Many enzymes mold their structures to enclose substrates in their active sites such that conformational remodeling may be required during each catalytic cycle. In adenylate kinase (AK), this involves a large-amplitude rearrangement of the enzyme's lid domain. Using our method of high-resolution single-molecule FRET, we directly followed AK's domain movements on its catalytic time scale. To quantitatively measure the enzyme's entire conformational distribution, we have applied maximum entropy-based methods to remove photon-counting noise from single-molecule data. This analysis shows unambiguously that AK is capable of dynamically sampling two distinct states, which correlate well with those observed by x-ray crystallography. Unexpectedly, the equilibrium favors the closed, active-site-forming configurations even in the absence of substrates. Our experiments further showed that interaction with substrates, rather than locking the enzyme into a compact state, restricts the spatial extent of conformational fluctuations and shifts the enzyme's conformational equilibrium toward the closed form by increasing the closing rate of the lid. Integrating these microscopic dynamics into macroscopic kinetics allows us to model lid opening-coupled product release as the enzyme's rate-limiting step.conformational equilibrium ͉ rate-limiting step ͉ single-molecule FRET ͉ adenylate kinase P roteins such as enzymes are flexible with a range of motions spanning from picoseconds for localized vibrations to seconds for concerted global conformational rearrangements (1). Despite their randomly fluctuating environment, in which stochastic collisions with solvent molecules drive changes in tertiary structure, enzymes have evolved to catalyze reactions efficiently and specifically. Indeed, conformational transitions have been postulated to play a central role in enzyme functions in a wide variety of ways, including direct contribution to catalysis (2), allosteric regulation (3), and large-scale conformational changes in response to ligand binding (4). Most of our current understanding of structural motions in solution comes from NMR experiments (5) as well as from molecular dynamics simulations (6), approaches that are best suited to study dynamics in the picoto millisecond time scales. Because catalysis in enzymes frequently occurs in the submillisecond to minute time regime, our current understanding of the relationship between enzyme function and conformational dynamics comes from NMR experiments involving relatively localized motions of active site forming loops on the submillisecond time scale (7-10). However, many enzymes contain active sites located in between domains in which large-amplitude, low-frequency domain motions are required to complete their Michaelis-Menten enzyme-substrate complexes. Even simple questions regarding these transitions remain generally unanswered: What is the number and range of conformational states accessible to enzymes during their catalytic cycle? How does the enzyme's conformation respond to interac...

Summary Many replication initiators form higher-order oligomers that process host origins to promote replisome formation. In addition to dedicated duplex DNA-binding domains, cellular initiators possess AAA+ (ATPases Associated with various cellular Activities) elements that drive functions ranging from protein assembly to origin recognition. In bacteria, the AAA+ domain of the initiator DnaA has been suggested to bind single-stranded DNA formed during origin melting. Here we show crystallographically and in solution that the ATP-dependent assembly of DnaA into a spiral oligomer creates a continuous surface that allows successive AAA+ domains to bind and extend single-stranded DNA segments. The mechanism of binding is unexpectedly similar to that of RecA, a homologous recombination factor, but it differs in that DnaA promotes a nucleic acid conformation that prevents pairing of a complementary strand. These findings, combined with strand-displacement assays, indicate that DnaA melts replication origins by a direct ATP-dependent stretching mechanism. Comparative studies reveal remarkable commonalities between the approach used by DnaA to engage DNA substrates and other, nucleic acid-dependent AAA+ systems.

The Escherichia coli DNA replication machinery has been used as a road map to uncover design rules that enable DNA duplication with high efficiency and fidelity. Although the enzymatic activities of the replicative DNA Pol III are well understood, its dynamics within the replisome are not. Here, we test the accepted view that the Pol III holoenzyme remains stably associated within the replisome. We use in vitro single-molecule assays with fluorescently labeled polymerases to demonstrate that the Pol III* complex (holoenzyme lacking the β2 sliding clamp), is rapidly exchanged during processive DNA replication. Nevertheless, the replisome is highly resistant to dilution in the absence of Pol III* in solution. We further show similar exchange in live cells containing labeled clamp loader and polymerase. These observations suggest a concentration-dependent exchange mechanism providing a balance between stability and plasticity, facilitating replacement of replisomal components dependent on their availability in the environment.DOI: http://dx.doi.org/10.7554/eLife.23932.001

Inflammasomes function as intracellular sensors of pathogen infection or cellular perturbation and thereby play a central role in numerous diseases. Given the high abundance of NLRP1 in epithelial barrier tissues, we screened a diverse panel of viruses for inflammasome activation in keratinocytes. We identified Semliki Forest virus (SFV), a positive-strand RNA virus, as a potent activator of human, but not murine NLRP1. SFV replication and the associated formation of double-stranded (ds) RNA was required to engage the NLRP1 inflammasome. Moreover, delivery of long dsRNA was sufficient to trigger activation. Biochemical studies revealed that NLRP1 binds dsRNA via its LRR, resulting in its NACHT domain gaining ATPase activity. Altogether, these results establish human NLRP1 as a direct sensor for dsRNA and thus RNA virus infection.

The initiation of DNA replication requires the melting of chromosomal origins to provide a template for replisomal polymerases. In bacteria, the DnaA initiator plays a key role in this process, forming a large nucleoprotein complex that opens DNA through a complex and poorly understood mechanism. Using structure-guided mutagenesis, biochemical, and genetic approaches, we establish an unexpected link between the duplex DNA-binding domain of DnaA and the ability of the protein to both self-assemble and engage singlestranded DNA in an ATP-dependent manner. Intersubunit cross-talk between this domain and the DnaA ATPase region regulates this link and is required for both origin unwinding in vitro and initiator function in vivo. These findings indicate that DnaA utilizes at least two different oligomeric conformations for engaging single-and double-stranded DNA, and that these states play distinct roles in controlling the progression of initiation.

The protein coat of the tobacco mosaic virus (TMV) has been explored extensively for the construction of nanoscale architectures. In previous work, we have reported efficient TMV-based light harvesting systems bearing chromophores in a hollow channel of the assembled protein. We have also reported an N-terminal transamination/oximation method that could be used to attach electrodes and catalytic groups to the exterior surface of the rods. To complement these techniques, we report herein a new circular permutant of the TMV capsid protein that repositions the N- and C-termini to the center of the assemblies. This protein can be produced in very high yield through E. coli expression and self-assembles into light harvesting rods that are much like those assembled from the wild-type protein. However, the disks formed from the permutant structure are stable over a significantly wider pH range, greatly improving the practicality of this assembled form for materials applications. The new position of the N-terminus allows functional groups to be installed in the inner pore of the disks, affording geometries reminiscent of natural photosynthetic systems. The permutant also shows the ability to coassemble with regular monomers, allowing the future generation of multicomponent rod structures that are modified on the exterior and interior surfaces, as well as in the internal RNA channel.

Voltage-gated calcium channels (CaVs) are large, multisubunit complexes that control cellular calcium entry. CaV pore-forming (CaValpha1) and cytoplasmic (CaVbeta) subunits associate through a high-affinity interaction between the CaValpha1 alpha interaction domain (AID) and CaVbeta alpha binding pocket (ABP). Here we analyze AID-ABP interaction thermodynamics using isothermal titration calorimetry. We find that commensurate with their strong sequence similarity, all CaV1 and CaV2 AID peptides bind CaVbeta with similar nanomolar affinities. Although the AID-ABP interface encompasses 24 side chains, alanine-scanning mutagenesis reveals that the binding energy is focused in two complementary hotspots comprising four deeply conserved residues. Electrophysiological experiments show that hotspot interaction disruption prevents trafficking and functional modulation of CaV1.2 by CaVbeta. Together, the data support the primacy of the AID-ABP interface for CaValpha1-CaVbeta association, underscore the idea that hotspots dominate protein-protein interaction affinities, and uncover a target for strategies to control cellular excitability by blocking CaValpha1-CaVbeta complex formation.

. (2016). Simultaneous real-time imaging of leading and lagging strand synthesis reveals the coordination dynamics of single replisomes. Molecular Cell, 64 (6), 1035-1047.Simultaneous real-time imaging of leading and lagging strand synthesis reveals the coordination dynamics of single replisomes AbstractThe molecular machinery responsible for DNA replication, the replisome, must efficiently coordinate DNA unwinding with priming and synthesis to complete duplication of both strands. Due to the anti-parallel nature of DNA, the leading strand is copied continuously, while the lagging strand is produced by repeated cycles of priming, DNA looping, and Okazaki-fragment synthesis. Here, we report a multidimensional single-molecule approach to visualize this coordination in the bacteriophage T7 replisome by simultaneously monitoring the kinetics of loop growth and leading-strand synthesis. We show that loops in the lagging strand predominantly occur during priming and only infrequently support subsequent Okazaki-fragment synthesis. Fluorescence imaging reveals polymerases remaining bound to the lagging strand behind the replication fork, consistent with Okazaki-fragment synthesis behind and independent of the replication complex. Individual replisomes display both looping and pausing during priming, reconciling divergent models for the regulation of primer synthesis and revealing an underlying plasticity in replisome operation. SummaryThe molecular machinery responsible for DNA replication, the replisome, must efficiently coordinate DNA unwinding with priming and synthesis to complete duplication of both strands. Due to the anti-parallel nature of DNA, the leading strand is copied continuously, while the lagging strand is produced by repeated cycles of priming, DNA looping, andOkazaki-fragment synthesis. Here, we report a multidimensional single-molecule approach to visualize this coordination in the bacteriophage T7 replisome by simultaneously monitoring the kinetics of loop growth and leading-strand synthesis. We show that loops in the lagging strand predominantly occur during priming and only infrequently support subsequent Okazaki-fragment synthesis. Fluorescence imaging reveals polymerases remaining bound to the lagging strand behind the replication fork, consistent with Okazakifragment synthesis behind and independent of the replication complex. Individual replisomes display both looping and pausing during priming, reconciling divergent models for the regulation of primer synthesis and revealing an underlying plasticity in replisome operation.3