W. eLife 3, e03583 (2014). 14. Bolla, J.R. et al. Nat. Commun. 6, 6874 (2015). 15. Su, C.C. et al. Cell Rep. 11, 61–70 (2015). 16. Hu, N.J., Iwata, S., Cameron, A.D. & Drew, D. Nature 478, 408–411 (2011). 17. Zhou, X. et al. Nature 505, 569–573 (2014). 18. Wohlert, D., Grotzinger, M.J., Kuhlbrandt, W. & Yildiz, O. eLife 4, (2015). 19. Slotboom, D.J. Nat. Rev. Microbiol. 12, 79–87 (2014).
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A new communication hub in the RNA world Megan Mayerle & Christine Guthrie During assembly of spliceosomal small nuclear ribonucleoproteins (snRNPs), the RNA-binding protein (RBP) Gemin5 recognizes the snRNP code and interacts with the large Gemin2–SMN complex. So et al. now find that Gemin2 also interacts with U1-70K, thereby conferring a preferential advantage on U1 snRNP assembly, and they extrapolate that SMN–Gemin2 serves a general ribonucleoprotein-exchange function.
The U1 snRNP has been best characterized regarding its role in precursor (pre)-mRNA splicing; however, its abundance in cells far exceeds that necessary for splicing regulation1. Furthermore, U1 is the only snRNP that localizes to intronless genes2, and it has been implicated in the regulation of transcription initiation as well as pre-mRNA length via its interactions with the polyadenylation machinery3,4. However, the mechanism through which cells maintain high levels of U1 relative to the other components of the spliceosome is not well understood. In this issue, So et al.5 reveal that small nuclear RNAs (snRNAs) compete for access to SMN, a protein complex required for snRNP maturation. They show that the unrelated RNA-binding protein (RBP) U1-70K gives U1 an advantage in this competition for SMN access, thus explaining the cellular overabundance of U1. Furthermore, these results hint that, rather than simply being a spliceosome-assembly factor, SMN may act as a hub for ribonucleoprotein (RNP) exchange, where a diverse set of RNPs swap their cargo Megan Mayerle and Christine Guthrie are at the Department of Biochemistry and Biophysics, University of California San Francisco, San Francisco, California, USA. e-mail: [email protected]
RNAs (Fig. 1). These findings may also explain the widespread perturbations in RNA metabolism observed in the neurological disease spinal muscle atrophy (SMA). So et al.5 have established that U1 snRNA has two potential pathways to interact with SMN: (i) the canonical pathway used by all snRNAs, which requires the RBP Gemin5, and (ii) a unique pathway that makes use of U1-70K. Through a series of pulldown assays designed to assess the roles of U1 stem-loop 1 and the snRNP code in assembly of the Sm core, the authors have demonstrated that depletion of Gemin5 abolishes U4 snRNA’s ability to interact with the SMN complex, as expected. In contrast, the interaction between U1 snRNA and SMN is only modestly affected by depletion of Gemin5, and, more surprisingly, U1 does not require the U1 Sm-binding sequence. Instead, the ability of pre-U1 or mature U1 snRNAs to interact with SMN largely depends on the presence of U1-70K. Moreover, a mutant form of U1 lacking an Sm-binding site is entirely dependent on U1-70K for association with SMN. In contrast, mutant U1 snRNA defective in U1-70K binding, or with a strengthened Sm-binding-site sequence, requires Gemin5 for interaction with SMN. The authors5 have further shown that access to SMN, which is required for the maturation
nature structural & molecular biology volume 23 number 3 March 2016
of multiple snRNAs, is competitive. They have found that knockdown of U1-70K, removing U1-specific SMN access, increases Sm-complex assembly on U2, U4 and U5 snRNAs at the expense of U1, presumably because SMN, which is normally occupied by U1, becomes available to the other snRNAs. The interaction between U1-70K and SMN is direct, requiring U1-70K amino acids 90–194 (which bind stem-loop 1 of U1 snRNA) and SMN’s C-terminal YG box (which is required for oligomerization). The authors have also touched upon the question of how the Sm core itself is built. Gemin2 has previously been shown to interact with and recruit Sm5 (ref. 6), an Sm assembly intermediate; however, formation of the complete Sm ring, Sm7, requires an Sm-binding site on RNA7. So et al.5 have shown that the N-terminal 99 amino acids of U1-70K, in combination with Gemin2, can form the complete Sm7 complex, a result consistent with observations made from recent SMN–Gemin2–Sm5 (ref. 8) and U1 snRNP crystal structures9,10. Thus, U1-70K helps mediate U1 assembly not only by helping it outcompete other snRNAs for access to SMN but also by recruiting the building blocks required by the SMN complex for snRNP maturation. Citing the unique pathways to SMN used by Gemin5 and U1-70K, So et al.5 have hypothesized 189
Elevating the alternating-access model Renae M Ryan & Robert J Vandenberg
npg
© 2016 Nature America, Inc. All rights reserved.
50 years ago, Jardetzky proposed the alternating-access model, which has shaped the theoretical understanding of how substrates are carried across cell membranes. Two studies now demonstrate that transporters from distinct families undergo unexpectedly large elevator-like movements and also suggest that an ‘elevate and twist’ mechanism is a common means of achieving alternating access across the membrane. Membrane transport proteins control the movement of ions, solutes, chemical messengers, nutrients and waste across the cell membrane. They make up ~10% of all encoded proteins in humans and are important not only as drug targets but also in facilitating drug transport across membranes. In 1966, Jardetzky proposed an alternatingaccess mechanism describing the movement of substrates across the membrane1. It has been proposed that a carrier (transport protein) alternates between an extracellular- and an intracellular-facing state in which the centrally located substrate-binding site is accessible to only one side of the membrane at a time. Until recently, it had been thought that most membrane transporters use the rocker-switch model of alternating access, in which substrate binds to a site deep within the membrane, and the protein rearranges to allow access of the substrate-binding site to the inside of the cell (Fig. 1a). This type of mechanism is used widely in the major facilitator superfamily of transporters, such as the archetypal lactose permease, LacY2. In 2009, Boudker and colleagues revealed that the glutamate transporter homolog GltPh uses a twisting elevator-like mechanism of transport in which the ‘transport domain’ containing all of the cargo to be transported undergoes a 16-Å vertical movement and a 37° rotation to deliver substrate to the inside of the cell, while the ‘scaffold domain’ that anchors the protein in the membrane remains relatively static3. It had previously been thought that this elevator Renae M. Ryan and Robert J. Vandenberg are at the Discipline of Pharmacology, Sydney Medical School, University of Sydney, Sydney, New South Wales, Australia. e-mail: [email protected]
mechanism (Fig. 1b) may have been unique to the glutamate transporter family, but two unrelated proteins have now been shown to use an elevator-like mechanism of transport. In this issue, Mindell and colleagues4 and Drew and colleagues5 demonstrate that the transport domains of the dicarboxylate transporter from Vibrio cholerae (VcINDY) and an N+/H+ antiporter from Thermus thermophilus (NapA) undergo vertical translocation and rotation relative to their respective scaffold domains during substrate transport.
a
b
The only crystal structure available for the divalent anion sodium symporter (DASS) family is the inward-facing inhibitor-bound structure of VcINDY, which is a dimer of two identical protomers6. Each protomer has a similar overall architecture to a protomer of GltPh3,7 and is divided into two domains: a transport domain that contains the binding sites for substrate and coupled Na+ ions and a scaffold domain that contains the interprotomer contacts along the dimer interface (Fig. 2a,b). In this issue, Mindell and colleagues4
Rocker-switch mechanism
Elevator mechanism Gate
Transport domain
Scaffold domain
Gate
Figure 1 Alternating-access mechanisms of substrate transport across the membrane. (a) The rockerswitch mechanism involves protein rearrangements around a central substrate-binding site (orange circle) that results in the substrate being alternately exposed to either side of the membrane. (b) The elevator mechanism of transport describes a process in which the substrate is picked up on one side of the membrane, and a ‘gate’ subsequently closes. Next, part of the protein (termed the transport or core domain) undergoes a vertical translation relative to the membrane plane to carry the cargo across the membrane. For the cargo to be delivered into the cell, another gate must open to release the substrate, and the elevator then returns to the outside of the cell and is ready to pick up the next passenger. The elevator can be empty, as is the case with symporters such as VcINDY, or it may carry another ion or substrate across the membrane if the transporter operates via an antiport mechanism, as does NapA. Adapted with permission from ref. 19, Nature Publishing Group.
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VcINDY Inward facing
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Figure 2 The two domains of elevator transporters. (a–c) The aspartate transporter GltPh (PDB 2WNX) (a), the succinate transporter VcINDY (PDB 4F35) (b) and the Na+/H+ exchanger NapA (PDB 4BWZ) (c), viewed from the extracellular side of the membrane. GltPh exists as a trimer (of three identical protomers), whereas VcINDY and NapA are dimers (of two identical protomers). Although these proteins are unrelated in sequence, the tertiary structure of all three transporters reveals two distinct domains: a transporter domain that contains all of the cargo to be transported across the membrane (purple helices) and a scaffold domain that anchors the protein in the membrane and contains the interprotomer contacts (also known as the oligomerization domain; blue helices). GltPh is shown with bound aspartate and two Na+ ions, and VcINDY is shown with bound citrate and one Na+ ion. Aspartate and citrate are shown as spheres, and Na+ ions are indicated in yellow. In NapA, the ion-binding residue (Asp157) is shown in orange spheres, and the ‘linker’ helix (transmembrane domain 6) is shown in gray. (d) VcINDY viewed in the plane of the membrane in the inward-facing conformation (left; PDB 4F35) and the outward-facing conformation (right; Protein Model Database PM0080216 (ref. 4)). (e) NapA in the inward-facing conformation (left; PDB 5BZ2 (ref. 5)) and outward-facing (right; PDB 4BWZ) conformation. In d and e, the scaffold domain (blue helices) and the transport domain (purple helices) are indicated, and the elevator movement of the transport domain is indicated by the movement of the transported species (VcINDY, Na+/citrate; NapA, Asp157). The linker helix (TM6) of NapA is shown in gray ribbon. Structures were prepared with PyMOL (http://www.pymol.org/).
use a repeat-swap method to predict the outward-facing structure of VcINDY. This method has successfully been used to predict the inward-facing structure of GltPh8,9. The outward-facing model of VcINDY reveals an ~15-Å vertical translation and an ~43° rotation of the transport domain relative to the scaffold domain (Fig. 2d). This twisting elevator movement has been verified by Mindell and colleagues4 through a series of cross-links between pairs of introduced cysteine residues, thus confirming that the transport domain indeed undergoes this large movement. When this elevator movement is restricted by cross-linking of the transport domain to the scaffold domain, the transport 188
process is inhibited, thus demonstrating that large movements of the transport domain are required for transport. In addition, crosslinks designed to bridge the scaffold domain at the dimer interface do not affect the ability of VcINDY to transport substrate, thus revealing that large-scale movements of the two protomers relative to each other are not compulsory for substrate translocation. There are several available crystal structures of Na+/H+ antiporters from different species and in different states of the transport cycle10–13. Although there is general agreement that these antiporters can be divided into two domains consisting of a transport (or core) domain and a scaffold (or dimerization) domain (Fig. 2c),
interpretation of the movements required for Na+/H+ antiport has been controversial. These antiporters have been proposed to operate via a traditional rocker-switch mechanism with small rearrangements of the protein around a central binding site13 and also via an elevatorlike mechanism similar to that of GltPh11. To clarify this issue, Drew and colleagues5 have solved the structure of NapA in the outward- and inward-facing states, revealing substantial movements of the core ion-binding domain relative to the dimer or scaffold domain in the same protein. In this study, with the aim of trapping the transporter in an inward-facing state, the authors introduced two cysteine residues. These two residues form a disulfide bond, and when they are cross-linked, the function of the transporter is inhibited. Drew and colleagues5 have crystallized this inward-facing disulfide-trapped protein and have compared the structure to the outward-facing structure determined in detergent11 and also in lipidic mesophase5. Using a combination of molecular dynamics and biochemical analysis, the authors conclude that the transport domain undergoes a rigid-body vertical translation (7–10 Å) relative to the scaffold domain, but with minimal twist (Fig. 2e). Nevertheless, it appears that NapA uses an elevator-like mechanism rather than a rocker-switch mechanism. There are several transporters with a domain architecture similar to that of both VcINDY14,15 and NapA16,17 that have also been predicted to use a twisting elevator mechanism, and it will be interesting to determine how many other transport proteins use this mechanism of transport. Given the important role of tertiary protein structure in determining transporter mechanism, it is likely that other members of the VcINDY and NapA families, and even transporters from unrelated families, will be found to use a similar mechanism for substrate transport. Indeed, a recent study has reported two structures of a citrate transporter (CitS): one in an inward-facing conformation and one in an outward-facing conformation18. Similarly to VcINDY and NapA, this unrelated protein also exists as a dimer with each protomer divided into a transport and scaffold domain. During the transport process, the transport domain undergoes a 16-Å translation and a 31° rotation and also appears to use a twisting elevator mechanism of transport. Although there is variability in the extent of the translation and rotation of the transport domains among these elevator transporters3–5,18, all of these examples reveal that the substrate-binding site undergoes a vertical translation during substrate translocation, and transport is not achieved solely through rearrangement of the protein around the
volume 23 number 3 March 2016 nature structural & molecular biology
news and views substrate-binding site, as occurs in the rockerswitch mechanism. On this 50th anniversary of Jardetzky’s alternating-access proposal, a wealth of available structural and biophysical information on many transport proteins is allowing researchers to visualize the physical reality of his theoretical proposal. Subtle variations in the complex process of membrane transport can now begin to be teased apart, and the alternating-access model can be refined to include translation and rotation of the protein—two properties that Jardetzky excluded from the ‘allosteric pump’ model1. In summary, these studies have provided two new examples of transporters that use an elevator mechanism. It is now clear
that this mechanism is not unique to the glutamate transporter family, and there may be many more transport proteins—unrelated in sequence and varied in biological function— that also use a twisting elevator to move their substrates across membranes. COMPETING FINANCIAL INTERESTS The authors declare no competing financial interests. 1. Jardetzky, O. Nature 211, 969–970 (1966). 2. Yan, N. Annu. Rev. Biophys. 44, 257–283 (2015). 3. Reyes, N., Ginter, C. & Boudker, O. Nature 462, 880–885 (2009). 4. Mulligan, C. et al. Nat. Struct. Mol. Biol. 23, 256–263 (2016). 5. Coincon, M. et al. Nat. Struct. Mol. Biol. 23, 248–255 (2016).
6. Mancusso, R., Gregorio, G.G., Liu, Q. & Wang, D.N. Nature 491, 622–626 (2012). 7. Boudker, O., Ryan, R.M., Yernool, D., Shimamoto, K. & Gouaux, E. Nature 445, 387–393 (2007). 8. Crisman, T.J., Qu, S., Kanner, B.I. & Forrest, L.R. Proc. Natl. Acad. Sci. USA 106, 20752–20757 (2009). 9. Vergara-Jaque, A., Fenollar-Ferrer, C., Kaufmann, D. & Forrest, L.R. Front. Pharmacol. 6, 183 (2015). 10. Hunte, C. et al. Nature 435, 1197–1202 (2005). 11. Lee, C. et al. Nature 501, 573–577 (2013). 12. Lee, C. et al. J. Gen. Physiol. 144, 529–544 (2014). 13. Paulino, C., Wöhlert, D., Kapotova, E., Yildiz, Ö. & Kühlbrandt, W. eLife 3, e03583 (2014). 14. Bolla, J.R. et al. Nat. Commun. 6, 6874 (2015). 15. Su, C.C. et al. Cell Rep. 11, 61–70 (2015). 16. Hu, N.J., Iwata, S., Cameron, A.D. & Drew, D. Nature 478, 408–411 (2011). 17. Zhou, X. et al. Nature 505, 569–573 (2014). 18. Wohlert, D., Grotzinger, M.J., Kuhlbrandt, W. & Yildiz, O. eLife 4, (2015). 19. Slotboom, D.J. Nat. Rev. Microbiol. 12, 79–87 (2014).
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© 2016 Nature America, Inc. All rights reserved.
A new communication hub in the RNA world Megan Mayerle & Christine Guthrie During assembly of spliceosomal small nuclear ribonucleoproteins (snRNPs), the RNA-binding protein (RBP) Gemin5 recognizes the snRNP code and interacts with the large Gemin2–SMN complex. So et al. now find that Gemin2 also interacts with U1-70K, thereby conferring a preferential advantage on U1 snRNP assembly, and they extrapolate that SMN–Gemin2 serves a general ribonucleoprotein-exchange function.
The U1 snRNP has been best characterized regarding its role in precursor (pre)-mRNA splicing; however, its abundance in cells far exceeds that necessary for splicing regulation1. Furthermore, U1 is the only snRNP that localizes to intronless genes2, and it has been implicated in the regulation of transcription initiation as well as pre-mRNA length via its interactions with the polyadenylation machinery3,4. However, the mechanism through which cells maintain high levels of U1 relative to the other components of the spliceosome is not well understood. In this issue, So et al.5 reveal that small nuclear RNAs (snRNAs) compete for access to SMN, a protein complex required for snRNP maturation. They show that the unrelated RNA-binding protein (RBP) U1-70K gives U1 an advantage in this competition for SMN access, thus explaining the cellular overabundance of U1. Furthermore, these results hint that, rather than simply being a spliceosome-assembly factor, SMN may act as a hub for ribonucleoprotein (RNP) exchange, where a diverse set of RNPs swap their cargo Megan Mayerle and Christine Guthrie are at the Department of Biochemistry and Biophysics, University of California San Francisco, San Francisco, California, USA. e-mail: [email protected]
RNAs (Fig. 1). These findings may also explain the widespread perturbations in RNA metabolism observed in the neurological disease spinal muscle atrophy (SMA). So et al.5 have established that U1 snRNA has two potential pathways to interact with SMN: (i) the canonical pathway used by all snRNAs, which requires the RBP Gemin5, and (ii) a unique pathway that makes use of U1-70K. Through a series of pulldown assays designed to assess the roles of U1 stem-loop 1 and the snRNP code in assembly of the Sm core, the authors have demonstrated that depletion of Gemin5 abolishes U4 snRNA’s ability to interact with the SMN complex, as expected. In contrast, the interaction between U1 snRNA and SMN is only modestly affected by depletion of Gemin5, and, more surprisingly, U1 does not require the U1 Sm-binding sequence. Instead, the ability of pre-U1 or mature U1 snRNAs to interact with SMN largely depends on the presence of U1-70K. Moreover, a mutant form of U1 lacking an Sm-binding site is entirely dependent on U1-70K for association with SMN. In contrast, mutant U1 snRNA defective in U1-70K binding, or with a strengthened Sm-binding-site sequence, requires Gemin5 for interaction with SMN. The authors5 have further shown that access to SMN, which is required for the maturation
nature structural & molecular biology volume 23 number 3 March 2016
of multiple snRNAs, is competitive. They have found that knockdown of U1-70K, removing U1-specific SMN access, increases Sm-complex assembly on U2, U4 and U5 snRNAs at the expense of U1, presumably because SMN, which is normally occupied by U1, becomes available to the other snRNAs. The interaction between U1-70K and SMN is direct, requiring U1-70K amino acids 90–194 (which bind stem-loop 1 of U1 snRNA) and SMN’s C-terminal YG box (which is required for oligomerization). The authors have also touched upon the question of how the Sm core itself is built. Gemin2 has previously been shown to interact with and recruit Sm5 (ref. 6), an Sm assembly intermediate; however, formation of the complete Sm ring, Sm7, requires an Sm-binding site on RNA7. So et al.5 have shown that the N-terminal 99 amino acids of U1-70K, in combination with Gemin2, can form the complete Sm7 complex, a result consistent with observations made from recent SMN–Gemin2–Sm5 (ref. 8) and U1 snRNP crystal structures9,10. Thus, U1-70K helps mediate U1 assembly not only by helping it outcompete other snRNAs for access to SMN but also by recruiting the building blocks required by the SMN complex for snRNP maturation. Citing the unique pathways to SMN used by Gemin5 and U1-70K, So et al.5 have hypothesized 189
