Legionella pneumophila macrophage infectivity potentiator protein appendage domains modulate protein dynamics and inhibitor binding.

Macrophage infectivity potentiator (MIP) proteins are widespread in human pathogens including Legionella pneumophila, the causative agent of Legionnaires' disease and protozoans such as Trypanosoma cruzi. All MIP proteins contain a FKBP (FK506 binding protein)-like prolyl-cis/trans-isomerase domain that hence presents an attractive drug target. Some MIPs such as the Legionella pneumophila protein (LpMIP) have additional appendage domains of mostly unknown function. In full-length, homodimeric LpMIP, the N-terminal dimerization domain is linked to the FKBP-like domain via a long, free-standing stalk helix. Combining X-ray crystallography, NMR and EPR spectroscopy and SAXS, we elucidated the importance of the stalk helix for protein dynamics and inhibitor binding to the FKBP-like domain and bidirectional crosstalk between the different protein regions. The first comparison of a microbial MIP and a human FKBP in complex with the same synthetic inhibitor was made possible by high-resolution structures of LpMIP with a [4.3.1]-aza-bicyclic sulfonamide and provides a basis for designing pathogen-selective inhibitors. Through stereospecific methylation, the affinity of inhibitors to L. pneumophila and T. cruzi MIP was greatly improved. The resulting X-ray inhibitor-complex structures of LpMIP and TcMIP at 1.49 and 1.34 Å, respectively, provide a starting point for developing potent inhibitors against MIPs from multiple pathogenic microorganisms.

Maintenance of repression control of the ilvGMEDA operon in a temperature-sensitive leucyl-transfer RNA synthetase mutant of Escherichia coli K-12 at a restrictive temperature.

The mechanism of L-leucine regulation of ilvGMEDA is thought to be by ribosome-mediated attenuation that is dependent upon the concentration of Leu-tRNA(Leu) which results from leucyl-tRNA synthetase (LeuRS) activity. The requirement for LeuRS activity in attenuation control was tested in an Escherichia coli K-12 strain containing a temperature-sensitive LeuRS and the ilvGMEDA operon with an active ilvGM. Growth of this strain at 30 degrees C followed by a shift to 37 degrees C to inactivate the LeuRS revealed that ilvGM expression decreased at the restrictive temperature whereas the downstream gene expression was slightly elevated. We suggest that ilvGM does not respond to a deattenuation signal, and that, possibly, a secondary repression/derepression mechanism exists.