Project Summary Mammalian cells have developed an elaborate network of immunoproteins that serve to identify and combat viral pathogens. Interferon-stimulated gene 15 (ISG15) is a 17.2 kDa tandem ubiquitin-like protein that is used by specific E1– E2–E3 ubiquitin cascade enzymes to interfere with the activity of viral proteins. Recent biochemical studies have demonstrated how the HECT and RCC1-containing protein 5 (HERC5) E3 ligase regulates ISG15 signaling in response to SARS-CoV-2, hepatitis C (HCV), influenza-A (IAV), human immunodeficiency virus (HIV), and other viral infections. While the immunological role of HERC5-dependent ISGylation is well established, the molecular mechanisms used by HERC5 to catalyze the specific and timely attachment of ISG15 to proteins in response to a viral infection remain unclear. It is paramount that we understand how HERC5 works at the atomic level to aid in the future development of therapies to treat viral infections and to enhance human health. The objective of this project is to understand the structural and biochemical basis for HERC5-dependent IGSylation. To date there have been no structural studies reported for HERC5. We will elucidate the unique mechanism used by HERC5 to attach ISG15 on to viral substrates. HERC5 is a unique member of the Homologous to E6AP C-Terminus (HECT) E3 ubiquitin ligases that contains the characteristic HECT domain, consisting of an N-terminal lobe and a C-terminal lobe, that is responsible for catalyzing the covalent attachment of ISG15 to a target protein. Currently the mechanism that HERC5 uses and the identities of specific residues in and around the active site required to catalyze the attachment of ISG15 with is unclear. The long-term scientific goal of the PI is to investigate the 3D structures and underlying enzymology for HERC5 to learn how this enzyme selectively and specifically attaches ISG15 to viral proteins as part of the host’s innate immune response. The major foci of this proposal will be to determine the catalytic mechanism of HERC5 using structural and biophysical approaches (Aim 1), and to examine HERC5 complex with ISG15 and the E2 enzyme UBE2L6 (Aim 2). Our preliminary studies using NMR spectroscopy and other biochemical approaches suggest that the novel mechanism of HERC5 is found exclusively in the HECT domain C-terminal lobe that contain the absolutely conserved catalytic cysteine required to ISGylate viral proteins. Building on our established track-record of examining the mechanisms of other members of the HECT E3 ubiquitin ligases, the inherent difference of HERC5 being ISG15-specific and not able to catalyze ubiquitin transfer provide an enticing opportunity to expand our current understanding of HECT-dependent activity and how their dysfunctions cause disease. Our findings will offer new insight into the molecular mechanisms used by the HECT E3 ligase HERC5 and help us learn how and why this enzyme works in response to a viral infection. Undergr...