David F. Savage

Substrate twinning activates the signal recognition particle and its receptor

Signal sequences target proteins for secretion from cells or for integration into cell membranes. As nascent proteins emerge from the ribosome, signal sequences are recognized by the signal recognition particle (SRP), which subsequently associates with its receptor (SR). In this complex, the SRP and SR stimulate each others GTPase activity, and GTP hydrolysis ensures unidirectional targeting of cargo through a translocation pore in the membrane. To define the mechanism of reciprocal activation, we determined the 1.9 Å structure of the complex formed between these two GTPases. The two partners form a quasi-two-fold symmetrical heterodimer. Biochemical analysis supports the importance of the extensive interaction surface. Complex formation aligns the two GTP molecules in a symmetrical, composite active site, and the 3′OH groups are essential for association, reciprocal activation and catalysis. This unique circle of twinned interactions is severed twice on hydrolysis, leading to complex dissociation after cargo delivery.

Architecture and selectivity in aquaporins: 2.5 Å X-ray structure of aquaporin Z

Aquaporins are a family of water and small molecule channels found in organisms ranging from bacteria to animals. One of these channels, the E. coli protein aquaporin Z (AqpZ), has been shown to selectively conduct only water at high rates. We have expressed, purified, crystallized, and solved the X-ray structure of AqpZ. The 2.5 Å resolution structure of AqpZ suggests aquaporin selectivity results both from a steric mechanism due to pore size and from specific amino acid substitutions that regulate the preference for a hydrophobic or hydrophilic substrate. This structure provides direct evidence on the molecular mechanisms of specificity between water and glycerol in this family of channels from a single species. It is to our knowledge the first atomic resolution structure of a recombinant aquaporin and so provides a platform for combined genetic, mutational, functional, and structural determinations of the mechanisms of aquaporins and, more generally, the assembly of multimeric membrane proteins.

Spatially Ordered Dynamics of the Bacterial Carbon Fixation Machinery

Carboxysomes in a Row The carboxysome is an organelle-like proteinaceous microcompartment that sequesters the enzymes of carbon fixation from the rest of the cytoplasm in cyanobacteria. Cyanobacterial carbon fixation is a major component of the global carbon cycle. Savage et al. (p. 1258) now show that carboxysomes are linearly arranged within the cytoplasm in a process that involves the bacterial cytoskeleton. This arrangement is important in carboxysome partitioning during cell division. When carboxysome partitioning is disrupted by interfering with the bacterial cytoskeleton, carbon fixation is impaired. Tight control of the spatial arrangement of carboxysome organelles optimizes carbon fixation in cyanobacterial cells. Cyanobacterial carbon fixation is a major component of the global carbon cycle. This process requires the carboxysome, an organelle-like proteinaceous microcompartment that sequesters the enzymes of carbon fixation from the cytoplasm. Here, fluorescently tagged carboxysomes were found to be spatially ordered in a linear fashion. As a consequence, cells undergoing division evenly segregated carboxysomes in a nonrandom process. Mutation of the cytoskeletal protein ParA specifically disrupted carboxysome order, promoted random carboxysome segregation during cell division, and impaired carbon fixation after disparate partitioning. Thus, cyanobacteria use the cytoskeleton to control the spatial arrangement of carboxysomes and to optimize the metabolic process of carbon fixation.

Engineering Cyanobacteria To Synthesize and Export Hydrophilic Products

ABSTRACT Metabolic engineering of cyanobacteria has the advantage that sunlight and CO2 are the sole source of energy and carbon for these organisms. However, as photoautotrophs, cyanobacteria generally lack transporters to move hydrophilic primary metabolites across membranes. To address whether cyanobacteria could be engineered to produce and secrete organic primary metabolites, Synechococcus elongatus PCC7942 was engineered to express genes encoding an invertase and a glucose facilitator, which mediated secretion of glucose and fructose. Similarly, expression of lactate dehydrogenase- and lactate transporter-encoding genes allowed lactate accumulation in the extracellular medium. Expression of the relevant transporter was essential for secretion. Production of these molecules was further improved by expression of additional heterologous enzymes. Sugars secreted by the engineered cyanobacteria could be used to support Escherichia coli growth in the absence of additional nutrient sources. These results indicate that cyanobacteria can be engineered to produce and secrete high-value hydrophilic products.

Modularity of a carbon-fixing protein organelle

Bacterial microcompartments are proteinaceous complexes that catalyze metabolic pathways in a manner reminiscent of organelles. Although microcompartment structure is well understood, much less is known about their assembly and function in vivo. We show here that carboxysomes, CO2-fixing microcompartments encoded by 10 genes, can be heterologously produced in Escherichia coli. Expression of carboxysomes in E. coli resulted in the production of icosahedral complexes similar to those from the native host. In vivo, the complexes were capable of both assembling with carboxysomal proteins and fixing CO2. Characterization of purified synthetic carboxysomes indicated that they were well formed in structure, contained the expected molecular components, and were capable of fixing CO2 in vitro. In addition, we verify association of the postulated pore-forming protein CsoS1D with the carboxysome and show how it may modulate function. We have developed a genetic system capable of producing modular carbon-fixing microcompartments in a heterologous host. In doing so, we lay the groundwork for understanding these elaborate protein complexes and for the synthetic biological engineering of self-assembling molecular structures.

Catalysis, Specificity, and ACP Docking Site of Streptomyces coelicolor Malonyl-CoA:ACP Transacylase

Malonyl-CoA:ACP transacylase (MAT), the fabD gene product of Streptomyces coelicolor A3(2), participates in both fatty acid and polyketide synthesis pathways, transferring malonyl groups that are used as extender units in chain growth from malonyl-CoA to pathway-specific acyl carrier proteins (ACPs). Here, the 2.0 A structure reveals an invariant arginine bound to an acetate that mimics the malonyl carboxylate and helps define the extender unit binding site. Catalysis may only occur when the oxyanion hole is formed through substrate binding, preventing hydrolysis of the acyl-enzyme intermediate. Macromolecular docking simulations with actinorhodin ACP suggest that the majority of the ACP docking surface is formed by a helical flap. These results should help to engineer polyketide synthases (PKSs) that produce novel polyketides.

Defossiling Fuel: How Synthetic Biology Can Transform Biofuel Production

A lthough crude oil production is predicted to peak soon, it is reasonable to assume that unconventional fossil fuel sources can continue to meet society’s increasing energy demands for many decades to come (1). The real challenge is sustainability: stabilizing and reversing global climate change, minimizing political and economic energy volatility, and smoothing the transition from fossil fuels in the distant future. In response to this challenge, many are looking to biotechnology to develop biofuels, such as ethanol, butanol, biodiesel, and hydrogen (H2), in which the energy ultimately derives from photosynthetic capture of sunlight. A fundamental issue with biofuels is efficiency. The pathway from sunlight through natural intermediates to final molecule is long, and biofuel production is perhaps the ultimate metabolic engineering problem (2). This challenge is made even greater by its inherent systems complexity, because any solutionmust be implemented in the context of an energy infrastructure with challenging engineering, economic, political, and environmental realities. Are biofuels sustainable? Consider U.S. transportation fuels, a market poised for impact. Biofuels derive their stored chemical energy from the sun viaphotosynthesis. Biofuel use is therefore a closed carbon cycle, as carbon released during combustion is sequestered during photosynthesis. Solar radiation is clearly a sustainable energy source on human time scales, and U.S. incident solar power ( 2300 TW) (3) greatly exceeds our transportation fuel usage ( 1.0 TW) (Table 1) (4). The reactions of photosynthesis impose a maximal efficiency of 12%, but final yields are significantly lower (2). Terrestrial plant efficiencies for solar to biomass conversion is maximally 2% (e.g., for the rapidly growingMiscanthus (5)), and the subsequent conversion into biofuels is 50% efficient (6). It would therefore require 4.3% of the U.S. land area to meet our transportation energy demands, which corresponds to 22% of current cropland. Thus, in an optimistic approximation (and ignoring social, political, and economic complexities), we can say biofuel production could be sustainable, albeit with significant challenges. These calculations suggest that a biofuelbased energy economy is feasible but that enhancements in the efficiency of any step in energy production would be favorable fromaneconomic and environmental standpoint. The upper bound on efficiency is set by photosynthesis—the challenge is therefore to come as close to this bound as possible. Put another way, how can one optimizemetabolism todirectmaximal flux from one set of metabolites to another, while still maintaining, at least partially, host fitness? Traditional industrial approaches have given us many such successes (e.g., beer) andwill play amajor role, but the tools of systems and synthetic biology promise to deliver a degree of optimization not previously attainable (7). A synthetic-biological redesign of the organisms that produce biofuels has the potential to significantly increase efficiency, *Corresponding author, pamela_silver@hms.harvard.edu.

A general protocol for the crystallization of membrane proteins for X-ray structural investigation

Protein crystallography is used to generate atomic resolution structures of protein molecules. These structures provide information about biological function, mechanism and interaction of a protein with substrates or effectors including DNA, RNA, cofactors or other small molecules, ions and other proteins. This technique can be applied to membrane proteins resident in the membranes of cells. To accomplish this, membrane proteins first need to be either heterologously expressed or purified from a native source. The protein has to be extracted from the lipid membrane with a mild detergent and purified to a stable, homogeneous population that may then be crystallized. Protein crystals are then used for X-ray diffraction to yield atomic resolution structures of the desired membrane protein target. Below, we present a general protocol for the growth of diffraction quality membrane protein crystals. The process of protein crystallization is highly variable, and obtaining diffraction quality crystals can require weeks to months or even years in some cases.

The Bacterial Carbon-Fixing Organelle Is Formed by Shell Envelopment of Preassembled Cargo

Background Cyanobacteria play a significant role in the global carbon cycle. In Synechococcus elongatus , the carbon-fixing enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) is concentrated into polyhedral, proteinaceous compartments called carboxysomes. Methodology/Principal Findings Using live cell fluorescence microscopy, we show that carboxysomes are first detected as small seeds of RuBisCO that colocalize with existing carboxysomes. These seeds contain little or no shell protein, but increase in RuBisCO content over several hours, during which time they are exposed to the solvent. The maturing seed is then enclosed by shell proteins, a rapid process that seals RuBisCO from the cytosol to establish a distinct, solvent-protected microenvironment that is oxidizing relative to the cytosol. These closure events can be spatially and temporally coincident with the appearance of a nascent daughter RuBisCO seed. Conclusions/Significance Carboxysomes assemble in a stepwise fashion, inside-to-outside, revealing that cargo is the principle organizer of this compartment’s biogenesis. Our observations of the spatial relationship of seeds to previously formed carboxysomes lead us to propose a model for carboxysome replication via sequential fission, polymerization, and encapsulation of their internal cargo.

Profiling of engineering hotspots identifies an allosteric CRISPR-Cas9 switch

The clustered, regularly interspaced, short palindromic repeats (CRISPR)-associated protein Cas9 from Streptococcus pyogenes is an RNA-guided DNA endonuclease with widespread utility for genome modification. However, the structural constraints limiting the engineering of Cas9 have not been determined. Here we experimentally profile Cas9 using randomized insertional mutagenesis and delineate hotspots in the structure capable of tolerating insertions of a PDZ domain without disruption of the enzymes binding and cleavage functions. Orthogonal domains or combinations of domains can be inserted into the identified sites with minimal functional consequence. To illustrate the utility of the identified sites, we construct an allosterically regulated Cas9 by insertion of the estrogen receptor-α ligand-binding domain. This protein showed robust, ligand-dependent activation in prokaryotic and eukaryotic cells, establishing a versatile one-component system for inducible and reversible Cas9 activation. Thus, domain insertion profiling facilitates the rapid generation of new Cas9 functionalities and provides useful data for future engineering of Cas9.