Allosteric effects in catalytic impaired variants of the enzyme cyclophilin A may be explained by changes in nano-microsecond time scale motions

Abstract

Abstract:Robust catalyst design requires clear understanding of the mechanisms by which molecular motions influence catalysis. This work investigates the connection between molecular motions and catalysis for the much debated enzyme Cyclophilin A (CypA) in wild-type (WT) form, and a variant that features a distal serine to threonine (S99T) mutation. Previous biophysical studies have proposed that conformational exchange between a 'major' active and a 'minor' inactive state on millisecond timescales plays a key role in catalysis. Here this hypothesis was addressed with molecular dynamics simulation techniques. The simulations reproduce well NMR-derived measurements of changes of activation barriers for the cis/trans amide group isomerization of a model substrate, and support X-ray crystallography derived evidence for a shift in populations of major and minor active site conformations between the wild-type and S99T mutant forms. Strikingly, exchange between major and minor active site conformations occurs at a rate that is 5 to 6 orders of magnitude faster than previously proposed. Further analyses indicate that the decreased catalytic activity of the S99T mutant is a result of weakened hydrogen bonding interactions between the substrate and several active site residues in the transition state ensemble.Weakened hydrogen bonding interactions in the S99T mutant are due to an overall increase in fast positional fluctuations of active site residues caused by poorer packing of sidechains.Therefore the previously described millisecond time scale coupled motions are not necessary to explain allosteric effects in the S99T Cyclophilin A mutant.

Temperature-jump solution X-ray scattering reveals distinct motions in a dynamic enzyme

“…However, there is a separation of timescales between the NMR results, which indicate ms dynamics in the "core" region, and the T-jump SAXS results here, which indicate μs dynamics. This discrepancy may reflect coupled processes that are related by a population shuffling mechanism 52 and agree with a broad timescale range of side chain dynamics in CypA uncovered by molecular dynamics experiments 53 . In contrast to the S99T mutant, the NH variant lacks the initial fast signal change (k 1 ) in our T-jump experiments, but clearly retains the slow (k 2 ) signal.…”

Temperature-Jump Solution X-ray Scattering Reveals Distinct Motions in a Dynamic Enzyme

“…However, there is a separation of timescales between the NMR results, which indicate ms dynamics in the "core" region, and the T-jump SAXS results here, which indicate µs dynamics. This discrepancy may reflect coupled processes that are related by a population shuffling mechanism (Smith et al, 2015) and agree with a broad timescale range of side chain dynamics in CypA uncovered by molecular dynamics experiments (Wapeesittipan et al, 2018). In contrast to the S99T mutant, the NH variant lacks the initial fast signal change (k1) in our T-jump experiments, but clearly retains the slow (k2) signal.…”

Transient association between proteins elicits alteration of dynamics at sites far away from interfaces

“…Differential dynamics of cyclophilin A likely plays a role in stabilizing HIV-1 capsid assembly The first example we describe is human cyclophilin A (CypA), a peptidyl-prolyl enzyme that catalyzes the isomerization of peptide bonds from trans to cis form and participates in various biological process such as protein folding, apoptosis, and signaling (Nigro et al, 2013). Many studies have reported dynamic allostery associated with CypA, which couples the active-site and distal residues, regulating the enzymatic activity (Agarwal, 2005;Rodriguez-Bussey et al, 2018;Wapeesittipan et al, 2019). In addition to the native functions, CypA plays an important role in (de)stabilization of HIV-1 capsid and hence is often recruited by HIV-1 during its life cycle in host cells (Lu et al, 2015;Thali et al, 1994).Although the structure of CypA and HIV-1 capsid (CA) have been available for a long time (Gamble et al, 1996), it was not clear how CypA modulates the CA stability until recently, when the cryoelectron microscopy (cryo-EM) structure of CypA-CA assembly was solved at 8 Å (Liu et al, 2016).…”