Dynamic Allostery Modulates Catalytic Activity by Modifying the Hydrogen Bonding Network in the Catalytic Site of Human Pin1

Abstract

Allosteric communication among domains in modular proteins consisting of flexibly linked domains with complimentary roles remains poorly understood. To understand how complementary domains communicate, we have studied human Pin1, a representative modular protein with two domains mutually tethered by a flexible linker: a WW domain for substrate recognition and a peptidyl-prolyl isomerase (PPIase) domain. Previous studies of Pin1 showed that physical contact between the domains causes dynamic allostery by reducing conformation dynamics in the catalytic domain, which compensates for the entropy costs of substrate binding to the catalytic site and thus increases catalytic activity. In this study, the S138A mutant PPIase domain, a mutation that mimics the structural impact of the interdomain contact, was demonstrated to display dynamic allostery by rigidification of the α2-α3 loop that harbors the key catalytic residue C113. The reduced dynamics of the α2-α3 loop stabilizes the C113–H59 hydrogen bond in the hydrogen-bonding network of the catalytic site. The stabilized hydrogen bond between C113 and H59 retards initiation of isomerization, which explains the reduced isomerization rate by ~20% caused by the S138A mutation. These results provide new insight into the interdomain allosteric communication of Pin1.

Activity and Affinity of Pin1 Variants

“…The S138E-PPIase construct had low expression in E. coli and was extremely unstable at room temperature, therefore it was unsuitable for NMR studies. These challenges were not present for S138A-PPIase mutant [46]. The S138A mutant caused minimal structural changes, but significantly altered the dynamics in the catalytic site.…”

“…C113A and C113S mutations were also tested. Interestingly, while a serine is the best mimetic for cysteine for having a similar size side-chain and being a hydrogen bond donor, no activity was actually detected of this mutant unlike aspartate and alanine [46]. This C113 study was performed on the isolated PPIase domain, so there is currently no evidence on if or how this mutation impacts the WW domain.…”

Activity and Affinity of Pin1 Variants

“…These challenges were not present for S138A-PPIase mutant [33]. The S138A mutant Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 6 November 2019 doi:10.20944/preprints201911.0050.v1 caused minimal structural changes, but significantly altered the dynamics in the catalytic site.…”

“…C113A and C113S mutations were also tested. Interestingly, while a serine is the best mimetic for cysteine for having a similar size side-chain and being a hydrogen bond donor, no activity was actually detected of this mutant unlike aspartate and alanine [33]. This C113 study was performed on the isolated PPIase domain, so there is currently no evidence on if or how this mutation impacts the WW domain.…”

“…Using 15 N-HSQC, NOE, and H/D exchange spectra, it is apparent that the catalytic site is slightly reorganized and the hydrogen-bonding network has been altered due to the S138A mutation. The S138A mutation in the isolated PPIase may be mimicking the impact of the presence of the WW domain [33]. This would help it explain why it has lower catalytic activity than the WT isolated PPIase.…”