Next-generation interaction proteomics for quantitative Jumbophage-bacteria interaction mapping.

Host-pathogen interactions (HPIs) are pivotal in regulating establishment, progression, and outcome of an infection. Affinity-purification mass spectrometry has become instrumental for the characterization of HPIs, however the targeted nature of exogenously expressing individual viral proteins has limited its utility to the analysis of relatively small pathogens. Here we present the use of co-fractionation mass spectrometry (SEC-MS) for the high-throughput analysis of HPIs from native viral infections of two jumbophages ( ϕ KZ and ϕ PA3) in Pseudomonas aeruginosa . This enabled the detection > 6000 unique host-pathogen and > 200 pathogen-pathogen interactions for each phage, encompassing > 50% of the phage proteome. Interactome-wide comparison across phages showed similar perturbed protein interactions suggesting fundamentally conserved mechanisms of phage predation within the KZ-like phage family. Prediction of novel ORFs revealed a ϕ PA3 complex showing strong structural and sequence similarity to ϕ KZ nvRNAp, suggesting ϕ PA3 also possesses two RNA polymerases acting at different stages of the infection cycle. We further expanded our understanding on the molecular organization of the virion packaged and injected proteome by identifying 23 novel virion components and 5 novel injected proteins, as well as providing the first evidence for interactions between KZ-like phage proteins and the host ribosome. To enable accessibility to this data, we developed PhageMAP, an online resource for network query, visualization, and interaction prediction ( https://phagemap.ucsf.edu/ ). We anticipate this study will lay the foundation for the application of co-fractionation mass spectrometry for the scalable profiling of hostpathogen interactomes and protein complex dynamics upon infection.

The ϕPA3 phage nucleus is enclosed by a self-assembling 2D crystalline lattice.

To protect themselves from host attack, numerous jumbo bacteriophages establish a phage nucleus-a micron-scale, proteinaceous structure encompassing the replicating phage DNA. Bacteriophage and host proteins associated with replication and transcription are concentrated inside the phage nucleus while other phage and host proteins are excluded, including CRISPR-Cas and restriction endonuclease host defense systems. Here, we show that nucleus fragments isolated from ϕPA3 infected Pseudomonas aeruginosa form a 2-dimensional lattice, having p2 or p4 symmetry. We further demonstrate that recombinantly purified primary Phage Nuclear Enclosure (PhuN) protein spontaneously assembles into similar 2D sheets with p2 and p4 symmetry. We resolve the dominant p2 symmetric state to 3.9 Šby cryo-EM. Our structure reveals a two-domain core, organized into quasi-symmetric tetramers. Flexible loops and termini mediate adaptable inter-tetramer contacts that drive subunit assembly into a lattice and enable the adoption of different symmetric states. While the interfaces between subunits are mostly well packed, two are open, forming channels that likely have functional implications for the transport of proteins, mRNA, and small molecules.

Next-generation proteomics for quantitative Jumbophage-bacteria interaction mapping.

Host-pathogen interactions are pivotal in regulating establishment, progression, and outcome of an infection. While affinity-purification mass spectrometry has become instrumental in characterizing such interactions, it suffers from limitations in scalability and biological authenticity. Here we present the use of co-fractionation mass spectrometry for high throughput analysis of host-pathogen interactions from native viral infections of two jumbophages (ϕKZ and ϕPA3) in Pseudomonas aeruginosa. This approach enabled the detection of > 6000 unique host-pathogen interactions for each phage, encompassing > 50% of their respective proteomes. This deep coverage provided evidence for interactions between KZ-like phage proteins and the host ribosome, and revealed protein complexes for previously undescribed phage ORFs, including a ϕPA3 complex showing strong structural and sequence similarity to ϕKZ non-virion RNA polymerase. Interactome-wide comparison across phages showed similar perturbed protein interactions suggesting fundamentally conserved mechanisms of phage predation within the KZ-like phage family. To enable accessibility to this data, we developed PhageMAP, an online resource for network query, visualization, and interaction prediction ( https://phagemap.ucsf.edu/ ). We anticipate this study will lay the foundation for the application of co-fractionation mass spectrometry for the scalable profiling of host-pathogen interactomes and protein complex dynamics upon infection.

Conserved structural elements specialize ATAD1 as a membrane protein extraction machine.

The mitochondrial AAA (ATPase Associated with diverse cellular Activities) protein ATAD1 (in humans; Msp1 in yeast) removes mislocalized membrane proteins, as well as stuck import substrates from the mitochondrial outer membrane, facilitating their re-insertion into their cognate organelles and maintaining mitochondria's protein import capacity. In doing so, it helps to maintain proteostasis in mitochondria. How ATAD1 tackles the energetic challenge to extract hydrophobic membrane proteins from the lipid bilayer and what structural features adapt ATAD1 for its particular function has remained a mystery. Previously, we determined the structure of Msp1 in complex with a peptide substrate (Wang et al., 2020). The structure showed that Msp1's mechanism follows the general principle established for AAA proteins while adopting several structural features that specialize it for its function. Among these features in Msp1 was the utilization of multiple aromatic amino acids to firmly grip the substrate in the central pore. However, it was not clear whether the aromatic nature of these amino acids were required, or if they could be functionally replaced by aliphatic amino acids. In this work, we determined the cryo-EM structures of the human ATAD1 in complex with a peptide substrate at near atomic resolution. The structures show that phylogenetically conserved structural elements adapt ATAD1 for its function while generally adopting a conserved mechanism shared by many AAA proteins. We developed a microscopy-based assay reporting on protein mislocalization, with which we directly assessed ATAD1's activity in live cells and showed that both aromatic amino acids in pore-loop 1 are required for ATAD1's function and cannot be substituted by aliphatic amino acids. A short α-helix at the C-terminus strongly facilitates ATAD1's oligomerization, a structural feature that distinguishes ATAD1 from its closely related proteins.