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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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Interaction of cyclophilin A with full-length HIV capsid. Cyclophilin catalyzes cis−trans isomerization of a -Gly-Pro- motif on the exposed loop structure of the HIV capsid (left). The binding site of cyclophilin (right) has a very hydrophobic pocket with the nonpolar residues shown as white, an arginine residue at the entrance of the pocket shown as blue surface, a histidine shown as cyan surface, and two asparagines shown as green. The side-chain ring of the proline residue fits very nicely into the hydrophobic pocket of the binding site.
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fig1: Interaction of cyclophilin A with full-length HIV capsid. Cyclophilin catalyzes cis−trans isomerization of a -Gly-Pro- motif on the exposed loop structure of the HIV capsid (left). The binding site of cyclophilin (right) has a very hydrophobic pocket with the nonpolar residues shown as white, an arginine residue at the entrance of the pocket shown as blue surface, a histidine shown as cyan surface, and two asparagines shown as green. The side-chain ring of the proline residue fits very nicely into the hydrophobic pocket of the binding site.

Mentions: Therefore, PPIases are one of the rare enzymes in biology that carry out their function in the absence of any actual bond formation and cleavage. How do the PPIases then achieve this remarkable speedup of more than 5 orders of magnitude? Several hypotheses have been proposed over the years that include the effect of substrate desolvation and the idea of preferential transition-state binding in the active site.(1) It was shown that the effect of removing the substrate from aqueous solution to the hydrophobic pocket of the PPIases, as shown in Figure 1, could result in a speedup of cis−trans isomerization. This effect is partly due to the weakening of the pseudo-double-bond character of C−N in nonaqueous environment, resulting in a small reduction of the transition barrier height by about 1.3 kcal/mol.(17) Similarly, a speedup of up to about 20-fold of the rate of cis−trans isomerization was later observed in micelles that also resulted in a small decrease in the barrier height by about 1.8 kcal/mol, assuming that the speedup is purely due to barrier reduction.(18) Likewise, we have previously observed a speedup in the rate of cis−trans isomerization using molecular dynamics simulations in the absence of explicit water molecules around the prolyl peptide bond due to a reduction in the effective roughness on the energy landscape that results in a change in the kinetic prefactor.(19) The kinetic prefactor depends on the diffusion coefficient on the landscape, which in turn depends on the effective roughness of the landscape. Also, the speedup can simply be a consequence of the change in the frictional drag experienced by the substrate in moving from an aqueous environment to the dry hydrophobic cavity of the binding site of the PPIases. However, these prefactor effects and slight reduction in barrier height due to the lack of aqueous medium could not account for the more than 5 orders of magnitude increase in the observed rate of cis−trans isomerization due to PPIases. Also, it has previously been shown that an increase in the rate of cis−trans isomerization of the ω angle can be achieved by constraining the peptide bond in a loop conformation,20,21 but the extent of the role of this phenomenon in the catalysis of cis−trans isomerization of the peptide bond by PPIases is not known.


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

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

Interaction of cyclophilin A with full-length HIV capsid. Cyclophilin catalyzes cis−trans isomerization of a -Gly-Pro- motif on the exposed loop structure of the HIV capsid (left). The binding site of cyclophilin (right) has a very hydrophobic pocket with the nonpolar residues shown as white, an arginine residue at the entrance of the pocket shown as blue surface, a histidine shown as cyan surface, and two asparagines shown as green. The side-chain ring of the proline residue fits very nicely into the hydrophobic pocket of the binding site.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig1: Interaction of cyclophilin A with full-length HIV capsid. Cyclophilin catalyzes cis−trans isomerization of a -Gly-Pro- motif on the exposed loop structure of the HIV capsid (left). The binding site of cyclophilin (right) has a very hydrophobic pocket with the nonpolar residues shown as white, an arginine residue at the entrance of the pocket shown as blue surface, a histidine shown as cyan surface, and two asparagines shown as green. The side-chain ring of the proline residue fits very nicely into the hydrophobic pocket of the binding site.
Mentions: Therefore, PPIases are one of the rare enzymes in biology that carry out their function in the absence of any actual bond formation and cleavage. How do the PPIases then achieve this remarkable speedup of more than 5 orders of magnitude? Several hypotheses have been proposed over the years that include the effect of substrate desolvation and the idea of preferential transition-state binding in the active site.(1) It was shown that the effect of removing the substrate from aqueous solution to the hydrophobic pocket of the PPIases, as shown in Figure 1, could result in a speedup of cis−trans isomerization. This effect is partly due to the weakening of the pseudo-double-bond character of C−N in nonaqueous environment, resulting in a small reduction of the transition barrier height by about 1.3 kcal/mol.(17) Similarly, a speedup of up to about 20-fold of the rate of cis−trans isomerization was later observed in micelles that also resulted in a small decrease in the barrier height by about 1.8 kcal/mol, assuming that the speedup is purely due to barrier reduction.(18) Likewise, we have previously observed a speedup in the rate of cis−trans isomerization using molecular dynamics simulations in the absence of explicit water molecules around the prolyl peptide bond due to a reduction in the effective roughness on the energy landscape that results in a change in the kinetic prefactor.(19) The kinetic prefactor depends on the diffusion coefficient on the landscape, which in turn depends on the effective roughness of the landscape. Also, the speedup can simply be a consequence of the change in the frictional drag experienced by the substrate in moving from an aqueous environment to the dry hydrophobic cavity of the binding site of the PPIases. However, these prefactor effects and slight reduction in barrier height due to the lack of aqueous medium could not account for the more than 5 orders of magnitude increase in the observed rate of cis−trans isomerization due to PPIases. Also, it has previously been shown that an increase in the rate of cis−trans isomerization of the ω angle can be achieved by constraining the peptide bond in a loop conformation,20,21 but the extent of the role of this phenomenon in the catalysis of cis−trans isomerization of the peptide bond by PPIases is not known.

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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