Differential Large-Amplitude Breathing Motions in the Interface of FKBP12–Drug Complexes

Abstract

The tight complexes FKBP12 forms with immunosuppressive drugs, such as FK506 and rapamycin, are frequently used as models for developing approaches to structure-based drug design. Although the interfaces between FKBP12 and these ligands are well-defined structurally and are almost identical in the X-ray crystallographic structures of various complexes, our nuclear magnetic resonance studies have revealed the existence of substantial large-amplitude motions in the FKBP12-ligand interfaces that depend on the nature of the ligand. We have monitored these motions by measuring the rates of Tyr and Phe aromatic ring flips, and hydroxyl proton exchange for residues clustered within the FKBP12-ligand interface. The results show that the rates of hydroxyl proton exchange and ring flipping for Tyr26 are much slower in the FK506 complex than in the rapamycin complex, whereas the rates of ring flipping for Phe48 and Phe99 are significantly faster in the FK506 complex than in the rapamycin complex. The apparent rate differences observed for the interfacial aromatic residues in the two complexes confirm that these dynamic processes occur without ligand dissociation. We tentatively attribute the differential interface dynamics for these complexes to a single hydrogen bond between the ζ-hydrogen of Phe46 and the C32 carbonyl oxygen of rapamycin, which is not present in the KF506 complex. This newly identified Phe46 ζ-hydrogen bond in the rapamycin complex imposes motional restriction on the surrounding hydrophobic cluster and subsequently regulates the dynamics within the protein-ligand interface. Such information concerning large-amplitude dynamics at drug-target interfaces has the potential to provide novel clues for drug design.

Slow ring flips in aromatic cluster of GB1 studied by aromatic 13C relaxation dispersion methods

“…Similar behaviour has been observed in BPTI and rapamycin-or FK506-bound FKBP12 (SI Fig. 11) (Wagner et al 1987;Weininger et al 2014b;Yang et al 2015). There are also examples of slow ring flips where both positions (δ and ε) do not display shift differences in 1 H and 13 C and thus are not accessible by relaxation dispersion methods (Weininger et al 2013).…”

“…The activation enthalpies for F52 (88 ± 11 kJ mol −1 ) and Y3 (87 ± 14 kJ mol −1 ) are virtually the same, no activation enthalpy could be determined for F30, but it is rather safe to conclude that it is not larger, whereas Y45 (129 ± 29 kJ mol −1 ) might also display the same activation enthalpy (within margin of error) or a slightly higher value. Activation entropies (126 ± 46 J mol −1 K −1 , 137 ± 38 J mol −1 K −1 and 275 ± 102 J mol −1 K −1 ) are somewhat higher than previously reported ones, which range between 16 and 96 J mol −1 K −1 (Hattori et al 2004;Weininger et al 2014b;Yang et al 2015). This reflects a higher loss in order in the transition state compared to the ground state of the aromatic cluster of GB1.…”

“…Additionally, many Phe and Tyr residues undergo frequent 180° rotations ("ring flips") of the χ 2 dihedral angle (around the imaginary C β -C γ -C ζ axis) (Campbell et al 1975;Hattori et al 2004;Hull and Sykes 1975;Wagner et al 1976Wagner et al , 1987Weininger et al 2013Weininger et al , 2014bWüthrich and Wagner 1975;Yang et al 2015). The requirement for a ring flip to occur is that the surrounding undergoes concerted "breathing" motions with relatively large activation volumes (Hattori et al 2004;Li et al 1999;Wagner 1980).…”

“…Experimental measurements of ring flips, however, have been limited so far to a handful of cases since their discovery in the 1970s (Campbell et al 1975;Hull and Sykes 1975;Wagner et al 1976). Recently, new cases have been reported (Weininger et al 2014b;Yang et al 2015) enabled by methodological advances in site-selective 13 C labeling (Lundström et al 2007;Teilum et al 2006), aromatic 13 C relaxation dispersion experiments (Weininger et al 2012(Weininger et al , 2014a and the understanding of strong 1 H-1 H couplings (Weininger et al 2013). Additionally, ring flips can be studied by long scale MD simulations (Shaw et al 2010) and extremely fast ring flips are shown to affect order parameters (Kasinath et al 2015).…”

On the role, ecology, phylogeny, and structure of dual-family immunophilins

“…I presume that the majority of DFIs are functional PPIases and may also have the expected chaperone activity. As a corollary, they may be explored as therapeutic targets (Schiene-Fischer 2015;Ünal and Steinert 2014), perhaps through the designing of selective bispecific drugs (Dunyak et al 2015;Yang et al 2015a).…”