From humans and eukaryotes to viruses and pathogens; how transition metals shape catalysis and allostery - Project Summary Metalloproteins play a crucial but poorly understood role in how viruses manipulate their hosts and how key cellular processes are regulated in eukaryotes, parasites, and viruses. Our proposed research program for the next five years aims to bridge the existing knowledge gap about how metal ions affect the function of enzymes that are at the nexus of host-pathogen interface, immune response, and dNTP homeostasis. Our strategy is a conceptualization of our ideas about how metal ions tune activity and regulation of two important enzyme super- families: viral proteases and SAMHD1 dNTPases. Viral proteases. Viral polyprotein complexes enclose proteases that not only release the non-structural proteins required for replication of the viral genome but also harbor moonlighting activities that intercept host protein function, ultimately derailing immune defense. A number of viral proteases coordinate an essential for function metallocofactor in a putative Zinc-finger motif that is located far from the proteolytic active site. We propose that this metallocofactor is an Fe-S cluster and that this center is a common but unrecognized element in viral proteases. We aim to dissect the metal-dependent activities of four proteases from viruses for which efficient treatments are lacking: the PLpro from Sars-Cov(2), the Nsp1α from the Porcine Reproductive and Respiratory Syndrome Virus, the putative cysteine protease (PCP) from the Hepatitis E Virus, and the 2Apro from Enterovirus 71 (hand-foot-mouth disease). Based on our results that these proteases interchangeably coordinate [4Fe-4S] clusters or Zn2+, we propose a revision of the well-accepted paradigm of Zn utilization in this class of enzymes. Our studies promise to resolve the apparent Fe-S cluster/Zn dilemma in viral proteases by establishing: i) the in vivo physiological cofactor and ii) the biological relevance of this metal binding promiscuity. SAMHD1 dNTPases. Sterile Alpha Motif and HD-domain containing protein 1 (SAMHD1) is the only hydrolase in humans that catalyzes the breakdown of cellular deoxynucleotides (dNTPs). SAMHD1 also occurs in phylogenetically diverse organisms, such as viruses, plants, and human pathogens. This proposal exposes significant conceptual and methodological gaps about how SAMHD1 dNTPases have evolved to regulate dNTP levels essential for organism fitness. In this respect our studies promise to delineate, for the first time, molecular and chemical details that govern SAMHD1 catalysis in the human enzyme as well as the untapped pool of eukaryotic and viral orthologs. In this proposal we will: i) establish the role of Fe and Mn in activation and catalysis of human SAMHD1, ii) map the functional repertoire of newly identified SAMHD1 orthologs, and iii) shed light into the evolutionary and functional diversification of SAMHD1 dNTPases. The projected outcome will broadly provide a molecular paradigm for dNTP regulation in plants, fungi and parasites that remains entirely unexplored.
