Our lab works on a variety of different biological systems including, allergens, DNA replication and repair, bacterial infection, and heparan sulfate biosynthesis. Listed below are the highlights from this past year: 1) In Collaboration with Dr. Myron Goodmans Lab at the University of Southern California we solved the crystal structure of activation-induced deoxycytidine deaminase better known as AID. AID is a protein that plays a critical role in immunoglobulin (Ig) diversification by initiating somatic hypermutation (SHM) and class-switch recombination (CSR) in B-Cells. AID initiates SHM and CSR by deaminating C -> U during transcription of Ig variable regions. These mutations generate diversity which results in high affinity antibodies. Deleterious mutations in AID contribute to a disease known as hyper IgM type 2 syndrome (HIGM-2). Despite the importance of this enzyme its structure had not been solved. Working with the Goodman group, we were able to utilize the MBP-fusion crystallization system we developed to obtain a crystal structure of a MBP-fusion construct of human AID. This study revealed important differences between AID and other APOBEC family members, specifically a unique specificity loop, as well as provided for a model to better understand DNA binding and the mutation in AID that lead to HIGM-2 syndrome. 2) The Group A pathogen Streptococcus pyogenes (GAS) is a leading cause of severe invasive infection including streptococcal toxic shock syndrome and necrotizing fasciitis, leading to over 160,000 deaths annually worldwide. Hyper-virulent stains of GAS have developed a number of defenses against the host immune system including the secretion of the nuclease Sda1 that degrades neutrophil extracellular traps (NETs) extruded by neutrophils which are rich in chromatin material. The ability to degrade these NETS protects GAS from the innate immune response. The fact that this nuclease is not an essential enzyme for bacterial but does promote virulence makes it an interesting drug target. Inhibiting of Sda1 could lead to reduced virulence allowing the host immune system to kill off the infection. Ideally a drug that targets Sda1 would not disrupt the delicate balance of the gut microbiome thus reducing many of the deleterious side effects associated with conventional antibiotics. In or lab, we determined the crystal structure of Sda1 in two different crystal forms and characterized a number of mutants to better understand how Sda1 functions. The crystal structure revealed a number of differences between Sda1 and other nuclease as well as provided a template on which to model DNA binding. From this structure we were able to identify key residues involved in catalysis as well as DNA binding. We are continuing to work on obtaining crystal structures of Sda1 with DNA bound to map potential drug binding sites. 3) In support of Dr. Londons lab at the NIEHS we determined the crystal structure of the ribonuclease domain from HIV-1 reverse transcriptase (RT) contributing to a better understanding of the maturation process of the active form of RT. RT is formed from two molecules of a p66 precursor. The active form of the enzyme is a heterodimer of the p66 protein and a proteolytically cleaved p51 that has lost its RNaseH domain. Despite having the same sequence (minus the RNaseH domain), the two molecules of the p66/p51 have different overall structures in the final functional enzyme. Our crystal structure of the RnaseH domain contained a swap of the C-termini between two molecules in the asymmetric unit. This domain swap is suggestive of the unfolding pathway required by the RNaseH domain to expose the proteolysis site allowing for conversion of a p66 molecule to p51. Understanding the maturation process that converts a p66 molecule to p51 provides a unique drug target to inhibit HIV that would ideally not alter the function of essential host proteins.
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