Conformational regulation of AP1 and AP2 clathrin adaptor complexes.

Heterotetrameric clathrin adaptor protein complexes (APs) orchestrate the formation of coated vesicles for transport among organelles of the cell periphery. AP1 binds membranes enriched for phosphatidylinositol 4-phosphate, such as the trans Golgi network, while AP2 associates with phosphatidylinositol 4,5-bisphosphate of the plasma membrane. At their respective membranes, AP1 and AP2 bind the cytoplasmic tails of transmembrane protein cargo and clathrin triskelions, thereby coupling cargo recruitment to coat polymerization. Structural, biochemical and genetic studies have revealed that APs undergo conformational rearrangements and reversible phosphorylation to cycle between different activity states. While membrane, cargo and clathrin have been demonstrated to promote AP activation, growing evidence supports that membrane-associated proteins such as Arf1 and FCHo also stimulate this transition. APs may be returned to the inactive state via a regulated process involving phosphorylation and a protein called NECAP. Finally, because antiviral mechanisms often rely on appropriate trafficking of membrane proteins, viruses have evolved novel strategies to evade host defenses by influencing the conformation of APs. This review will cover recent advances in our understanding of the molecular inputs that stimulate AP1 and AP2 to adopt structurally and functionally distinct configurations.

Structural characterization of Class 2 OLD family nucleases supports a two-metal catalysis mechanism for cleavage.

Overcoming lysogenization defect (OLD) proteins constitute a family of uncharacterized nucleases present in bacteria, archaea, and some viruses. These enzymes contain an N-terminal ATPase domain and a C-terminal Toprim domain common amongst replication, recombination, and repair proteins. The in vivo activities of OLD proteins remain poorly understood and no definitive structural information exists. Here we identify and define two classes of OLD proteins based on differences in gene neighborhood and amino acid sequence conservation and present the crystal structures of the catalytic C-terminal regions from the Burkholderia pseudomallei and Xanthamonas campestris p.v. campestris Class 2 OLD proteins at 2.24 Å and 1.86 Å resolution respectively. The structures reveal a two-domain architecture containing a Toprim domain with altered architecture and a unique helical domain. Conserved side chains contributed by both domains coordinate two bound magnesium ions in the active site of B. pseudomallei OLD in a geometry that supports a two-metal catalysis mechanism for cleavage. The spatial organization of these domains additionally suggests a novel mode of DNA binding that is distinct from other Toprim containing proteins. Together, these findings define the fundamental structural properties of the OLD family catalytic core and the underlying mechanism controlling nuclease activity.

Influenza A Virus Infections Sense Host Membrane Tension to Dynamically Tune Assembly.

Enveloped viruses often exhibit a pleomorphic morphology, ranging in size from 100nm spheres to tens-of-micron long filaments. For influenza A virus (IAV), spheres enable rapid replication and minimize metabolic cost, while filaments resist effects of antibodies or other cell-entry pressures. The current paradigm is that virion shape changes require genetic adaptation; however, a virus evolved to alter its shape phenotypically would outperform one that relies on genetic selection. Using a novel quantitative flow virometry assay to characterize virion shape dynamics we find that IAV rapidly tunes its shape distribution to favor spheres under optimal, and filaments under attenuating conditions including the presence of antibodies. We identify membrane tension as a key cue sensed by IAV determining shape distributions. This phenotypic shift outpaces genetic change and serves to enable additional life cycles under pressure. Our work expands knowledge of the complex host-virus interplay to include viral responses to the local environment by optimizing its structure to maximize replication and ultimately host-host transmission.

Structural basis of an endocytic checkpoint that primes the AP2 clathrin adaptor for cargo internalization.

Clathrin-mediated endocytosis (CME) is the main route of internalization from the plasma membrane. It is known that the heterotetrameric AP2 clathrin adaptor must open to simultaneously engage membrane and endocytic cargo, yet it is unclear how transmembrane cargos are captured to catalyze CME. Using cryogenic-electron microscopy, we discover a new way in which mouse AP2 can reorganize to expose membrane- and cargo-binding pockets, which is not observed in clathrin-coated structures. Instead, it is stimulated by endocytic pioneer proteins called muniscins, which do not enter vesicles. Muniscin-engaged AP2 is primed to rearrange into the vesicle-competent conformation on binding the tyrosine cargo internalization motif (YxxΦ). We propose adaptor priming as a checkpoint to ensure cargo internalization.

A structural mechanism for phosphorylation-dependent inactivation of the AP2 complex.

Endocytosis of transmembrane proteins is orchestrated by the AP2 clathrin adaptor complex. AP2 dwells in a closed, inactive state in the cytosol, but adopts an open, active conformation on the plasma membrane. Membrane-activated complexes are also phosphorylated, but the significance of this mark is debated. We recently proposed that NECAP negatively regulates AP2 by binding open and phosphorylated complexes (Beacham et al., 2018). Here, we report high-resolution cryo-EM structures of NECAP bound to phosphorylated AP2. The site of AP2 phosphorylation is directly coordinated by residues of the NECAP PHear domain that are predicted from genetic screens in C. elegans. Using membrane mimetics to generate conformationally open AP2, we find that a second domain of NECAP binds these complexes and cryo-EM reveals both domains of NECAP engaging closed, inactive AP2. Assays in vitro and in vivo confirm these domains cooperate to inactivate AP2. We propose that phosphorylation marks adaptors for inactivation.

NECAPs are negative regulators of the AP2 clathrin adaptor complex.

Eukaryotic cells internalize transmembrane receptors via clathrin-mediated endocytosis, but it remains unclear how the machinery underpinning this process is regulated. We recently discovered that membrane-associated muniscin proteins such as FCHo and SGIP initiate endocytosis by converting the AP2 clathrin adaptor complex to an open, active conformation that is then phosphorylated (Hollopeter et al., 2014). Here we report that loss of ncap-1, the sole C. elegans gene encoding an adaptiN Ear-binding Coat-Associated Protein (NECAP), bypasses the requirement for FCHO-1. Biochemical analyses reveal AP2 accumulates in an open, phosphorylated state in ncap-1 mutant worms, suggesting NECAPs promote the closed, inactive conformation of AP2. Consistent with this model, NECAPs preferentially bind open and phosphorylated forms of AP2 in vitro and localize with constitutively open AP2 mutants in vivo. NECAPs do not associate with phosphorylation-defective AP2 mutants, implying that phosphorylation precedes NECAP recruitment. We propose NECAPs function late in endocytosis to inactivate AP2.