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.

The chemical biology of immunophilin ligands.

The immunophilin ligands cyclosporin A, FK506 and rapamycin are best known for their immunosuppressive properties and their clinical use in transplantation medicine. These compounds or their analogs are also clinically used or investigated in various types of cancer, coronary angioplasty, dermatology, hepatitis C infections, and neuroprotection. Furthermore, the role of immunophilins in various pathologies is increasingly being recognized, supporting the preclinical drug development for novel immunophilin targets. Finally, immunophilin ligands are widely used as sophisticated tools in chemical biology. This review shows the progress on three major areas made in the last five years. An update of the immunosuppressive ligands and their clinical applications is discussed in the first part of the review, followed by a discussion about the emerging immunophilin targets and their respective ligands. The final section gives a detailed assessment of immunophilin ligand-based tools.

Multifunctional DNA conjugates for the in vitro selection of new catalysts.

DNA-substrate conjugates are required for the direct in vitro selection of novel DNA catalysts for reactions between two small reactants. Here we describe the introduction of all necessary features into ssDNA by a novel, multifunctional primer containing a flexible PEG spacer, an o-nitrobenzyl moiety allowing for selective photocleavage, and anthracene as a reactant, a fluorescence label and/or an immobilization tag. These components were checked individually and by a mock selection.

Libraries of multifunctional RNA conjugates for the selection of new RNA catalysts.

An in vitro selection system was developed for the selection of RNA molecules catalyzing bimolecular reactions between small reactants. The system is based on the direct selection protocol and involves libraries of multifunctional RNA conjugates rather than unmodified RNA transcripts. For the preparation of RNA conjugate libraries, a dinucleotide analog has been designed and synthesized containing a poly(ethylene glycol) linker with an embedded photocleavage site and a terminal attachment site for coupling potential reactants. Reactants are first coupled to the dinucleotide analog by activated ester chemistry and then ligated to the 3'-ends of enzymatically prepared RNA pool molecules, giving libraries of complex conjugates. Species that become attached to biotin on incubation with a biotinylated partner are isolated using streptavidin-derivatized matrices and then subjected to a photocleavage step. Selective cleavage of the linker releases only those RNA species in which reaction has taken place at the linker-coupled reactant, while products with the biotin attached to internal positions of the RNA part remain immobilized. Efficient photocleavage is achieved by laser irradiation at 355 nm, and the released RNAs are intact and amplifiable by reverse transcription. All steps are shown to be compatible with the overall selection procedure, as was shown by performing a model selection cycle. Besides allowing a broader scope of reaction types to be selected for, the strategy relieves the RNA from the requirement to possess substrate properties as well as catalytic activity, and the use of a cleavable linker will suppress the selection of catalysts for side reactions.