MARCO TEÓRICO:

A notable characteristic of how cyclophilin speeds up prolyl isomerization is by limiting the con- formational freedom of the substrate. The active sites of cyclophilin PPIase domains make use of several intermolecular hydrogen bonds with the substrate that form along the substrate peptide backbone. It is well known that for CypA, R55 and N102 form hydrogen bonds with the substrate that are critical to

substrate turnover.(13, 33, 42, 70, 198, 199) In addition to these, Q63 and W121 also participate in in- termolecular hydrogen bonds, which also contribute to the binding affinity of the substrate to CypA. (35, 197, 199)This hydrogen bonding pattern is also fully conserved in the active site of CypB. The side chain amine of Q63/65 forms a hydrogen bond with the carbonyl oxygen of the residue preceding the X-Pro motif, near the N-terminus. N102/104 forms two hydrogen bonds: (1) from the backbone carbonyl oxy- gen of N102/104 to the backbone amine hydrogen of the X residue in the X-Pro motif and (2) from the backbone amine hydrogen of N102/104 to the backbone carbonyl oxygen of the X residue in the X-Pro motif. The guanidinium group of R55/57 forms a bifurcated hydrogen bond with the carbonyl oxygen of the substrate proline. Lastly, the side chain amine group on the imidazole ring of W121/123 forms a hy- drogen bond with the carbonyl oxygen of the residue following the X-Pro motif, near the C-terminus. We have measured the probability of each hydrogen bond distance for CypA and CypB complexed to each of the three configurations of substrate (Fig. 28). All of these hydrogen bonds are well formed when com- plexed to the transition state, which indicates one component of how both CypA and CypB proceed with transition state stabilization. Also, hydrogen bonds tend to be better formed for the cis complex of cy- clophilin as compared to the trans complex of cyclophilin, which can also contribute to cyclophilin hav- ing better affinity for the cis substrate over the trans. (13, 31, 67)

When the substrate is not bound to the enzyme, it can populate two transition states (ω = +90˚) as it converts between the cis (ω = 0˚) and trans (ω = +180˚) conformers. Once the catalytic step pro- ceeds, the cleft of the enzyme prevents the ω-bond from rotating freely and only positive ω-bond angle values are sampled. We have used accelerated MD to sample the phi and psi torsional backbone angles of the X-Pro substrate motif over five average simulations (Fig. 29). Accelerated MD allows for the en- zyme-substrate complex to undergo cis/trans interconversion by making the cis and trans energetic wells more shallow, thereby facilitating barrier transitions. When the substrate is not bound to the en

Figure 28

Figure 28. Intermolecular hydrogen bonding pattern for CypA and CypB trans (red), transition state (green), and cis (blue) enzyme-substrate complexes.

These include the hydrogen bonds formed by enzyme residues Arg 55/57, Gln 63/65, Asn 102/104, and Trp 121/123, for CypA/CypB respectively, with the participating atoms labeled in parentheses.

Figure 29

Figure 29. Ramachandran space of substrate residues Ala and Pro sampled for the (A) free substrate, (B) CypA complex, and (C) CypB complex, generated using accelerated MD.

Accelerated MD allows each enzyme complex to sample the trans, transition state, and cis conformations of the substrate. Ala and Pro are consecutive in sequence, which make up the X-Pro motif recognized by cyclophilin. En- ergy levels are Boltzmann-weighted with a new contour line every 4.5 kcal/mol.

zyme, it can sample both α-helical and β-sheet regions of Ramachandran space (Fig. 29A). For alanine of the Ala-Pro motif, this includes both the left-handed and right-handed α-helical regions. Proline also samples the β-sheet and right-handed α-helix conformations while unbound. However, when the sub- strate is bound to cyclophilin, the β-sheet region is preferred. For the CypA complex (Fig. 29B), the α- helical region can be sampled at higher energies, similar to the energies of the barrier height of the reac- tion. (41) In the CypB complex (Fig. 29C), the β-sheet region is more strictly sampled. It has previously been suggested that the mechanism of all immunophilins involve specificity for the β-sheet substrate conformation. (200)

The volume of the proline binding pocket was measured in both CypA and CypB for the apo en- zyme and the enzyme-substrate complex with the substrate in the trans, cis, and transition state config- urations using a previously established algorithm known as pocket volume measurer (POVME). Per this calculation, the volume of the proline binding pocket rarely exceeds 100 Å3 (Fig. D11). This is a reasona- ble result since the volume of the proline ring is approximately 50 Å3. Volumes less than 10 Å3 occur when the cavity becomes shallow. In apo cyclophilin, the proline binding pocket becomes shallow when the stochastic dynamic motions of the residues cause the pocket to be ill-formed. Occasionally, the phe- nyl ring of F113/115, which forms the base of the pocket, flips outward approximately 90˚ to fill the pocket. Other atoms of the active site residues occasionally drift8 toward the center of the proline bind- ing pocket in the apo enzymes as well, such as the methyl group at the terminal end of M61/63 or the imidazole side chain of H126/128. The concept of a shallow pocket can easily be conceived for the apo enzymes, but not as easily for the bound trajectories which can also sample low pocket volumes. The pocket volumes become shallow for the bound trajectories when the enzyme and substrate form strong polar contacts in solution, but the nonpolar contacts are weak and near dissolution. Moreover, the pro- line ring can also lie roughly orthogonal to its classic orientation in the pocket where the rings of proline and H126/128 lie on top of each other, which is also coupled with shallow pocket volumes. This is par-

ticularly true in the cis complex of cyclophilin when the substrate analogue takes on a curved geometry compared to the linear conformation of the trans substrate. The pocket tends to sample volumes larger than expected when certain distortions cause the pocket not to be well formed, such as the side chains of R55/57 or F60/62 being oriented away from the pocket.