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X-ray crystallographic studies of two bacterial antibiotic resistance enzymes: Aminoglycoside Phosphotransferase (2’’)-Ic and GES-1 β-lactamase Laura Byrnes Rensselaer Polytechnic Institute Stanford Linear Accelerator Center August 15th, 2007 Background Antibiotic resistance has been around for eons Widespread use, poor patient compliance have pushed along resistance Structures of antibiotic resistance enzymes contain valuable information for getting around resistance May 23, 2017 Laura Byrnes SULI 2 Why bother? Reaction mechanisms much clearer Hydrogen bonds Hydrogen atoms (at ultra-high resolution) Illustrate and characterize proteinsubstrate interactions Structure-based drug discovery May 23, 2017 Laura Byrnes SULI 3 Goals Characterize interactions between GES-1 and inhibitor complex, imipenem Solve the structure of APH(2’’)-Ic May 23, 2017 Laura Byrnes SULI 4 Enzymatic Action GES-1 a β-lactamase Penicillin APH(2’’)-Ic May 23, 2017 an aminoglycoside phosphotransferase Laura Byrnes SULI Gentamicin 5 What do we need? X-rays! May 23, 2017 Laura Byrnes SULI 6 Why crystals and X-rays? Molecules arranged in a regular, repeating pattern in crystals Electrons in the molecules bend an incident X-ray beam into thousands of diffracted beams Multiple copies of the same molecule in the same orientation amplify the diffraction peaks The diffraction pattern contains all of the information necessary to determine the structure! May 23, 2017 Laura Byrnes SULI 7 Different crystallization methods Vapor Diffusion Hanging drop Sitting drop Dialysis We used the hanging drop method to grow the APH(2’’)-Ic crystals used to collect data May 23, 2017 Laura Byrnes SULI 8 The Protein Crystal Cookbook You wish! What can we do… ? May 23, 2017 Laura Byrnes SULI 9 What’s Next? Vary conditions May 23, 2017 Salt/metal ions (trace) Precipitant (e.g. PEG -- polyethylene glycol, alcohols, high salt concentration) pH Buffer types Temperature Protein concentration Organic compounds (trace) Laura Byrnes SULI 10 Obstacles May 23, 2017 Protein showers Salty! Some proteins just won’t crystallize Unique problems; APH(2’’)-Ic crystals grow as flat sheets Laura Byrnes SULI 11 Tools of the Trade May 23, 2017 Laura Byrnes SULI 12 So you think you have crystals? Crystals mounted on a nylon fiber loop (~200μm) Keep it cold! Cryoprotectant May 23, 2017 Laura Byrnes SULI Our crystals were grown in 20% PEG 13 Leave it to the robot… May 23, 2017 Laura Byrnes SULI 14 X-ray Diffraction, in a Nutshell Bragg’s Law: May 23, 2017 n 2 * d sin Laura Byrnes SULI 15 The Reciprocal Lattice 1 1 a* d (100) a 1 1 b* d (010) b 1 1 c* d (001) c May 23, 2017 Laura Byrnes SULI 16 Ewald Sphere May 23, 2017 Laura Byrnes SULI 17 What does it all add up to? Water lines Diffraction pattern May 23, 2017 Laura Byrnes SULI 18 Molecular Replacement Use a related protein as a structural model We used APH(2’’)-Ib Doesn’t always work out Other options: Multiple isomorphous replacement Soak crystals in heavy metal solutions Modify substrates or cofactors with heavy atoms Collect X-ray Absorption edge, collect diffraction data at that wavelength May 23, 2017 Laura Byrnes SULI 19 Fitting the Data Specially designed programs (e.g. COOT) and hardware are used to refine the structure in 3D! May 23, 2017 Laura Byrnes SULI 20 The Beautiful Result Blue: 2Fobs-Fcalc Green: (+) Fobs-Fcalc Red: (-) Fobs-Fcalc May 23, 2017 Laura Byrnes SULI Something missing Atoms in wrong place Model outlined inside density map 21 Conclusions: GES-1 GES-1 crystals bound imipenem showed ~65% occupancy for the inhibitor in the active site Some interactions observed: May 23, 2017 Covalent bonding between inhibitor and key active site residue Multiple hydrogen bonding partners Salt bridge Laura Byrnes SULI 22 GES-1 bound Imipenem May 23, 2017 Laura Byrnes SULI 23 Conclusions: APH(2’’)-1c The APH(2’’)-Ib structure, with ~25% sequence identity to APH(2’’)-Ic, not an ideal model Future studies May 23, 2017 Try soaking with 8-bromo-ATP Crystallize other mutants and WT Co-crystallize with substrates Laura Byrnes SULI 24 Acknowledgments Clyde Smith, Apurva Mehta, Stanford Linear Accelerator Center Stanford Linear Accelerator Center SULI Program coordinators at SLAC Office of Science, Department of Energy May 23, 2017 Laura Byrnes SULI 25