Structural studies of ricin toxin A-chain and C subunit of ATP synthase
Goldman, Joshua D.
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Ricin toxin A-chain (RTA) is the catalytic subunit of the ribosomal inhibition protein, ricin. It depurinates adenonine-4324 (numbering according to the rat) within the sarcin/ricin loop (S/RL) of the 28S ribosomal ribonucleic acid (rRNA) a component of the 60S ribosomal subunit, halting protein synthesis and eventually leading to cell death. To act as a rescue agent against both accidental exposure and to limit side-effects associated with medicinal application, inhibitors that mimic the tetraloop of the S/RL structure have been investigated. However, inhibitor design has been hindered by limited structural information about the contacts made between RTA and the tetraloop. We have taken a two pronged structural biology approach involving both nuclear magnetic resonance (NMR) spectroscopy and x-ray crystallography to investigate the structure of RTA with a tetraloop mimic. For both techniques, new protocols were developed to overcome many of the technical challenges, which eventually lead to the first structure of an N-ribohydrolase bound to a tetraloop (RTA-cyclic G(9-DA)GA 2'-OMe) as determined by x-ray crystallography to 2.0A resolution.;The ion-translocating c subunit of F1FO ATP synthase forms a helix-loop-helix structure and resides within the transmembrane portion of the FO subunit. Darylquinolines (DARQ), the first new class of drugs against Mycobacterium tuberculosis in 40 years, have exquisite specificity to the c subunit of mycobacteria, suggesting a unique micro-environment exists within the c subunit of this genus. The piCa value of all acidic residues and complete NMR residue specific assignments were determined for M. tuberculosis c subunit and the well studied Escherichia coli c subunit in a lyso-myristoyl-phosphatidylglycerol (LMPG) based micelle. We also performed titrations with known and suspected c subunit inhibitors but could not elicit binding to the M. tuberculosis or E. coli c subunit monomer in an LMPG micelle.