Unraveling Lactococcal Phage Baseplate Assembly by Mass Spectrometry

Abstract

Bacteriophages belonging to the Caudovirales order possess a tail acting as a molecular machine used during infection to recognize the host and ensure high-efficiency genome delivery to the cell cytoplasm. They bear a large and sophisticated multiprotein organelle at their distal tail end, either a baseplate or a tail-tip, which is the control center for infectivity. We report here insights into the baseplate assembly pathways of two lactoccocal phages (p2 and TP901-1) using electrospray ionization-mass spectrometry. Based on our "block cloning" strategy we have expressed large complexes of their baseplates as well as several significant structural subcomplexes. Previous biophysical characterization using size-exclusion chromatography coupled with on-line light scattering and refractometry demonstrated that the overproduced recombinant proteins interact with each other to form large (up to 1.9 MDa) and stable assemblies. The structures of several of these complexes have been determined by x-ray diffraction or by electron microscopy. In this contribution, we demonstrate that electrospray ionization-mass spectrometry yields accurate mass measurements for the different baseplate complexes studied from which their stoichiometries can be discerned, and that the subspecies observed in the spectra provide valuable information on the assembly mechanisms of these large organelles. Molecular & Cellular Proteomics

Crystal Structure of pb9, the Distal Tail Protein of Bacteriophage T5: a Conserved Structural Motif among All Siphophages

“…As for pb9, expression of isolated Dit genes TP901-1 orf46, p2 orf15, and Tuc2009 orf49 yielded monomeric proteins in solution (31,32), while only SPP1 gp19.1 was crystallized as a dodecamer (two head-to-tail hexameric rings) (8). For the former phages, hexamerization of the Dit protein is induced by its interaction with the Tal trimer, as evidenced by mass spectrometry (33) and crystallography (5,31,33). This is a common phenomenon among phage proteins, where oligomerization of a protein is regulated by the interaction with its partners (see, e.g., reference 28).…”

Visualizing a Complete Siphoviridae Member by Single-Particle Electron Microscopy: the Structure of Lactococcal Phage TP901-1

“…Structural studies of the host adsorption apparatus of the Lactococcus lactis phages p2 and TP901-1 revealed distinct baseplate architectures and diverse strategies used by the two virions to initiate infection (6)(7)(8)(9)(10)(11). The phage p2 baseplate undergoes large conformational changes in the presence of Ca 2ϩ ions to appropriately orient its RBPs and establish multiple interactions with host saccharides at the onset of infection (8).…”

Viral infection modulation and neutralization by camelid nanobodies

“…In recent years, we have reported on the structures of the RBPs and the baseplate of two phages, p2 and TP901-1, which infect the Gram + bacterium L. lactis, and we have identified their different strategies used to ensure infection (6,8,(14)(15)(16)(17)(18)(19). Although Ca 2+ ions trigger large conformational changes of phage p2 baseplate to orientate the RBPs to the host (15), the RBPs of phage TP901-1 baseplate point to the appropriate direction without the need for conformational change or Ca 2+ requirement (6).…”