Elucidation of the Mechanisms of Inter-domain Coupling in the Monomeric State of Enzyme I by High-pressure NMR

Abstract

The catalytic cycle of Enzyme I (EI), a phosphotransferase enzyme responsible for converting phosphoenolpyruvate (PEP) into pyruvate, is characterized by a series of local and global conformational rearrangements. This multistep process includes a monomer-to-dimer transition, followed by an open-to-closed rearrangement of the dimeric complex upon PEP binding. In the present study, we investigate the thermodynamics of EI dimerization using a range of high-pressure solution NMR techniques complemented by SAXS experiments. ¹H-¹⁵N TROSY and ¹H-¹³C methyl TROSY NMR spectra combined with ¹⁵N relaxation measurements revealed that a native-like engineered variant of full-length EI fully dissociates into stable monomeric state above 1.5 kbar. Conformational ensembles of EI monomeric state were generated via a recently developed protocol combining coarse-grained molecular simulations with experimental backbone residual dipolar coupling measurements. Analysis of the structural ensembles provided detailed insights into the molecular mechanisms driving formation of the catalytically competent dimeric state, and reveals that each step of EI catalytical cycle is associated with a significant reduction in either inter- or intra-domain conformational entropy. Altogether, this study completes a large body work conducted by our group on EI and establishes a comprehensive structural and dynamical description of the catalytic cycle of this prototypical multidomain, oligomeric enzyme.

Keywords: Enzyme I; NMR relaxation; high-pressure NMR; phosphotransferase system; protein dynamics.

Figure 1.. EI conformational equilibria.

(A) Schematic representation of the synergistic coupling of multiple intra- and interdomain conformational equilibria that regulate EI. Specifically, EI undergoes (i) a monomer–dimer equilibrium (19), (ii) a compact-to-expanded equilibrium within EIC (31), (iii) a g+-to-g− equilibrium within the rotameric state of His 189 in EIN (32), (iv) a state A-to-state B equilibrium within EIN (33), and (v) an open-to-close equilibrium describing a reorientation of EIN relative to EIC (33). EINα, EINα/β, EIC, and PEP are colored blue, light blue, red, and green, respectively. Pressure titration monitored via (B) ¹H-¹⁵N TROSY and (C) ¹H-¹³C methyl TROSY NMR experiments for representative cross-peaks that report on the monomer/dimer equilibrium of wt-EI (left) and 3m-EIC (right). Color of individual cross-peaks refers to the pressure at which the experiments have been conducted, from dark blue (1 bar) to red (2.5 kbar).

Figure 2.. Monitoring dimer-to-monomer transition via NMR and SAXS.

(A-B) Intensity of individual amide ¹H-¹⁵N cross-peaks measured at 1 bar (blue) and 2.5 kbar (red) reported as a function of residue index for (A) wt-EI, and (B) 3m-EI. (C-D) ¹⁵N relaxation rates ratio R₂/R₁ measured at 1 bar (blue) and 2.5 kbar (red) reported as a function of residue index for (C) wt-EI, and (D) 3m-EI. Solid horizontal lines indicate average R₂/R₁ values calculated over each subdomain. Transparent blue, light blue and pink boxes highlight residues from the EINα, EINα/β, and EIC domains, respectively. (E) SAXS pair distance distribution calculated for wt-EI (solid line, blue: 1 bar, red: 2.4 kbar) and 3m-EI (dashed line, blue: 1 bar, red: 2.4 kbar). (F) Collective fit of NMR cross-peak intensities (amide and methyl resonances) via two-state model shows for 3m-EI a sharp transition around 1 kbar between a dimeric state (black circle) and a monomeric state (gray circle). No major transition is observed for wt-EI (black triangle). Datapoints are the normalized averages over the peak intensities of all peaks reporting on the dimer-to-monomer equilibrium in 3m-EI. Error bars were set to one standard deviations. The fits are shown as solid black lines.

Figure 3.. EI monomeric and dimeric ensembles.

(A-B) Overlay of the ensembles generated through cgMD/RDC for (A) EI monomeric state and (B) EI dimeric complex. EINα, EINα/β, and EIC are shown in blue, light blues, and salmon, respectively. The structures for each member of the generated ensembles are shown in Fig. S5. Agreement of the experimental RDC data with the RDC values back-calculated from the generated ensembles for (C) EI monomeric state and (D) EI dimeric complex. Data from EINα, EINα/β, and EIC are colored blue, light blue, and red, respectively.

Figure 4.. Change in conformational entropy along EI catalytic cycle.

Formation of the EI catalytically competent dimeric, closed state is accompanied by multiple steps of reduction in conformational entropy. The first step is characterized by the reduction of interdomain flexibility and EIC intradomain disorder upon formation of an open state dimer. It is followed by a reduction of local disorder in EIC upon binding of PEP. Finally, formation of the catalytically competent closed state is accompanied by a further reduction of interdomain flexibility.