Cryo-EM Elucidation of the Structure of Bacteriophage P22 Virions after Genome Release
To our knowledge, we report the first cryo-EM asymmetric reconstruction of bacteriophage P22 after it has released its packaged double-stranded DNA chromosome. The phage P22 portal protein is known to have different conformations depending on the maturation state of the virus, but it remained plausible that the conformation of the portal protein might have an additional change in empty particles since interactions with DNA would be lost upon genome ejection. Could the portal C-terminal barrel invert to extend through the tail and be the protective structure that delivers the genome through the protective barriers of Salmonella? Here, our results demonstrate that the portal structure remains essentially unchanged inside the capsid after DNA release and does not revert to the conformation it adopts in procapsids. These conformational changes in the fivefold vertex occur in the portal and tail machine. The reconstruction shows tube-like protein density extending from the center of the tail assembly.
Publication: McNulty R†, Cardone G, Gilcrease EB, Baker TS, Casjens SR, Johnson JE†. Cryo-EM Elucidation of the Structure of Bacteriophage P22 Virions after Genome Release. Biophys J. 2018;114(6):1295-301.
Architecture of the Complex Formed by Large and Small Terminase Subunits from Bacteriophage P22.
Viral packaging motors are fundamental molecular machines that power the delivery of viral genomes into preformed procapsid shells. Despite growing interest in this field of biology, a plethora of individual S-terminase and L-terminase subunit crystal structures, and recent advances in single-molecule biophysical analysis of packaging motors, not even moderate resolution information exists for a terminase complex. In this study, we purified an in vivo assembled complex of P22 terminase subunits and characterized its architecture using biochemical and hybrid structural methods. We found that P22 terminase subunits assemble preferentially into a stable complex containing one nonameric S-terminase bound to two L-terminase subunits (1S-terminase:2L-terminase).
Publication: McNulty R*†, Lokareddy RK*, Roy A, Yang Y, Lander GC, Heck AJ, Johnson JE, Cingolani G†. Architecture of the Complex Formed by Large and Small Terminase Subunits from Bacteriophage P22. J Mol Biol. 2015;427(20):3285-99.
Structural basis of photosensitivity in a bacterial light-oxygen-voltage/helix-turn-helix (LOV-HTH) DNA-binding protein
Light-oxygen-voltage (LOV) domains are blue light-activated signaling modules integral to a wide range of photosensory proteins. Upon illumination, LOV domains form internal protein-flavin adducts that generate conformational changes which control effector function. Here we advance our understanding of LOV regulation with structural, biophysical, and biochemical studies of EL222, a light-regulated DNA-binding protein. The dark-state crystal structure reveals interactions between the EL222 LOV and helix-turn-helix domains that we show inhibit DNA binding. Solution biophysical data indicate that illumination breaks these interactions, freeing the LOV and helix-turn-helix domains of each other. This conformational change has a key functional effect, allowing EL222 to bind DNA in a light-dependent manner. Our data reveal a conserved signaling mechanism among diverse LOV-containing proteins, where light-induced conformational changes trigger activation via a conserved interaction surface.
Publication: McNulty R, Nash AI, Shillito ME, Swartz TE, Bogomolni RA, Luecke H, Gardner KH. Structural basis of photosensitivity in a bacterial light-oxygen-voltage/helix-turn-helix (LOV-HTH) DNA-binding protein. PNAS 2011;108(23):9449-54.
Structure of the proton-gated urea channel from the gastric pathogen Helicobacter pylori
This first three-dimensional structure of a channel from the AmiS/UreI superfamily reveals a previously unobserved fold with a unique channel architecture able to filter polar organic solutes selectively. As the structure of a validated target for H. pylori eradication it may guide the discovery of small-molecule inhibitors, providing the possibility of monotherapy without the use of conventional antibiotics.
Publication: McNulty R*, Strugatsky D*, Munson K*, Chen CK, Soltis SM, Sachs G, Luecke H. Structure of the proton-gated urea channel from the gastric pathogen Helicobacter pylori. Nature. 2013;493(7431):255-8.