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Mechanistic insight into the role of transition-state stabilization in cyclophilin A.

Hamelberg D, McCammon JA - J. Am. Chem. Soc. (2009)

Bottom Line: Here we have carried out several accelerated molecular dynamics simulations with explicit solvent, and we have provided a detailed description of cis-trans isomerization of the free and cyclophilin A-catalyzed process.The stability of the enzyme-substrate complex is directly correlated with the interaction the substrate makes with a highly conserved arginine residue.Finally, we show that catalysis is achieved through the rotation of the carbonyl oxygen on the N-terminal of the prolyl peptide bond in a predominately unidirectional fashion.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, Georgia State University, Atlanta, Georgia 30302-4098, USA.

ABSTRACT
Peptidyl prolyl cis-trans isomerases (PPIases) are ubiquitous enzymes in biology that catalyze the cis-trans isomerization of the proline imide peptide bond in many cell signaling pathways. The local change of the isomeric state of the prolyl peptide bond acts as a switching mechanism in altering the conformation of proteins. A complete understanding of the mechanism of PPIases is still lacking, and current experimental techniques have not been able to provide a detailed atomistic picture. Here we have carried out several accelerated molecular dynamics simulations with explicit solvent, and we have provided a detailed description of cis-trans isomerization of the free and cyclophilin A-catalyzed process. We show that the catalytic mechanism of cyclophilin is due mainly to the stabilization and preferential binding of the transition state that is achieved by a favorable hydrogen bond interaction with a backbone NH group. We also show that the substrate in the transition state interacts more favorably with the enzyme than the cis isomer, which in turn interacts more favorably than the trans isomer. The stability of the enzyme-substrate complex is directly correlated with the interaction the substrate makes with a highly conserved arginine residue. Finally, we show that catalysis is achieved through the rotation of the carbonyl oxygen on the N-terminal of the prolyl peptide bond in a predominately unidirectional fashion.

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cis−trans Isomerization of the ω bond of the -Gly-Pro- motif of the free substrate (A) and enzyme−substrate bound complex (B) and the corresponding free energy profile after reweighting of the distribution (C) for the free substrate (black) and enzyme−substrate complex (red).
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fig2: cis−trans Isomerization of the ω bond of the -Gly-Pro- motif of the free substrate (A) and enzyme−substrate bound complex (B) and the corresponding free energy profile after reweighting of the distribution (C) for the free substrate (black) and enzyme−substrate complex (red).

Mentions: The catalytic process of cyclophilin does not involve any bond formation or cleavage. Therefore, we have used classical molecular mechanics to study the catalytic mechanism of this enzyme. We observed cis−trans isomerization of the -Gly-Pro- ω bond of the free substrate Ace-His-Ala-Gly-Pro-Ile-Ala-Nme from the accelerated molecular dynamics simulations in explicit water as shown in Figure 2A. The substrate is derived from the loop region of the HIV capsid (Figure 1) that is regulated by cyclophilin A. In addition to the crystal structures of cyclophilin complexed with the whole HIV capsid, cyclophilin has also been cocrystallized with the short piece taken from the full-length capsid.29,30 The free energy profile along the ω bond was estimated as shown in Figure 2C, after reweighting the distribution of the peptide ω angle of -Gly-Pro-. The free energy barriers are similar regardless of the direction of rotation, with a barrier height of about 16.5 ± 1.2 kcal/mol going from the trans to cis isomer and about 12.8 ± 1.5 kcal/mol going from the cis to trans isomer. The similarity of the free energy profile in both directions can be attributed to the lack of the side chain in the preceding glycine residue, thus allowing for almost equal probability of undergoing clockwise and anticlockwise rotations. This result contrasts with our previous study of the -Ser-Pro- motif that has an asymmetric free energy profile, which could be attributed to the side chain of serine hindering the trans-to-cis clockwise rotation.(27)


Mechanistic insight into the role of transition-state stabilization in cyclophilin A.

Hamelberg D, McCammon JA - J. Am. Chem. Soc. (2009)

cis−trans Isomerization of the ω bond of the -Gly-Pro- motif of the free substrate (A) and enzyme−substrate bound complex (B) and the corresponding free energy profile after reweighting of the distribution (C) for the free substrate (black) and enzyme−substrate complex (red).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2651649&req=5

fig2: cis−trans Isomerization of the ω bond of the -Gly-Pro- motif of the free substrate (A) and enzyme−substrate bound complex (B) and the corresponding free energy profile after reweighting of the distribution (C) for the free substrate (black) and enzyme−substrate complex (red).
Mentions: The catalytic process of cyclophilin does not involve any bond formation or cleavage. Therefore, we have used classical molecular mechanics to study the catalytic mechanism of this enzyme. We observed cis−trans isomerization of the -Gly-Pro- ω bond of the free substrate Ace-His-Ala-Gly-Pro-Ile-Ala-Nme from the accelerated molecular dynamics simulations in explicit water as shown in Figure 2A. The substrate is derived from the loop region of the HIV capsid (Figure 1) that is regulated by cyclophilin A. In addition to the crystal structures of cyclophilin complexed with the whole HIV capsid, cyclophilin has also been cocrystallized with the short piece taken from the full-length capsid.29,30 The free energy profile along the ω bond was estimated as shown in Figure 2C, after reweighting the distribution of the peptide ω angle of -Gly-Pro-. The free energy barriers are similar regardless of the direction of rotation, with a barrier height of about 16.5 ± 1.2 kcal/mol going from the trans to cis isomer and about 12.8 ± 1.5 kcal/mol going from the cis to trans isomer. The similarity of the free energy profile in both directions can be attributed to the lack of the side chain in the preceding glycine residue, thus allowing for almost equal probability of undergoing clockwise and anticlockwise rotations. This result contrasts with our previous study of the -Ser-Pro- motif that has an asymmetric free energy profile, which could be attributed to the side chain of serine hindering the trans-to-cis clockwise rotation.(27)

Bottom Line: Here we have carried out several accelerated molecular dynamics simulations with explicit solvent, and we have provided a detailed description of cis-trans isomerization of the free and cyclophilin A-catalyzed process.The stability of the enzyme-substrate complex is directly correlated with the interaction the substrate makes with a highly conserved arginine residue.Finally, we show that catalysis is achieved through the rotation of the carbonyl oxygen on the N-terminal of the prolyl peptide bond in a predominately unidirectional fashion.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, Georgia State University, Atlanta, Georgia 30302-4098, USA.

ABSTRACT
Peptidyl prolyl cis-trans isomerases (PPIases) are ubiquitous enzymes in biology that catalyze the cis-trans isomerization of the proline imide peptide bond in many cell signaling pathways. The local change of the isomeric state of the prolyl peptide bond acts as a switching mechanism in altering the conformation of proteins. A complete understanding of the mechanism of PPIases is still lacking, and current experimental techniques have not been able to provide a detailed atomistic picture. Here we have carried out several accelerated molecular dynamics simulations with explicit solvent, and we have provided a detailed description of cis-trans isomerization of the free and cyclophilin A-catalyzed process. We show that the catalytic mechanism of cyclophilin is due mainly to the stabilization and preferential binding of the transition state that is achieved by a favorable hydrogen bond interaction with a backbone NH group. We also show that the substrate in the transition state interacts more favorably with the enzyme than the cis isomer, which in turn interacts more favorably than the trans isomer. The stability of the enzyme-substrate complex is directly correlated with the interaction the substrate makes with a highly conserved arginine residue. Finally, we show that catalysis is achieved through the rotation of the carbonyl oxygen on the N-terminal of the prolyl peptide bond in a predominately unidirectional fashion.

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