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.
BackgroundMalate synthase, one of the two enzymes unique to the glyoxylate cycle, is found in all three domains of life, and is crucial to the utilization of two-carbon compounds for net biosynthetic pathways such as gluconeogenesis. In addition to the main isoforms A and G, so named because of their differential expression in E. coli grown on either acetate or glycolate respectively, a third distinct isoform has been identified. These three isoforms differ considerably in size and sequence conservation. The A isoform (MSA) comprises ~530 residues, the G isoform (MSG) is ~730 residues, and this third isoform (MSH-halophilic) is ~430 residues in length. Both isoforms A and G have been structurally characterized in detail, but no structures have been reported for the H isoform which has been found thus far only in members of the halophilic Archaea.ResultsWe have solved the structure of a malate synthase H (MSH) isoform member from Haloferax volcanii in complex with glyoxylate at 2.51 Å resolution, and also as a ternary complex with acetyl-coenzyme A and pyruvate at 1.95 Å. Like the A and G isoforms, MSH is based on a β8/α8 (TIM) barrel. Unlike previously solved malate synthase structures which are all monomeric, this enzyme is found in the native state as a trimer/hexamer equilibrium. Compared to isoforms A and G, MSH displays deletion of an N-terminal domain and a smaller deletion at the C-terminus. The MSH active site is closely superimposable with those of MSA and MSG, with the ternary complex indicating a nucleophilic attack on pyruvate by the enolate intermediate of acetyl-coenzyme A.ConclusionsThe reported structures of MSH from Haloferax volcanii allow a detailed analysis and comparison with previously solved structures of isoforms A and G. These structural comparisons provide insight into evolutionary relationships among these isoforms, and also indicate that despite the size and sequence variation, and the truncated C-terminal domain of the H isoform, the catalytic mechanism is conserved. Sequence analysis in light of the structure indicates that additional members of isoform H likely exist in the databases but have been misannotated.
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