Structure and function studies of B. cereus Metallo-β-lactamase 5/B/6 and synaptotagmin 1 C2 domain AD3 mutants



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β-lactam antibiotics are the most heavily prescribed antibacterial, chemotherapeutic agents in clinical use in the world today. These highly effective drugs are quickly becoming ineffective, due to the emergence of antibiotic resistant bacterial strains. The major mechanism in which β-lactam antibiotics are rendered ineffective is through the production of a family of proteins called β-lactamases. The most problematic of these are the class B, metal dependent metallo-β-lactamases that can catalyze the hydrolysis of many types of β-lactam antibiotics. These metallo-β-lactamases have structures and mechanisms that are different from those of metal independent beta-lactamases (classes A, C and D). To this date, no clinically useful inhibitors of these enzymes are available. Our group has previously developed an effective 10-residue single-stranded DNA aptamer that inhibits the Bacillus cereus 5/B/6 metallo-β-lactamase in vitro to a very effective nanomolar range. Structural information obtained about the holoenzyme and enzyme-aptamer complex can provide details on the mechanism of inhibition. This is best done through the most popular structural determination method, X-ray crystallography.
Synaptotagmin 1 (Syt 1) is a member of a family of proteins that play a crucial role in facilitating Ca2+-mediated vesicle fusion in neurotransmitter release. They are comprised of two tandem C2 domains, C2A and C2B, which are tethered to the membrane by a single transmembrane helix. Generally, the C2 domain motif is found in a broad range of proteins that bridge the interaction between protein and membrane. A single-point mutation (Y311N) in a highly conserved region of the C2B domain of Drosophila synaptotagmin 1 has the ability to cripple Ca2+-mediated vesicle fusion, despite the protein’s ability to fold correctly and target the membrane. In addition to this mutation, we introduce another point mutation (Y311F) to combat the hindered thermodynamic stability of the domain from the original mutation. In this study, we will show that the AD3 locus appears to regulate a delicate balance between the structural stability of the general C2 fold and its Ca2+ sensitivity by acting as a “brace” through the combination of steric bulk and hydrogen bonding potential. We aim to elucidate the importance of the AD3 locus of Synaptotagmin 1 through the use of X-ray crystallography, isothermal titration calorimetry (ITC), guanidine denaturation coupled to circular dichroism (CD), stopped-flow spectrometry, and co-sedimentation assay.



Enzyme, Metallo-β-lactamase, Structure, Crystallography, Synaptotagmin, AD3, C2 domain