Crystallographic Proof for an Extended Hydrogen-Bonding Network in Small Prolyl Isomerases

Abstract

Parvulins compose a family of small peptidyl-prolyl isomerases (PPIases) involved in protein folding and protein quality control. A number of amino acids in the catalytic cavity are highly conserved, but their precise role within the catalytic mechanism is unknown. The 0.8 Å crystal structure of the prolyl isomerase domain of parvulin Par14 shows the electron density of hydrogen atoms between the D74, H42, H123, and T118 side chains. This threonine residue has previously not been associated with catalysis, but a corresponding T152A mutant of Pin1 shows a dramatic reduction of catalytic activity without compromising protein stability. The observed catalytic tetrad is strikingly conserved in Pin1- and parvulin-type proteins and hence constitutes a common feature of small peptidyl prolyl isomerases.

Allosteric Breakage of the Hydrogen Bond within the Dual-Histidine Motif in the Active Site of Human Pin1 PPIase

“…17 The results are consistent with the tautomers of the corresponding histidines identified in the high-resolution crystal structure of Par14, a PPIase homologous to Pin1. 16 Our NMR results showed that the hydrogen bond network exists as in the Par14 crystal structure; however, NMR could not directly observe the sample if C113 was in the thiolate or thiol form. 17 As described in the Results, the unusual 1 H− 15 N and 1 H− 13 C HSQC spectra for the imidazole rings in the mutants could represent chemical exchange between the different protonation states of the H59 imidazole ring ( Figure 9B,C).…”

“…The histidines in the dual-histidine motif engage in the hydrogen bond network, in which H59 and H157 are in ε-and δ-tautomers, respectively. 16,17 In our previous work, we showed that impairing the hydrogen bond between H59 and H157 destabilized the structure. 17 The change in the protonation states of the histidines, which is theoretically predicted to occur according to the state of C113, 20 should have a significant impact on the structure and dynamics of these residues.…”

“…Both mutants showed similar Δδ profiles, demonstrating the obvious spectral changes for residues in the β1 strand, β3−β4 loop, and β4 strand, which includes residues engaged in the C113−H59−H157−T152 hydrogen bond network (Figure 2, right). 16 This observation shows that structural perturbation by the C113 mutation, as evidenced by the spectral changes, propagates through the hydrogen bond network. Thus, the C113 mutation disturbs all residues in the network, irrespective of their spatial distance from the mutation site in the α2−α3 loop.…”

“…16 The residues in the hydrogen bond network are structurally superpositioned to C113, H59, H157, and T152 (listed in the connecting order in the hydrogen bonding network) in Pin1 (in Scheme 1, the residue numbers in Pin1 are in parentheses). 16 Par14 has aspartate instead of cysteine at position 113. 16 A C113D mutant Pin1 PPIase, mimicking the Par14 active site, was shown to have a reduced rate of isomerization relative to that of the wild type.…”

“…16 Par14 has aspartate instead of cysteine at position 113. 16 A C113D mutant Pin1 PPIase, mimicking the Par14 active site, was shown to have a reduced rate of isomerization relative to that of the wild type. 17 Nuclear magnetic resonance (NMR) analysis showed that D113 formed a tighter hydrogen bond with H59 via the carboxylate oxygen in D113 while the hydrogen bond between H59 and H157 was destabilized.…”