Structural investigations of three different proteins by X-ray crystallography and NMR Leucine aminopeptidase ppLAP from Pseudomonas putida Competence regulator MecA from Bacillus subtilis Actin-regulatory protein SWAP-70 from Mus musculus

Chapter 1 presents a short review on leucine aminopeptidases. These are zinc-dependent proteases are widespread and occur in all kingdoms of life. Generally, they take care of protein degradation and peptide recycling in the cell. The review discusses the classification of these enzymes and focuses on what we currently know about their three-dimensional (3D) structures and catalytic mechanisms. The crystal structure determination of ppLAP, a leucine aminopeptidase from Pseudomonas putida, is presented in chapter 2. Three different structures were determined, in a substrate-free, metal-bound state at pH 9.5, in a substrate-free, metal-free state at pH 5.2, and in an inhibitor-bound, metal-bound state at pH 9. Chapter 3 offers a simplified description of the complex phenomenon of genetic competence development in the Gram-positive bacterium Bacillus subtilis. The review focuses on adapter switch, MecA, and on what is currently known about its interactions with ComK, ComS, and ClpC. Our efforts to determine a detailed structure of MecA are presented in chapter 4. The failure in obtaining crystals has compelled us try and solve its 3D structure by using NMR. The NMR data did not allow a full 3D structure determination. Explanations are proposed for this recalcitrant behaviour of MecA. Finally, chapter 5 describes the structural and functional studies of SWAP-70, a 70-kDa protein from mouse, which represents a novel Rac-dependent guanine exchange factor (GEF). Various crystallization trials using different constructs of full length SWAP-70 and of SWAP-70 fragments were set up, albeit without success. Efforts to produce a complex between SWAP-70 and a Rac protein were also unsuccessful. Suggestions are offered of experiments which could move this project forward.