The encapsidation protein from Lactococcus lactis asccφ28 as a novel system for the study of molecular motors

Motor proteins, molecules capable of converting the cell’s free energy into mechanical work, are prevalent in life. However, despite their pervasiveness, our understanding of these molecular machines remains inadequate. Some of the simplest examples of molecular motors are found in many bacteriophages and some eukaryotic viruses. For example, many phages package their genomes into preformed empty protein shells, or procapsids, in a highly thermodynamically-unfavorable process. This process is thus driven by encapsidation proteins within large macromolecular motor complexes with energy derived from the hydrolysis of ATP. Indeed, Bacillus subtilis bacteriophage φ29 has served for many years as a model system for the study of phage motor complexes for its relative structural simplicity and ease of its in vitro packaging assay. However, despite its many advantages, the motor protein of φ29, the encapsidation protein (EP), has proven difficult to work with when isolated from the rest of the complex due to its low solubility. In fact, the low solubility of the φ29 EP has hampered crystallization trials, as well as other biochemical and biophysical characterization assays. We thus opted to take a fresh new approach by working on a closely related ortholog which could help us overcome the φ29 EP’s low solubility. A BLAST search was conducted for φ29 EP orthologs which yielded a number of potential candidates, but only one stood out with a favorable score in XtalPred, a crystallizability predicting algorithm. Hence, a recombinant form of the gene product from open reading frame 11 of the Lactococcus lactis phage asccφ28, the putative encapsidation protein, has been expressed in Escherichia coli and purified to homogeneity (55.6% sequence similarity, Smith-Waterman score). Size-exclusion chromatography suggests that the recombinant protein forms a single oligomeric species in solution, while analytical ultracentrifugation and small-angle x-ray studies reveal a decameric stoichiometry. Surprisingly, preliminary crystallographic, as well as cryo-electron tomography (cryo-ET) and cryo-electron microscopy (cryo-EM) studies, suggest the decameric assembly is in fact composed of a dimer of pentamers, akin to the widely investigated and closest related EP from phage φ29, which forms pentameric rings when attached to viral proheads. The dimer of pentamers assembly of the asccφ28 encapsidation protein is a highly soluble, very stable, and active ATPase. Thus, this recombinant form of the putative encapsidation protein from the Lactococcus lactis phage asccφ28 shows great promise as a novel model system for in vitro mechanistic studies of this isolated motor protein component of phage dsDNA-packaging motors.