news & views to optimize the reaction conditions for each transformation within the process; assisted tandem catalysis has the lowest overall reaction efficiency but holds the advantage of reaction-condition optimization for each intermediate generated within the process. Of the three subcategories, auto tandem catalysis is the most efficient, and several examples of such processes have been developed. They are typically challenging, however, as both the interactions between reaction intermediates and considerations of catalysts’ compatibility must be taken into account. Nonetheless, some very interesting work has come out of such efforts4–7, with some cases even including asymmetric tandem catalysed processes8,9. The simultaneous occurrence of multiple catalytic cycles make investigation and analysis of these processes rather complicated; as the rates of each step in the sequence are interrelated, the reaction substrates need be selected carefully for a particular transformation in the presence of others, and a single catalyst that can carry out the specific sequence of transformations needs to be chosen. The recent work by Li and Herzon on separating catalytic activities in time addresses such drawbacks in auto tandem catalysed processes3. Temporal separation was achieved by considering a catalyst that, for each cycle, would have a resting state — consisting of a substrate–metal complex — which would have increasing energy along

the reaction profile. In this manner, the catalyst would be limited to carrying out the first reaction cycle until all of the starting substrate is consumed — that is, it would be more energetically favourable to carry out the transformations sequentially. Building on previous work on an orthogonal tandem catalysed process10, Li and Herzon have applied this temporal separation approach to devise a tandem catalytic process in which a piano stool ruthenium catalyst carries out an antiMarkovnikov alkyne hydration followed by transfer hydrogenation (Fig. 1). The method proceeds in good yields and has a broad scope. Although this is an important transformation in its own right, the particularly interesting aspect of this work was that the researchers were not only able to optimize the reaction conditions for both transformations, but also to investigate and analyse the transformations over time. Indeed, the reaction profile unambiguously showed this separation in time between both catalytic cycles. As seen in Fig. 1, conversion of the alkyne (red) into the intermediate aldehyde (green) takes place first, and only once this process is complete does the second process take place to give the final alcohol (yellow) product. The added benefit of this temporal separation is the accumulation of intermediates during one step — these can be further functionalized by introduction of a stoichiometric reagent to divert the

system towards other reaction pathways. This functionalization possibility further increases the scope and efficiency of the catalyst. Demonstrating this approach, Li and Herzon were able to block the transfer hydrogenation pathway of their reaction and redirect it towards a Wittig olefination followed by enone reduction, in a triple-cascade tandem-catalysed system. Although this may not be the first instance in which temporal separation in tandem catalysis has occurred, it is the first time that it has been recognized and investigated. Overall, this approach provides a good strategy for developing tandem catalysis transformations by identifying suitable catalysts, modifying their reaction pathways to achieve a wider scope, and increasing the overall reaction efficiency. ❐ Sarah Abou-Shehada and Jonathan M. J. Williams are in the Department of Chemistry, University of Bath, Bath BA2 7AY, UK. e-mail: [email protected] References 1. Sheldon, R. A. Chem. Commun. 3352–3365 (2008). 2. Fogg, D. E. & dos Santos, E. N. Coord. Chem. Rev. 248, 2365–2379 (2004). 3. Li, L. & Herzon, S. B. Nature Chem. 6, 22–27 (2014). 4. Edwards, M. G. & Williams, J. M. J. Angew. Chem. Int. Ed. 41, 4740–4743 (2002). 5. Chen, J.-R. et al. Angew. Chem. Int. Ed. 47, 2489–2492 (2008). 6. Yadav, A. K. et al. Org. Lett. 15, 1060–1063 (2013). 7. Fleischer, I. et al. Angew. Chem. Int. Ed. 52, 2949–2953 (2013). 8. Kanbayashi, N,. Takenaka, K,. Okamura, T.-a. & Onitsuka, K. Angew. Chem. Int. Ed. 52, 4897–4901 (2013). 9. Chapman, C. J. & Frost, C. G. Synthesis-Stuttgart 1–21 (2007). 10. Li, L. & Herzon, S. B. J. Am. Chem. Soc. 134, 17376–17379 (2012).

BIOPHYSICAL CHEMISTRY

Strength in numbers

Replication of the HIV-1 viral genome can be inhibited by a protein known as APOBEC3G, via two seemingly contradictory mechanisms. Now, the molecular conundrum behind these two processes has been resolved.

Graeme A. King and Gijs J. L. Wuite

M

ammals have evolved a series of complex early-defence mechanisms to protect their cells against retroviral infections. These defensive processes typically focus on either attacking the invading viral capsid or destabilizing the viral genome1. The latter is mediated by a class of enzymes known as APOBEC3. One member of this family, APOBEC3G (A3G), can inhibit the HIV-1 retrovirus by restricting the replication of the viral genome2; however, the molecular mechanism behind the antiviral activity of A3G is not yet fully understood. Evidence suggests that A3G can

inhibit the HIV-1 retrovirus by two separate methods. One involves enzymatically editing the viral DNA sequence. The other requires binding to the HIV-1 genome and forming a roadblock to physically obstruct viral DNA synthesis. Whereas the first process relies on rapid binding and unbinding of the protein from the genome to swiftly sample a large number of reaction sites, the second requires that A3G is bound more strongly to the viral DNA — to ensure the roadblock remains in place. This creates a molecular conundrum: how can A3G dissociate from the viral

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genome both rapidly and slowly at the same time? As they report in Nature Chemistry, Mark C. Williams and co-workers have now resolved this issue3. Using a singlemolecule approach, they demonstrate that A3G binds and unbinds from singlestranded DNA rapidly at first, but then converts through oligomerization to a slowly dissociating complex. Williams and co-workers analysed the kinetics of A3G binding to single-stranded DNA using an optical tweezers assay in which a double-stranded DNA molecule is held between two micro-sized beads. 13

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Monomers or dimers

k 1c

k2 Oligomers

k–1

k–2

Figure 1 | Schematic of the proposed model for the binding of A3G to single-stranded DNA in vitro3. In the first step, monomers or dimers bind with forward rate k1c and backward rate k–1. These rates are of similar timescales and sufficiently fast that binding reaches equilibrium before A3G can form oligomers on the DNA. With time, however, oligomers of A3G are created on the DNA, but the timescale for their dissociation is significantly longer than for their formation (k–2 versus k2).

Pulling on one of the beads while the other is held in an optical trap applies a force to the DNA molecule; application of sufficient force induces melting of the DNA double-helix, creating regions of single-stranded DNA. Plotting the applied force as a function of DNA extension establishes a characteristic force–distance profile, which can be used as a benchmark to compare the effect of potential DNA ligands and different solution conditions4. Repeating this measurement in the presence of A3G showed that the protein induces minimal deviation from the reference force–extension profile, suggesting that it has low affinity for double-stranded DNA. Once the applied force was sufficient to melt the DNA double-helix, Williams and coworkers released the tension on the DNA, which should have enabled the double helix to reform; however they found that the relative length of the DNA was greater than expected. This is evidence that A3G binds to some fraction of the melted DNA. In the next test the DNA molecule was held at zero tension for up to two hours to see whether A3G remains bound to the single-stranded DNA once the applied force has been reduced. If the protein unbinds at low tension, melted DNA regions will re-hybridize, forming double-stranded DNA. When the researchers repeated the stretching experiment after two hours they observed that the force–distance profile was intermediate between that of the reference double-stranded curve and the force-release measurement in which A3G was still bound 14

to melted DNA regions. This demonstrates that some A3G had dissociated from the single-stranded DNA at low tension while the rest remained bound. Williams and co-workers then considered the timeframe for A3G binding. To investigate this, the melted DNA molecule was exposed to A3G for progressively longer time periods (50 seconds to 35 minutes) before the applied tension was released. This experiment revealed that, as the melted DNA is exposed to A3G for longer time periods, the fraction of bound A3G that unbinds once the DNA tension is released becomes smaller. Therefore, the DNA–protein interaction becomes progressively more stable over time. Based on these observations, the researchers suggest that the binding of A3G to single-stranded DNA can be modelled as a two-step process. The first step represents the rapid binding and unbinding of A3G, whereas the second reflects a unimolecular reaction from a fast-dissociating protein– DNA complex to a more strongly bound arrangement. Using this model, and extracting the fraction of melted DNA that is bound by A3G from experiments, they were able to quantify the dual-mode kinetics of the system. In another crucial test they showed that a mutant of A3G, which cannot form oligomers on the genome, only shows rapid dissociation from single-stranded DNA. This encouraged Williams and co-workers to explain the two-state model of HIV-1 restriction as follows: monomers or dimers

of A3G bind and unbind rapidly, but over time the DNA-bound proteins progressively form oligomers with a notably slower unbinding rate (Fig. 1). This dual-mode mechanism is proposed to underpin the potency of A3G as a restrictor for retroviral replication. The HIV-1 virus has in turn evolved a viral infectivity factor to counter the role of A3G. Nonetheless, appreciating the cellular mechanisms behind the action of A3G is vital for understanding the immune response to HIV-1 and other retroviruses. This work also raises the question whether the regulation of enzymatic activity, via the oligomerization mechanism displayed by A3G, will turn out to be a general property of other APOBEC family members that inhibit replication of retroviruses and retrotransposons. The experiments reported by Williams and co-workers both showcase and re-affirm the power of optical tweezers in probing DNA–protein interactions. The force–extension measurements, in combination with kinetic models, have been able to unravel the kinetics that regulate the enzymatic function of this important protein. Moreover, this has been achieved without recourse to either DNA or protein labelling, offering a pleasingly clean, yet insightful singlemolecule approach. At the same time, this study of A3G provides a timely reminder of the importance of protein–protein interactions on DNA. In vivo, DNA is often densely coated with proteins, and thus inter-protein interactions on DNA are commonplace. We must question more generally, therefore, to what extent protein crowding — both in solution and on DNA — may influence the thermodynamics of proteins bound to DNA. Recent studies have highlighted, for instance, that protein search mechanisms5 and diffusion rates6 can be highly sensitive to protein concentration. Future work must surely address the issue of protein crowding on DNA more extensively, and is likely to require the utilization of multifaceted experimental approaches. ❐ Graeme A. King and Gijs J. L. Wuite are in the Department of Physics and Astronomy, VU University Amsterdam, De Boelelaan 1081, 1081 HV, Amsterdam, The Netherlands. e-mail: [email protected]; [email protected] References 1. Bieneasz, P. D. Nature Immunol. 5, 1109–1115 (2004). 2. Harris R. S. & Liddament M. T. Nature Rev. Immunol. 4, 868–877 (2004). 3. Chaurasiya, K. R. et al. Nature Chem. 6, 28–33 (2014). 4. Chaurasiya, K. R., Paramanathan, T., McCauley, M. J. & Williams, M. C. Phys. Life Rev. 7, 299–341 (2010). 5. Wang, F. et al. Nature Struct. Mol. Biol. 20, 174–181 (2013). 6. Heller, I. et al. Nature Methods 10, 910–920 (2013).

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Biophysical chemistry: Strength in numbers.

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