Structures of PKA-phospholamban complexes reveal a mechanism of familial dilated cardiomyopathy.

Several mutations identified in phospholamban (PLN) have been linked to familial dilated cardiomyopathy (DCM) and heart failure, yet the underlying molecular mechanism remains controversial. PLN interacts with sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) and regulates calcium uptake, which is modulated by the protein kinase A (PKA)-dependent phosphorylation of PLN during the fight-or-flight response. Here, we present the crystal structures of the catalytic domain of mouse PKA in complex with wild-type and DCM-mutant PLNs. Our structures, combined with the results from other biophysical and biochemical assays, reveal a common disease mechanism: the mutations in PLN reduce its phosphorylation level by changing its conformation and weakening its interactions with PKA. In addition, we demonstrate that another more ubiquitous SERCA-regulatory peptide, called another-regulin (ALN), shares a similar mechanism mediated by PKA in regulating SERCA activity.

Guest Sequence Can Influence RNA Encapsulation by an Engineered Cationic Protein Capsid.

Specific encapsulation of RNA in a cell is a challenging molecular recognition problem that has important implications for virology and drug delivery. An engineered variant of the Aquifex aeolicus lumazine synthase capsid that possesses a positively supercharged interior (AaLS-pos) has previously been shown to encapsulate a mixture of cellular RNAs in bacteria via charge complementarity. To investigate the influence of nucleotide sequence on encapsulation, eight reporter RNAs with the same charge but with highly diverse arbitrary sequence regions (ASRs) were coproduced with AaLS-pos in Escherichia coli cells. The ASRs cause significant differences in the yields of encapsulated full-length reporter RNA, measured by q-RT-PCR. Four of the reporters also have the Broccoli-F30 aptamer, which hinders encapsulation compared with reporters with an alternative sequence in place of Broccoli-F30. In all, the encapsulation yield of the best reporter was ∼200 times higher than the worst. The reporters also differed by up to ∼28 000 times in their partitioning between the capsid and the bulk cellular environment, with those lacking Broccoli-F30 showing higher enrichments in AaLS-pos. The detection of Broccoli-F30 by a fluorescence assay showed higher levels than that determined by q-RT-PCR, suggesting that partially degraded forms of the reporters are encapsulated more frequently than the full-length versions. For the reporters lacking the Broccoli-F30 aptamer, the encapsulation yield shows inverse correlations with both their expression levels and the predicted secondary structural stabilities of their ASRs. These observations suggest that in this system, guest selection depends on the RNA flexibility and the shape complementarity with the capsid.

A Protein-Capsid-Based System for Cell Delivery of Selenocysteine.

Selenocysteine (Sec) has received a lot of attention as a potential anticancer drug. However, its broad cytotoxicity limits its therapeutic usefulness. Thus, Sec is an attractive candidate for targeted drug delivery. Here, we demonstrate for the first time that an engineered version of the capsid formed by Aquifex aeolicus lumazine synthase (AaLS) can act as a nanocarrier for delivery of Sec to cells. Specifically, a previously reported variant of AaLS (AaLS-IC), which contains a single cysteine per subunit that projects into the capsid interior, was modified by reaction with the diselenide dimer of Sec (Sec2) to generate a selenenylsulfide conjugate between the capsid and Sec (AaLS-IC-Sec). Importantly, it was determined that the structural context of the reactive cysteine was important for efficient capsid loading. Further, the encapsulated Sec could be quantitatively released from AaLS-IC-Sec by reducing agents such as glutathione or dithiothreitol. To assess cellular penetrance capabilities of AaLS-IC-Sec and subsequent cytotoxic response, six different cells line models were examined. Across the cell lines analyzed, cytotoxic sensitivity correlated with cellular uptake and intracellular trafficking patterns. Together these findings suggest that the engineered AaLS-IC capsid is a promising vehicle for targeted cell delivery of Sec.

Encapsulation and Controlled Release of Protein Guests by the Bacillus subtilis Lumazine Synthase Capsid.

In Bacillus subtilis, the 60-subunit dodecahedral capsid formed by lumazine synthase (BsLS) acts as a container for trimeric riboflavin synthase (BsRS). To test whether the C-terminal sequence of BsRS is responsible for its encapsulation by BsLS, the green fluorescent protein (GFP) was fused to either the last 11 or the last 32 amino acids of BsRS, yielding variant GFP11 or GFP32, respectively. After purification, BsLS capsids that had been co-produced in bacteria with GFP11 and GFP32 are 15- and 6-fold more fluorescent, respectively, than BsLS co-produced with GFP lacking any BsRS fragment, indicating complex formation. Enzyme-linked immunosorbent assay experiments confirm that GFP11 is localized within the BsLS capsid. In addition, fusing the last 11 amino acids of BsRS to the C-terminus of the Abrin A chain also led to its encapsulation by BsLS at a level similar to that of GFP11. Together, these results demonstrate that the C-terminal tail of BsRS can act as an encapsulation tag capable of targeting other proteins to the BsLS capsid interior. As with the natural BsLS-BsRS complex, mild changes in pH and buffer identity trigger dissociation of the GFP11 guest, accompanied by a substantial expansion of the BsLS capsid. This system for protein encapsulation and release provides a novel tool for bionanotechnology.

In vivo encapsulation of nucleic acids using an engineered nonviral protein capsid.

In Nature, protein capsids function as molecular containers for a wide variety of molecular cargoes. Such containers have great potential for applications in nanotechnology, which often require encapsulation of non-native guest molecules. Charge complementarity represents a potentially powerful strategy for engineering novel encapsulation systems. In an effort to explore the generality of this approach, we engineered a nonviral, 60-subunit capsid, lumazine synthase from Aquifex aeolicus (AaLS), to act as a container for nucleic acid. Four mutations were introduced per subunit to increase the positive charge at the inner surface of the capsid. Characterization of the mutant (AaLS-pos) revealed that the positive charges lead to the uptake of cellular RNA during production and assembly of the capsid in vivo. Surprisingly, AaLS-pos capsids were found to be enriched with RNA molecules approximately 200-350 bases in length, suggesting that this simple charge complementarity approach to RNA encapsulation leads to both high affinity and a degree of selectivity. The ability to control loading of RNA by tuning the charge at the inner surface of a protein capsid could illuminate aspects of genome recognition by viruses and pave the way for the development of improved RNA delivery systems.

Conversion of a dodecahedral protein capsid into pentamers via minimal point mutations.

Protein self-assembly relies upon the formation of stabilizing noncovalent interactions across subunit interfaces. Identifying the determinants of self-assembly is crucial for understanding structure-function relationships in symmetric protein complexes and for engineering responsive nanoscale architectures for applications in medicine and biotechnology. Lumazine synthases (LS's) comprise a protein family that forms diverse quaternary structures, including pentamers and 60-subunit dodecahedral capsids. To improve our understanding of the basis for this difference in assembly, we attempted to convert the capsid-forming LS from Aquifex aeolicus (AaLS) into pentamers through a small number of rationally designed amino acid substitutions. Our mutations targeted side chains at ionic (R40), hydrogen bonding (H41), and hydrophobic (L121 and I125) interaction sites along the interfaces between pentamers. We found that substitutions at two or three of these positions could reliably generate pentameric variants of AaLS. Biophysical characterization indicates that this quaternary structure change is not accompanied by substantial changes in secondary or tertiary structure. Interestingly, previous homology-based studies of the assembly determinants in LS's had identified only one of these four positions. The ability to control assembly state in protein capsids such as AaLS could aid efforts in the development of new systems for drug delivery, biocatalysis, or materials synthesis.

Directed evolution of a protein container.

Confinement of enzymes in protein nanocompartments represents a potentially powerful strategy for controlling catalytic activity in cells. By using a simple electrostatically based tagging system for protein encapsulation, we successfully sequestered HIV protease, a toxic enzyme when produced cytoplasmically, within an engineered lumazine synthase capsid. The growth advantage resulting from protecting the Escherichia coli host from the protease enabled directed evolution of improved capsids. After four rounds of mutagenesis and selection, we obtained a variant with a 5- to 10-fold higher loading capacity than the starting capsid, which permitted efficient growth even at high intracellular concentrations of HIV protease. The superior properties of the evolved capsid can be ascribed to multiple mutations that increase the net negative charge on its luminal surface and thereby enhance engineered Coulombic interactions between host and guest. Such structures could be used for diverse biotechnological applications in living cells.

Diselenides as universal oxidative folding catalysts of diverse proteins.

Small-molecule diselenides show considerable potential as catalysts of oxidative protein folding. To explore their scope, diselenide-containing redox buffers were used to promote the folding of proteins that varied in properties such as size, overall tertiary structure, number of disulfide bonds, pI value, and difficulty of in vitro folding. Diselenides are able to catalyze the oxidative folding of all proteins tested, providing significant increases in both rate and yield relative to analogous disulfides. Compared to the disulfide-linked dimer of glutathione (the most commonly used oxidant for in vitro protein folding), selenoglutathione provided markedly improved efficiencies in the folding of biotechnologically important proteins such as hirudin, lysozyme, human epidermal growth factor and interferon α-2a. Selenoglutathione also enhances the renaturation of more challenging targets such as bovine serum albumin, whose native state contains 17 disulfide bonds, and the Fab fragment of an antibody. In the latter case, micromolar amounts of selenoglutathione are able to match the modest yield provided by a previously optimized redox buffer, which contains millimolar levels of glutathione. Taken together, the folding reactions of these diverse proteins exemplify the advantages and limitations of diselenide catalysts.

Small-molecule diselenides catalyze oxidative protein folding in vivo.

Prokaryotic cells normally rely on periplasmic oxidoreductases to promote oxidative protein folding. Here we show that simple diselenides can also facilitate the conversion of dithiols to disulfides in vivo, functionally replacing one such oxidoreductase, DsbA, in the oxidative folding of diverse proteins. Structurally analogous disulfides provide no detectable effect when used at concentrations that gave optimal activity with diselenides, and even at 100- to 1000-fold higher levels they show only partial activity. The low concentrations of diselenides needed to fully negate typical DsbA knockout phenotypes suggest catalysis in vivo, a property that sets these additives apart from other small molecules used in chemical biology. Supplementing growth media with cell-permeable organocatalysts provides a potentially general and operationally simple means of fine-tuning the cellular redox environment.

Relative tolerance of an enzymatic molten globule and its thermostable counterpart to point mutation.

Enzyme structures reflect the complex interplay between the free energy of unfolding (DeltaG) and catalytic efficiency. Consequently, the effects of point mutations on structure, stability, and function are difficult to predict. It has been proposed that the mutational robustness of homologous enzymes correlates with a higher initial DeltaG. To examine this issue, we compared the tolerance of a natural thermostable chorismate mutase and an engineered molten globular variant to targeted mutation. These mutases possess similar sequence, structure, and catalytic efficiency but dramatically different DeltaG values. We find that analogous point mutations can have widely divergent effects on catalytic activity in these scaffolds. In a set of five rationally designed single-amino acid changes, the thermostable scaffold suffers activity losses ranging from 50-fold smaller, for an aspartate-to-glycine substitution at the active site, to 2-fold greater, for a phenylalanine-to-tryptophan substitution in the hydrophobic core, versus that of the molten globular scaffold. However, biophysical characterization indicates that the variations in catalytic efficiency are not caused by losses of either secondary structural integrity or thermodynamic stability. Rather, the activity differences between variant pairs are very much context-dependent and likely stem from subtle changes in the fine structure of the active site. Thus, in many cases, it may be more productive to focus on changes in local conformation than on global stability when attempting to understand and predict how enzymes respond to point mutations.

Catalysis of oxidative protein folding by small-molecule diselenides.

The production of recombinant, disulfide-containing proteins often requires oxidative folding in vitro. Here, we show that diselenides, such as selenoglutathione, catalyze oxidative protein folding by O 2. Substantially lower concentrations of a redox buffer composed of selenoglutathione and the thiol form of glutathione can consequently be used to achieve the same rate and yield of folding as a standard glutathione redox buffer. Further, the low p K a of selenols extends the pH range for folding by selenoglutathione to acidic conditions, where glutathione is inactive. Harnessing the catalytic power of diselenides may thus pave the way for more efficient oxidative protein folding.

Selenoglutathione: efficient oxidative protein folding by a diselenide.

Diselenide bonds are intrinsically more stable than disulfide bonds. To examine how this stability difference affects reactivity, we synthesized selenoglutathione (GSeSeG), an analogue of the oxidized form of the tripeptide glutathione that contains a diselenide bond in place of the natural disulfide. The reduction potential of this diselenide bond was determined to be -407 +/- 9 mV, a value which is 151 mV lower than that of the disulfide bond in glutathione (GSSG). Thus, the diselenide bond of GSeSeG is 7 kcal/mol more stable than the disulfide bond of GSSG. Nonetheless, we found that GSeSeG can be used to oxidize cysteine residues in unfolded proteins, a process that is driven by the gain in protein conformational stability upon folding. Indeed, the folding of both ribonuclease A (RNase A) and bovine pancreatic trypsin inhibitor (BPTI) proceeded efficiently using GSeSeG as an oxidant, in the former case with a 2-fold rate increase relative to GSSG and in the latter case accelerating conversion of a stable folding intermediate to the native state. In addition, GSeSeG can also oxidize the common biological cofactor NADPH and is a good substrate for the NADPH-dependent enzyme glutathione reductase (kcat = 69 +/- 2 s-1, Km = 54 +/- 7 microM), suggesting that diselenides can efficiently interact with the cellular redox machinery. Surprisingly, the greater thermodynamic stability of diselenide bonds relative to disulfide bonds is not matched by a corresponding decrease in reactivity.

Minimalist active-site redesign: teaching old enzymes new tricks.

Although nature evolves its catalysts over millions of years, enzyme engineers try to do it a bit faster. Enzyme active sites provide highly optimized microenvironments for the catalysis of biologically useful chemical transformations. Consequently, changes at these centers can have large effects on enzyme activity. The prediction and control of these effects provides a promising way to access new functions. The development of methods and strategies to explore the untapped catalytic potential of natural enzyme scaffolds has been pushed by the increasing demand for industrial biocatalysts. This Review describes the use of minimal modifications at enzyme active sites to expand their catalytic repertoires, including targeted mutagenesis and the addition of new reactive functionalities. Often, a novel activity can be obtained with only a single point mutation. The many successful examples of active-site engineering through minimal mutations give useful insights into enzyme evolution and open new avenues in biocatalyst research.

Novel enzymes through design and evolution.

The generation of enzymes with new catalytic activities remains a major challenge. So far, several different strategies have been developed to tackle this problem, including site-directed mutagenesis, random mutagenesis (directed evolution), antibody catalysis, computational redesign, and de novo methods. Using these techniques, a broad array of novel enzymes has been created (aldolases, decarboxylases, dehydratases, isomerases, oxidases, reductases, and others), although their low efficiencies (10 to 100 M(-1) s(-l)) compared to those of the best natural enzymes (10(6) to 10(8) M(-1) s(-1)) remains a significant concern. Whereas rational design might be the most promising and versatile approach to generating new activities, directed evolution seems to be the best way to optimize the catalytic properties of novel enzymes. Indeed, impressive successes in enzyme engineering have resulted from a combination of rational and random design.

A simple tagging system for protein encapsulation.

Molecular containers that encapsulate specific cargo can be useful for many natural and non-natural processes. We report a simple system, based on charge complementarity, for the encapsulation of appropriately tagged proteins within an engineered, proteinaceous capsid. Four negative charges per monomer were added to the lumazine synthase from Aquifex aeolicus (AaLS). The capsids formed by the engineered AaLS associate with green fluorescent protein bearing a positively charged deca-arginine tag upon coproduction in Escherichia coli. Analytical ultracentrifugation and scanning force microscopy studies indicated that the engineered AaLS retains the ability to form capsids, but that their average size was substantially increased. The success of this strategy demonstrates that both the container and guest components of protein-based encapsulation systems can be convergently designed in a straightforward manner, which may help to extend their versatility.

Tunnel plasticity and quaternary structural integrity of a pentameric protein ring.

Cyclic protein oligomers are common in cells. However, the importance of the residues that line the central tunnel of protein rings for overall architectural integrity is not well understood. To investigate the role of tunnel positions in protein assembly and stability, we prepared variants of the homo-pentameric lumazine synthase (LS) from Saccharomyces cerevisiae in which the three residues that line the middle of the tunnel were simultaneously changed. As a consequence of symmetry, these mutations cause a total of 15 changes in the structure of the pentameric complex. Detailed characterization of the variants indicates that they retain quaternary structural integrity, even in cases where the mutations induce considerable secondary structure alterations. The tunnels of symmetric ring-shaped proteins, such as LS, may consequently represent an overlooked site for protein engineering.

Recombination of fragmented proteins.

Directed evolution is a powerful method for generating novel molecules with desirable properties. In developing a new sensor to screen for protein-protein interactions, Tafelmeyer et al. report a clever strategy to evolve heterodimeric "split proteins" from a monomer in this issue of Chemistry & Biology.

Deciphering enzymes. Genetic selection as a probe of structure and mechanism.

The efficient engineering of enzymes with novel activities remains an ongoing challenge. Towards this end, genetic selection techniques provide a method for finding rare solutions to catalytic problems that requires only a limited foreknowledge of structure-function relationships. We have used genetic selections to extensively probe the structure and mechanism of chorismate mutases. The insights gained from these investigations will aid future enzyme design efforts.

Catalysis of protein folding by an immobilized small-molecule dithiol.

The isomerization of non-native disulfide bonds often limits the rate of protein folding. Small-molecule dithiols can catalyze this process. Here, a symmetric trithiol, tris(2-mercaptoacetamidoethyl)amine, is designed on the basis of criteria known to be important for efficient catalysis of oxidative protein folding. The trithiol is synthesized and attached to two distinct solid supports via one of its three sulfhydryl groups. The resulting immobilized dithiol has an apparent disulfide E degrees ' = -208 mV, which is close to that of protein disulfide isomerase (E degrees ' = -180 mV). Incubation of the dithiol immobilized on a TentaGel resin with a protein containing non-native disulfide bonds produced only a 2-fold increase in native protein. This dithiol appeared to be inaccessible to protein. In contrast, incubation of the dithiol immobilized on styrene-glycidyl methacrylate microspheres with the non-native protein produced a 17-fold increase in native protein. This increase was 1.5-fold greater than that of a monothiol immobilized on the microspheres. Thus, the choice of both the solid support and thiol can affect catalysis of protein folding. The use of dithiol-decorated microspheres is an effective new strategy for preparative protein folding in vitro.