news & views to potential misses of high-value leads and inadvertently creating longer detours in the quest for best-in-class drugs6. This double uncertainty, i.e., that uncertain binding kinetics spells uncertain thermodynamic potency, should be studiously guarded against throughout all phases of drug discovery. Fortunately, high-throughput BK methods for in vitro kinetic profiling are now available7,8; the challenge ahead is to design in vitro BK assays relevant and translatable to in vivo settings where targets
may be present at high local concentrations in diffusion-restricted microenvironments, leading to potentially enhanced rebinding ■ phenomenon9. Rumin Zhang is in the Department of Pharmacology, Merck Research Laboratories, Kenilworth, New Jersey, USA. e-mail: [email protected] Published online 20 April 2015 doi:10.1038/nchembio.1795
References
1. Walkup, G.K. et al. Nat. Chem. Biol. doi:10.1038/nchembio.1796 (20 April 2015). 2. Copeland, R.A. et al. Expert Opin. Drug Discov. 5, 305–310 (2010). 3. Swinney, D.C. Curr. Top. Med. Chem. 6, 461–478 (2006). 4. Swinney, D.C. Pharmaceut. Med. 22, 23–34 (2008). 5. Louizos, C. J. Pharm. Pharm. Sci. 17, 34–91 (2014). 6. Zhang, R. & Monsma, F. Expert Opin. Drug Discov. 5, 1023–1029 (2010). 7. Zhang, R. & Windsor, W.T. Methods Mol. Biol. 1030, 59–79 (2013) 8. Schiele, F. et al. Anal. Biochem. 468, 42–49 (2015). 9. Vauquelin, G. Expert Opin. Drug Discov. 5, 927–941 (2010).
Competing financial interests The author declares no competing financial interests.
MEMBRANE FUSION
npg
© 2015 Nature America, Inc. All rights reserved.
A new role for lipid domains?
Boundaries between ordered and disordered membrane domains may be the site of HIV fusion protein insertion into its target membrane.
Erwin London
M
embrane fusion is a complex process, and the transient bilayer fusion event remains beyond the range of direct observation at the level of the individual lipid molecule. Nevertheless, it has been one of the triumphs of modern biology, biophysics and structural biology that membrane fusion no longer seems the mysterious process it once did. The crucial role of the proteins at the center of the fusion process in bringing the membranes that will fuse into intimate contact is now reasonably well understood1–3. Cellular fusion is a carefully orchestrated process involving several protein players. In contrast, viruses, with their limited protein profiles, do not have the luxury, or perhaps even the need, to over-orchestrate the fusion process and so have only a single fusion protein in charge. Because the viral fusion protein encodes the fusion machinery and so is important to infection (and thus is a target in blocking infection), it is not surprising that studies of viral fusion have concentrated on its functional role2–4. The report of Yang et al.5 in this issue of Nature Chemical Biology indicates that there is a new player to consider: the boundaries between disordered (Ld) domains and ordered (Lo) lipid domains, the latter often referred to as lipid rafts. Yang et al. started by measuring the extent of fusion after mixing HIV fusion peptide, the portion of the HIV fusion protein Env that triggers fusion, with two sets of model membranes5. Fusion between the sets of model membranes was more efficient when they contained coexisting
Ld and Lo domains than when they were fully Ld or fully Lo or had coexisting gel and Ld phases. Higher cholesterol concentration also enhanced fusion. Fusion after pre-mixing of fusion peptide with a first set of vesicles (host vesicles) lacking individual domains showed that coexistence of Lo and Ld in the second set of vesicles (target vesicles) enhanced fusion. The investigators then showed that the fusion peptide both bound preferentially to vesicles with coexisting Lo and Ld domains (Lo+d) and bound preferentially at the boundaries between the Lo and Ld domains. Further studies showed that when both host and target membranes contained Lo+d domains, the fusion events occurred most efficiently between lipids at the Lo-Ld boundary of the host and the Lo-Ld boundary of the target membrane. As these studies involved isolated fusion peptides, it might be argued that they are of biophysical interest but potentially irrelevant to the biological process of membrane fusion. However, Yang et al. took their studies one step further5. They prepared HIV pseudovirus particles, with intact HIV Env (gp41–gp120) protein in their membranes, and found that upon activation to expose fusion peptide, pseudovirus bound to the Lo-Ld boundaries of supported bilayers, a strong indication that intact Env protein targets boundaries in a fashion similar to that of isolated fusion peptide. Perhaps this is one clue to the biological importance of the well-studied role
nature chemical biology | VOL 11 | JUNE 2015 | www.nature.com/naturechemicalbiology
of Lo lipids and domains in HIV membranes and assembly6. The authors reasonably postulate that the energetic penalty of the discontinuities at the boundary between Lo and Ld domains favors interaction with peptide and fusion (see Fig. 1 for possible mechanisms). However, much remains to be done before the exact role of membrane domains in fusion can be firmly established. Although most investigators would concede that there is strong evidence that under certain conditions eukaryotic, and even prokaryotic, membranes can contain coexisting disordered and ordered domains7,8, whether such domains are constitutive or universal and whether they are sufficiently persistent to control biological functions remain controversial. In terms of membrane fusion, the issue of domain size could have special impact. In the nanodomains that may predominate in domain-containing eukaryotic cell membranes, the physical difference between Lo and Ld domains, and thus the energetic penalty at the boundaries between them, may be significantly less than in the easily visualized large domains studied in this report. On the other hand, as the authors point out, relative to a large domain, an equal amount of lipids in the form of nanodomains would have a much higher ratio of boundary lipids to total domain lipids, which would enhance the influence of boundaries upon fusion. It is also important to consider the possible biomedical implications of these studies. Variations in cellular lipid 383
news & views
a
Fusion
Ld
Lo
Ld
npg
© 2015 Nature America, Inc. All rights reserved.
b
Ld
Lo
Ld
Fusion peptide
composition between individuals might contribute to differential susceptibility to infection. Even more intriguing are the potential implications for the susceptibility of T cells to HIV infection. There is relatively strong evidence that Lo and Ld domains coexist in T cells and that T cell domains rearrange, and likely enlarge, upon T cell activation9–11. Thus, it is possible that activated T cells are particularly vulnerable to infection. Whatever the reader’s bias with regard to the existence and properties of raft-like membrane domains, it seems clear that, for good reason, their role in membrane fusion is likely to be much studied in the future. ■ Erwin London is in the Department of Biochemistry and Cell Biology, Stony Brook University, Stony Brook, New York, USA. e-mail: [email protected] References
Ld
Lo
Ld
Lo
Ld
Südhof, T.C. & Rothman, J.E. Science 323, 474–477 (2009). Carr, C.M. & Kim, P.S. Cell 73, 823–832 (1993). Skehel, J.J. & Wiley, D.C. Annu. Rev. Biochem. 69, 531–569 (2000). Kielian, M. & Rey, F.A. Nat. Rev. Microbiol. 4, 67–76 (2006). Yang, S.-T., Kiessling, V., Simmons, J.A., White, J.M. & Tamm, L.K. Nat. Chem. Biol. 11, 424–431 (2015). 6. Ono, A. & Freed, E.O. Proc. Natl. Acad. Sci. USA 98, 13925–13930 (2001). 7. Toulmay, A. & Prinz, W.A. J. Cell Biol. 202, 35–44 (2013). 8. LaRocca, T.J. et al. PLoS Pathog. 9, e1003353 (2013). 9. Zech, T. et al. EMBO J. 28, 466–476 (2009). 10. Harder, T., Rentero, C., Zech, T. & Gaus, K. Curr. Opin. Immunol. 19, 470–475 (2007). 11. Mahammad, S., Dinic, J., Adler, J. & Parmryd, I. Biochim. Biophys. Acta 1801, 625–634 (2010). 1. 2. 3. 4. 5.
Lo
Figure 1 | How HIV fusion peptide interacts with domain boundaries. (a) Impact on the fusion process. Unactivated virion (left) has fusion protein (tan); upon activation (arrow), fusion peptides (green) are inserted at domain boundaries, after which the system proceeds to fusion (right). (b) Models for the way that HIV fusion peptide binds and inserts. Left, insertion at domain boundaries in which hydrocarbon chains are exposed because of a hydrophobic mismatch between Lo- and Ld-domain thicknesses; center and right, extension of Ld-domain lipids in contact with Lo domain alters local packing and headgroup spacing of lipids adjacent to domain boundaries (center and right) in a manner favorable for peptide insertion.
Competing financial interests The author declares no competing financial interests.
ENZYME PATHWAYS
C1 metabolism redesigned
One-carbon metabolic pathways create new opportunities for metabolic engineering, but natural pathways have limitations in catalytic efficiency and interspecies transferability. Now a computationally designed enzyme, formolase, enables the construction of a synthetic metabolic pathway in Escherichia coli for assimilation of formate into a glycolytic intermediate in only five reaction steps.
Yi-Shu Tai & Kechun Zhang
A
dvances in metabolic engineering and synthetic biology have enabled the biomanufacturing of diverse sustainable products from various carbon feedstocks1. Recently there has been increasing interest in using one-carbon compounds such as CH4 or CO2 as substrates because of their abundance and their role in the greenhouse effect2. However, relevant carbon fixation pathways do not naturally exist in industrial microorganisms 384
such as E. coli and Saccharomyces cerevisiae, and incorporating heterologous enzymes is challenging in regard to generating efficient pathways in a new host. To address such difficulties, Siegel et al. have constructed an artificial formate assimilation pathway aided by a computationally designed enzyme3. A total of nine one-carbon fixation pathways have been discovered in nature3. The CO2-based pathways include the Calvin-Benson cycle, the
reductive tricarboxylic acid (TCA) cycle, the reductive acetyl-CoA pathway, the 3-hydroxypropionate cycle, the 3-hydroxypropionate–4-hydroxybutyrate cycle and the dicarboxylate–4hydroxybutyrate cycle. These pathways require energy input from sunlight or hydrogen, and they are limited by low catalytic rate, oxygen sensitivity and complicated regulatory mechanisms. Because formate can be electrochemically
nature chemical biology | VOL 11 | JUNE 2015 | www.nature.com/naturechemicalbiology
may be present at high local concentrations in diffusion-restricted microenvironments, leading to potentially enhanced rebinding ■ phenomenon9. Rumin Zhang is in the Department of Pharmacology, Merck Research Laboratories, Kenilworth, New Jersey, USA. e-mail: [email protected] Published online 20 April 2015 doi:10.1038/nchembio.1795
References
1. Walkup, G.K. et al. Nat. Chem. Biol. doi:10.1038/nchembio.1796 (20 April 2015). 2. Copeland, R.A. et al. Expert Opin. Drug Discov. 5, 305–310 (2010). 3. Swinney, D.C. Curr. Top. Med. Chem. 6, 461–478 (2006). 4. Swinney, D.C. Pharmaceut. Med. 22, 23–34 (2008). 5. Louizos, C. J. Pharm. Pharm. Sci. 17, 34–91 (2014). 6. Zhang, R. & Monsma, F. Expert Opin. Drug Discov. 5, 1023–1029 (2010). 7. Zhang, R. & Windsor, W.T. Methods Mol. Biol. 1030, 59–79 (2013) 8. Schiele, F. et al. Anal. Biochem. 468, 42–49 (2015). 9. Vauquelin, G. Expert Opin. Drug Discov. 5, 927–941 (2010).
Competing financial interests The author declares no competing financial interests.
MEMBRANE FUSION
npg
© 2015 Nature America, Inc. All rights reserved.
A new role for lipid domains?
Boundaries between ordered and disordered membrane domains may be the site of HIV fusion protein insertion into its target membrane.
Erwin London
M
embrane fusion is a complex process, and the transient bilayer fusion event remains beyond the range of direct observation at the level of the individual lipid molecule. Nevertheless, it has been one of the triumphs of modern biology, biophysics and structural biology that membrane fusion no longer seems the mysterious process it once did. The crucial role of the proteins at the center of the fusion process in bringing the membranes that will fuse into intimate contact is now reasonably well understood1–3. Cellular fusion is a carefully orchestrated process involving several protein players. In contrast, viruses, with their limited protein profiles, do not have the luxury, or perhaps even the need, to over-orchestrate the fusion process and so have only a single fusion protein in charge. Because the viral fusion protein encodes the fusion machinery and so is important to infection (and thus is a target in blocking infection), it is not surprising that studies of viral fusion have concentrated on its functional role2–4. The report of Yang et al.5 in this issue of Nature Chemical Biology indicates that there is a new player to consider: the boundaries between disordered (Ld) domains and ordered (Lo) lipid domains, the latter often referred to as lipid rafts. Yang et al. started by measuring the extent of fusion after mixing HIV fusion peptide, the portion of the HIV fusion protein Env that triggers fusion, with two sets of model membranes5. Fusion between the sets of model membranes was more efficient when they contained coexisting
Ld and Lo domains than when they were fully Ld or fully Lo or had coexisting gel and Ld phases. Higher cholesterol concentration also enhanced fusion. Fusion after pre-mixing of fusion peptide with a first set of vesicles (host vesicles) lacking individual domains showed that coexistence of Lo and Ld in the second set of vesicles (target vesicles) enhanced fusion. The investigators then showed that the fusion peptide both bound preferentially to vesicles with coexisting Lo and Ld domains (Lo+d) and bound preferentially at the boundaries between the Lo and Ld domains. Further studies showed that when both host and target membranes contained Lo+d domains, the fusion events occurred most efficiently between lipids at the Lo-Ld boundary of the host and the Lo-Ld boundary of the target membrane. As these studies involved isolated fusion peptides, it might be argued that they are of biophysical interest but potentially irrelevant to the biological process of membrane fusion. However, Yang et al. took their studies one step further5. They prepared HIV pseudovirus particles, with intact HIV Env (gp41–gp120) protein in their membranes, and found that upon activation to expose fusion peptide, pseudovirus bound to the Lo-Ld boundaries of supported bilayers, a strong indication that intact Env protein targets boundaries in a fashion similar to that of isolated fusion peptide. Perhaps this is one clue to the biological importance of the well-studied role
nature chemical biology | VOL 11 | JUNE 2015 | www.nature.com/naturechemicalbiology
of Lo lipids and domains in HIV membranes and assembly6. The authors reasonably postulate that the energetic penalty of the discontinuities at the boundary between Lo and Ld domains favors interaction with peptide and fusion (see Fig. 1 for possible mechanisms). However, much remains to be done before the exact role of membrane domains in fusion can be firmly established. Although most investigators would concede that there is strong evidence that under certain conditions eukaryotic, and even prokaryotic, membranes can contain coexisting disordered and ordered domains7,8, whether such domains are constitutive or universal and whether they are sufficiently persistent to control biological functions remain controversial. In terms of membrane fusion, the issue of domain size could have special impact. In the nanodomains that may predominate in domain-containing eukaryotic cell membranes, the physical difference between Lo and Ld domains, and thus the energetic penalty at the boundaries between them, may be significantly less than in the easily visualized large domains studied in this report. On the other hand, as the authors point out, relative to a large domain, an equal amount of lipids in the form of nanodomains would have a much higher ratio of boundary lipids to total domain lipids, which would enhance the influence of boundaries upon fusion. It is also important to consider the possible biomedical implications of these studies. Variations in cellular lipid 383
news & views
a
Fusion
Ld
Lo
Ld
npg
© 2015 Nature America, Inc. All rights reserved.
b
Ld
Lo
Ld
Fusion peptide
composition between individuals might contribute to differential susceptibility to infection. Even more intriguing are the potential implications for the susceptibility of T cells to HIV infection. There is relatively strong evidence that Lo and Ld domains coexist in T cells and that T cell domains rearrange, and likely enlarge, upon T cell activation9–11. Thus, it is possible that activated T cells are particularly vulnerable to infection. Whatever the reader’s bias with regard to the existence and properties of raft-like membrane domains, it seems clear that, for good reason, their role in membrane fusion is likely to be much studied in the future. ■ Erwin London is in the Department of Biochemistry and Cell Biology, Stony Brook University, Stony Brook, New York, USA. e-mail: [email protected] References
Ld
Lo
Ld
Lo
Ld
Südhof, T.C. & Rothman, J.E. Science 323, 474–477 (2009). Carr, C.M. & Kim, P.S. Cell 73, 823–832 (1993). Skehel, J.J. & Wiley, D.C. Annu. Rev. Biochem. 69, 531–569 (2000). Kielian, M. & Rey, F.A. Nat. Rev. Microbiol. 4, 67–76 (2006). Yang, S.-T., Kiessling, V., Simmons, J.A., White, J.M. & Tamm, L.K. Nat. Chem. Biol. 11, 424–431 (2015). 6. Ono, A. & Freed, E.O. Proc. Natl. Acad. Sci. USA 98, 13925–13930 (2001). 7. Toulmay, A. & Prinz, W.A. J. Cell Biol. 202, 35–44 (2013). 8. LaRocca, T.J. et al. PLoS Pathog. 9, e1003353 (2013). 9. Zech, T. et al. EMBO J. 28, 466–476 (2009). 10. Harder, T., Rentero, C., Zech, T. & Gaus, K. Curr. Opin. Immunol. 19, 470–475 (2007). 11. Mahammad, S., Dinic, J., Adler, J. & Parmryd, I. Biochim. Biophys. Acta 1801, 625–634 (2010). 1. 2. 3. 4. 5.
Lo
Figure 1 | How HIV fusion peptide interacts with domain boundaries. (a) Impact on the fusion process. Unactivated virion (left) has fusion protein (tan); upon activation (arrow), fusion peptides (green) are inserted at domain boundaries, after which the system proceeds to fusion (right). (b) Models for the way that HIV fusion peptide binds and inserts. Left, insertion at domain boundaries in which hydrocarbon chains are exposed because of a hydrophobic mismatch between Lo- and Ld-domain thicknesses; center and right, extension of Ld-domain lipids in contact with Lo domain alters local packing and headgroup spacing of lipids adjacent to domain boundaries (center and right) in a manner favorable for peptide insertion.
Competing financial interests The author declares no competing financial interests.
ENZYME PATHWAYS
C1 metabolism redesigned
One-carbon metabolic pathways create new opportunities for metabolic engineering, but natural pathways have limitations in catalytic efficiency and interspecies transferability. Now a computationally designed enzyme, formolase, enables the construction of a synthetic metabolic pathway in Escherichia coli for assimilation of formate into a glycolytic intermediate in only five reaction steps.
Yi-Shu Tai & Kechun Zhang
A
dvances in metabolic engineering and synthetic biology have enabled the biomanufacturing of diverse sustainable products from various carbon feedstocks1. Recently there has been increasing interest in using one-carbon compounds such as CH4 or CO2 as substrates because of their abundance and their role in the greenhouse effect2. However, relevant carbon fixation pathways do not naturally exist in industrial microorganisms 384
such as E. coli and Saccharomyces cerevisiae, and incorporating heterologous enzymes is challenging in regard to generating efficient pathways in a new host. To address such difficulties, Siegel et al. have constructed an artificial formate assimilation pathway aided by a computationally designed enzyme3. A total of nine one-carbon fixation pathways have been discovered in nature3. The CO2-based pathways include the Calvin-Benson cycle, the
reductive tricarboxylic acid (TCA) cycle, the reductive acetyl-CoA pathway, the 3-hydroxypropionate cycle, the 3-hydroxypropionate–4-hydroxybutyrate cycle and the dicarboxylate–4hydroxybutyrate cycle. These pathways require energy input from sunlight or hydrogen, and they are limited by low catalytic rate, oxygen sensitivity and complicated regulatory mechanisms. Because formate can be electrochemically
nature chemical biology | VOL 11 | JUNE 2015 | www.nature.com/naturechemicalbiology
