Transcript Crystallographic studies of two bacterial antibiotic

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
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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
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Why bother?
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Reaction mechanisms much clearer
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Hydrogen bonds
Hydrogen atoms (at ultra-high resolution)
Illustrate and characterize proteinsubstrate interactions
Structure-based drug discovery
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Goals
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Characterize interactions between
GES-1 and inhibitor complex,
imipenem
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Solve the structure of APH(2’’)-Ic
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Enzymatic Action
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GES-1
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a β-lactamase
Penicillin
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APH(2’’)-Ic
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an aminoglycoside
phosphotransferase
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Gentamicin
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What do we need?
X-rays!
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Why crystals and X-rays?
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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!
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Different crystallization
methods
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Vapor Diffusion
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Hanging drop
Sitting drop
Dialysis
We used the hanging drop method to grow
the APH(2’’)-Ic crystals used to collect data
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The Protein Crystal Cookbook
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You wish!
What can we do… ?
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What’s Next?
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Vary conditions
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Salt/metal ions (trace)
Precipitant (e.g. PEG -- polyethylene glycol,
alcohols, high salt concentration)
pH
Buffer types
Temperature
Protein concentration
Organic compounds (trace)
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Obstacles
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Protein showers
Salty!
Some proteins just won’t crystallize
Unique problems; APH(2’’)-Ic crystals
grow as flat sheets
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Tools of the Trade
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So you think you have
crystals?
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Crystals mounted
on a nylon fiber
loop (~200μm)
Keep it cold!
Cryoprotectant
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Our crystals were
grown in 20% PEG
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Leave it to the robot…
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X-ray Diffraction, in a Nutshell
Bragg’s Law:
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n  2 * d sin 
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The Reciprocal Lattice
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1
a* 
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d (100) a
1
1
b* 
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d (010) b
1
1
c* 

d (001) c
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Ewald Sphere
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What does it all add up to?
Water lines
Diffraction
pattern
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Molecular Replacement
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Use a related protein as a structural model
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We used APH(2’’)-Ib
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Doesn’t always work out
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Other options:
Multiple isomorphous replacement
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Soak crystals in heavy metal solutions
Modify substrates or cofactors with heavy atoms
Collect X-ray Absorption edge, collect diffraction data at that
wavelength
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Fitting the Data
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Specially designed
programs (e.g.
COOT) and
hardware are used
to refine the
structure in 3D!
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The Beautiful Result
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Blue: 2Fobs-Fcalc
Green: (+) Fobs-Fcalc
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Red: (-) Fobs-Fcalc
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Something missing
Atoms in wrong place
Model outlined
inside density map
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Conclusions: GES-1
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GES-1 crystals bound imipenem
showed ~65% occupancy for the
inhibitor in the active site
Some interactions observed:
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Covalent bonding between inhibitor and
key active site residue
Multiple hydrogen bonding partners
Salt bridge
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GES-1 bound Imipenem
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Conclusions: APH(2’’)-1c
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The APH(2’’)-Ib structure, with ~25%
sequence identity to APH(2’’)-Ic, not an
ideal model
Future studies
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Try soaking with 8-bromo-ATP
Crystallize other mutants and WT
Co-crystallize with substrates
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Acknowledgments
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Clyde Smith,
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Apurva Mehta,
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Stanford Linear Accelerator Center
Stanford Linear Accelerator Center
SULI Program coordinators at SLAC
Office of Science, Department of
Energy
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