This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. A recently discovered family of eukaryotic-like Ser/Thr protein kinases (STPKs) in Mycobacterium tuberculosis (M. tb.) are candidate regulators of the cell cycle, and there is growing evidence for their involvement in the critical stages of growth, latency, and persistence within human alveolar macrophages. PknB, a conserved transmembrane receptor STPK in M. tb, is essential for growth3 and is assumed to mediate a switch between growth and division in M. tb. The structure of the intracellular kinase domain has been solved by our lab. Because the structure globally resembles eukaryotic homologs, we have screened a small library of known eukaryotic kinase inhibitors with various STPKs in M. tb. Although we have found no strong hits for PknB, we have successfully used active-site mutants of PknB to tightly bind inhibitors that bind STPK paralogs in M. tb. We have performed co-crystallization studies of PknB surrogates with kinase inhibitors such as staurosporine, Bis1, and JNK inhibitor sp600125. We have co-crystallized PknB with sp600125 and other eukaryotic inhibitors and initial diffraction has been observed from 2.0-2.9 A. We are currently optimizing crystals of PknB mutants with several inhibitor ligands under similar crystallization conditions, adding to several other crystals our lab has solved using PknB, including PknB-ATPgammaS and a bump-hole complex of PknB M92A-1NMPP1. These structures will highlight the conservation of prokaryotic and eukaryotic protein kinases and enable the design of improved inhibitors to aid functional as well as drug discovery efforts. Currently, the use of PknB surrogates for their selective inhibition by eukaryotic kinase inhibitors in vivo is underway in order to glean functional information on PknB using a chemical genetics approach. Our proposed structural studies will shed insight on how to improve selective inhibition of eukaryotic-like STPKs in M. tb and enable a chemical genetics approach to validate the STPKs.