Hydrogen Bonding Network and Aromatic Interactions In the Active ...
Graduate Category: Health Sciences Degree Level: PhD Abstract ID# 413
Hydrogen Bonding Network and Aromatic Interactions In the Active Site of MGL: Mutagenesis, Kinetics and NMR Studies Girija Rajarshi, Sergiy Tyukhtenko, Ioannis Karageorgos, Nikolai Zvonok, and Alexandros Makriyannis Center For Drug Discovery, Northeastern University, Boston, MA 02115 Steady-state kinetic parameters for the hydrolysis of hMGL native substrate 2-AG by wild ABSTRACT RESULTS type and mutant enzymes at pH 7.4 Monoacylglycerol Lipase (MGL) is a serine protease, primarily responsible for the hydrolysis of the neuromodulatory endocannabinoid 2-arachidonyl glycerol (2-AG). Endocannabinoids play important roles in the regulation of appetite, pain, and cancer and therefore, the enzymes targeting their catabolism have become critical pharmacological targets. The amino acid residues that comprise the catalytic triad of MGL are immediately responsible for the mechanism by which 2-AG is hydrolyzed. However, there is a network of hydrogen-bonds and aromatic stacking interactions that extends away from the catalytic site that is equally important to the function of the enzyme. Structurally, MGL contains a lid that regulates access to the catalytic site. We have found that this network of hydrogen-bonds and aromatic interactions is responsible for opening and closing of the lid. Although crystal structures of the open and closed forms of the enzyme exist, these interactions are not obvious. We have drawn our conclusions from the observation of downfield NMR resonances of MGL directly arising from critical hydrogen-bond interactions. These resonances are sensitive to specific amino acid mutations that either eliminate proposed intramolecular hydrogen bonds, and/or modify the geometry around the active site. We correlated perturbations of NMR resonances with modulations of the enzyme turnover rates caused by each individual mutation. Combination of methods allowed us to identify hydrogen-bonding network and aromatic interactions responsible for an information relay from the catalytic site, to the distal protein regions. The knowledge of such pathways provides for new drug design possibilities.
Effect of pH on the equilibrium between open and closed conformations of MGL In solutions Keq≈1 at pH 9.6 (a)
(b)
The downfield side chain NMR resonances of apo hMGL (T=310K) with varying pH demonstrate exchange between two conformers which is slow on the NMR time scale.
Relative populations of the open and closed hMGL conformations calculated from the area measurements of H54 (a) and H269 (b) resonances.
Long-range interactions between W289 and residues within the catalytic triad
Effect of Hydrogen Bonding Network (HBN) modification within the catalytic triad S122/H269/D239 on enzyme kinetics (a) and NMR spectra (b) (a)
(a) Substitution of W289 with Leu leads to the modification of H-bonding pattern (a) and loss of enzyme activity (b) We found that this local perturbation was transmitted as structural changes to distal residues as far as 20 A away.
(b)
INTRODUCTION (b)
Substituted amino acids for identification of functionally important residues and understanding details of hMGL catalytic mechanism
MGL is responsible for inactivating 2-AG, a primary endocannabinoid lipid messenger in brain. MGL inhibitors hold therapeutic potential for managing pain and treating inflammatory, neurodegenerative diseases by enhancing tissue protective 2-AG tone. Here, we employ a combination of sitedirected mutagenesis, enzyme kinetics and NMR to get insights into the mechanism of MGL catalysis.
Open MGL conformation stabilized by cation –π interaction between R57 and H272 (a) and closed conformation (b) with R57 flipped out. Effect of conservative and non-conservative mutations on the population of conformers (c) and catalytic efficiency of enzyme (d) (a)
(c)
Open
(b)
(d)
CONCLUSIONS 1. Observation of hMGL downfield NMR resonances provides insights into functionally important structural rearrangements under a variety of conditions including pH, temperature and substitutions of residues. 2. Hydrogen bonds formed by an amide group of L241 and C242 are responsible for rigid and optimal positioning of D239/H269 dyad and any substitution perturbing this pre-organized geometry results in reduced catalytic efficiency. 3. Aromatic interactions between Y58, H272 and R57 residues contribute significantly to the regulation of conformational equilibrium between open and closed form. 4. Our results highlight the critical role of both hydrogen bonding and aromatic interactions in the mechanism of hMGL catalysis. Understanding the catalytic mechanism may provide for new drug design possibilities.
REFERENCE 1.
1H
NMR downfield resonance assignments based on the mutagenesis study
1H-15N
Labar, G., Bauvois, C., Borel, F., Ferrer, J.-L., Wouters, J., and Lambert, D.M. (2010) Crystal structure of the human monoacylglycerol lipase, a key actor in endocannabinoid signaling. Chembiochem 11, 218-227
ACKNOWLEDGEMENTS
HSQC spectrum covering detection of histidine side
chains Closed
This work was supported by NIH grants DA003801 (A.M.), DA007215 (A.M.) and DA007312 (A.M.) from the National Institute on Drug Abuse.
Hydrogen Bonding Network and Aromatic Interactions In the Active Site of MGL: Mutagenesis, Kinetics and NMR Studies Girija Rajarshi, Sergiy Tyukhtenko, Ioannis Karageorgos, Nikolai Zvonok, and Alexandros Makriyannis Center For Drug Discovery, Northeastern University, Boston, MA 02115 Steady-state kinetic parameters for the hydrolysis of hMGL native substrate 2-AG by wild ABSTRACT RESULTS type and mutant enzymes at pH 7.4 Monoacylglycerol Lipase (MGL) is a serine protease, primarily responsible for the hydrolysis of the neuromodulatory endocannabinoid 2-arachidonyl glycerol (2-AG). Endocannabinoids play important roles in the regulation of appetite, pain, and cancer and therefore, the enzymes targeting their catabolism have become critical pharmacological targets. The amino acid residues that comprise the catalytic triad of MGL are immediately responsible for the mechanism by which 2-AG is hydrolyzed. However, there is a network of hydrogen-bonds and aromatic stacking interactions that extends away from the catalytic site that is equally important to the function of the enzyme. Structurally, MGL contains a lid that regulates access to the catalytic site. We have found that this network of hydrogen-bonds and aromatic interactions is responsible for opening and closing of the lid. Although crystal structures of the open and closed forms of the enzyme exist, these interactions are not obvious. We have drawn our conclusions from the observation of downfield NMR resonances of MGL directly arising from critical hydrogen-bond interactions. These resonances are sensitive to specific amino acid mutations that either eliminate proposed intramolecular hydrogen bonds, and/or modify the geometry around the active site. We correlated perturbations of NMR resonances with modulations of the enzyme turnover rates caused by each individual mutation. Combination of methods allowed us to identify hydrogen-bonding network and aromatic interactions responsible for an information relay from the catalytic site, to the distal protein regions. The knowledge of such pathways provides for new drug design possibilities.
Effect of pH on the equilibrium between open and closed conformations of MGL In solutions Keq≈1 at pH 9.6 (a)
(b)
The downfield side chain NMR resonances of apo hMGL (T=310K) with varying pH demonstrate exchange between two conformers which is slow on the NMR time scale.
Relative populations of the open and closed hMGL conformations calculated from the area measurements of H54 (a) and H269 (b) resonances.
Long-range interactions between W289 and residues within the catalytic triad
Effect of Hydrogen Bonding Network (HBN) modification within the catalytic triad S122/H269/D239 on enzyme kinetics (a) and NMR spectra (b) (a)
(a) Substitution of W289 with Leu leads to the modification of H-bonding pattern (a) and loss of enzyme activity (b) We found that this local perturbation was transmitted as structural changes to distal residues as far as 20 A away.
(b)
INTRODUCTION (b)
Substituted amino acids for identification of functionally important residues and understanding details of hMGL catalytic mechanism
MGL is responsible for inactivating 2-AG, a primary endocannabinoid lipid messenger in brain. MGL inhibitors hold therapeutic potential for managing pain and treating inflammatory, neurodegenerative diseases by enhancing tissue protective 2-AG tone. Here, we employ a combination of sitedirected mutagenesis, enzyme kinetics and NMR to get insights into the mechanism of MGL catalysis.
Open MGL conformation stabilized by cation –π interaction between R57 and H272 (a) and closed conformation (b) with R57 flipped out. Effect of conservative and non-conservative mutations on the population of conformers (c) and catalytic efficiency of enzyme (d) (a)
(c)
Open
(b)
(d)
CONCLUSIONS 1. Observation of hMGL downfield NMR resonances provides insights into functionally important structural rearrangements under a variety of conditions including pH, temperature and substitutions of residues. 2. Hydrogen bonds formed by an amide group of L241 and C242 are responsible for rigid and optimal positioning of D239/H269 dyad and any substitution perturbing this pre-organized geometry results in reduced catalytic efficiency. 3. Aromatic interactions between Y58, H272 and R57 residues contribute significantly to the regulation of conformational equilibrium between open and closed form. 4. Our results highlight the critical role of both hydrogen bonding and aromatic interactions in the mechanism of hMGL catalysis. Understanding the catalytic mechanism may provide for new drug design possibilities.
REFERENCE 1.
1H
NMR downfield resonance assignments based on the mutagenesis study
1H-15N
Labar, G., Bauvois, C., Borel, F., Ferrer, J.-L., Wouters, J., and Lambert, D.M. (2010) Crystal structure of the human monoacylglycerol lipase, a key actor in endocannabinoid signaling. Chembiochem 11, 218-227
ACKNOWLEDGEMENTS
HSQC spectrum covering detection of histidine side
chains Closed
This work was supported by NIH grants DA003801 (A.M.), DA007215 (A.M.) and DA007312 (A.M.) from the National Institute on Drug Abuse.
