Project Grant F30AI202840

FUNCTIONAL CHARACTERIZATION OF NONCANONICAL NUCLEOTIDES AS SECOND MESSENGERS IN IMMUNE SIGNALING - PROJECT SUMMARY BACTERIAL IMMUNE SYSTEMS ARE ANCIENT DEFENSES THAT PREDATE MANY PATHWAYS IN HUMAN INNATE IMMUNITY. A RECURRING THEME IN THESE PATHWAYS IS THE USE OF NUCLEOTIDE SECOND MESSENGERS TO DETECT INFECTION AND TRIGGER IMMUNE DEFENSE. A NEWLY DISCOVERED ANTIPHAGE SYSTEM, KONGMING, USES AN UNUSUAL MESSENGER, DEOXYINOSINE TRIPHOSPHATE (DITP), TO SENSE PHAGE INFECTION AND DRIVE ABORTIVE INFECTION THAT PROTECTS THE BACTERIAL COMMUNITY. KONGMING COMPRISES KOMA, AN ADENOSINE DEAMINASE; KOMB, A HAM1-LIKE PURINE PYROPHOSPHATASE; AND KOMC, A SIR2-LIKE NAD HYDROLASE. UPON PHAGE INFECTION, KOMA AND PHAGE ENZYMES GENERATE DITP, WHICH ACTIVATES THE KOMB-KOMC (KOMBC) COMPLEX TO DEPLETE CELLULAR NAD AND INDUCE CELL DEATH. HOW KOMBC ASSEMBLES, RECOGNIZES DITP WITH HIGH SPECIFICITY, AND CONVERTS THIS SIGNAL INTO NAD DEPLETION REMAINS UNKNOWN. THE OVERALL OBJECTIVE OF THIS F30 PROPOSAL IS TO DEFINE THE STRUCTURAL AND MECHANISTIC BASIS OF DITP SENSING AND EFFECTOR ACTIVATION IN THE KONGMING SYSTEM. MY CENTRAL HYPOTHESIS IS THAT KOMBC FORMS A HIGHER-ORDER COMPLEX IN WHICH KOMB SELECTIVELY BINDS DITP AND ALLOSTERICALLY ACTIVATES KOMC TO CATALYZE NAD HYDROLYSIS. THIS HYPOTHESIS IS SUPPORTED BY PRELIMINARY CRYO-EM STRUCTURES OF APO AND DITP-BOUND KOMBC THAT REVEAL NUCLEOTIDE-DEPENDENT CONFORMATIONAL CHANGES. IN AIM 1, I WILL DETERMINE HOW KOMC OLIGOMERIZATION AND ACTIVE-SITE RESIDUES ENABLE EFFICIENT NAD HYDROLYSIS AND ANTIPHAGE DEFENSE BY COMBINING CRYO-EM, MUTAGENESIS, AND ENZYMOLOGY. IN AIM 2, I WILL DISSECT HOW KOMB SPECIFICALLY RECOGNIZES DITP AND HOW THE KOMB-KOMC INTERFACE RELAYS THIS SIGNAL TO ACTIVATE KOMC, USING QUANTITATIVE NUCLEOTIDE-BINDING ASSAYS, STRUCTURE-GUIDED MUTAGENESIS, AND PHAGE INFECTION ASSAYS. THESE STUDIES WILL ESTABLISH DITP AS A BONA FIDE SECOND MESSENGER, UNCOVER GENERAL PRINCIPLES OF NUCLEOTIDE-DRIVEN IMMUNE SIGNALING, AND INFORM THE DESIGN OF SYNTHETIC DEFENSE CIRCUITS FOR PHAGE THERAPY. THIS WORK WILL ALSO PROVIDE COMPREHENSIVE TRAINING IN STRUCTURAL BIOLOGY, BIOPHYSICS, AND HOST-PATHOGEN INTERACTIONS, PREPARING ME FOR AN INDEPENDENT CAREER AS A PHYSICIAN SCIENTIST.