A structural genomics approach to the structure determination of macrophage proteins

Macrophages are cells differentiated from circulating blood monocytes that represent the first line of defence against pathogen invasion. Macrophages are widely distributed throughout the body, which comprising 15% - 20% of cell populations in many organs and are particularly abundant at the route of pathogen entry. They play a critical role in immune defence by initiating, promoting, preventing, suppressing or terminating immune responses. Macrophages act as a key player in both the pro-inflammatory and anti-inflammatory responses. Characterisation of important macrophage proteins could helps to understand the molecular mechanisms of macrophages in these responses. We established a high-throughput pipeline at the University of Queensland to investigate the structures and functions of novel macrophage proteins. This PhD project began with the selection of 12 novel, biologically interesting and crystallization-feasible targets that were then designed into 96 different constructs. Processing of the 96 constructs was performed in parallel using simple automated applications of ligation-independent cloning, small-scale bacterial expression, small-scale purification and solubility assessment. After processing these 12 targets, I found that 16 constructs of three targets yielded soluble protein. From the three soluble targets, I focused on two proteins BinCARD and Fam96a, because in the meantime the third target was solved by another group. Bcl10 interacting CARD protein (BinCARD) is a CARD-domain containing protein that was reported to interact with Bcl10 through CARD-CARD interactions to down-regulate NF-κB transcription factor activation. The crystal structure of BinCARD solved at 1.5 Å resolution revealed six anti-parallel α-helices, showing that this protein is similar to other CARDs of known structures. Before progress toward the interaction study between BinCARD and Bcl10, I also addressed the bottleneck of expressing Bcl10 as soluble protein. Several rescue strategies were used including expression using different constructs and bacterial strains, insect cell system, and matrix-assisted refolding strategy. Carma1 was introduced as a positive control for the BinCARD and Bcl10 interaction study. GST-pull down assay suggests that dimerisation of Bcl10 is required for its interaction with Carma1. However, I have not yet been able confirm an interaction between BinCARD and Bcl10 interaction. Family with sequence similarity 96, member a (Fam96a) is a novel DUF59 domain containing protein that belongs to a group of diverse proteins with no function characterised yet. The crystal structures of Fam96a at 1.8 Å resolution revealed two different types of novel domain swapped-dimer conformation, a major dimer and a minor dimer. I also confirmed by chemical crosslinking and size-exclusion chromatography that Fam96a forms a complex with cytosolic iron-sulfur protein assembly 1 (Ciao1). Crystals of the Fam96a and Ciao1 complex were obtained and diffracted to 7.0 Å resolution