Preferential Regulation of Transient Protein-Protein Interaction by the Macromolecular Crowders

Abstract

The environmental condition is a critical regulation factor for protein behavior in solution. Several studies have shown that macromolecular crowders can modulate protein structures, interactions, and functions. Recent publications described the regulation of specific interaction by macromolecular crowders. However, the other category of protein-protein interaction, namely, the transient interaction, is rarely investigated, especially from the perspective of protein structure to study transient interactions between proteins. Here, we used nuclear magnetic resonance and small-angle X-ray/neutron scattering methods to structurally investigate the ensemble of the protein complex in dilute buffer and crowded environments. Histidine phosphocarrier protein (HPr) and the N-terminal domain of enzyme I (EIN) are the important components of the bacterial phosphotransfer system. Our results show that the addition of Ficoll-70 promotes HPr molecules to form the encounter complex with EIN maintained by long-range electrostatic interaction. However, when macromolecular crowder BSA is used, the soft interaction between BSA and HPr perturbs the active site of HPr, driving HPr to form an encounter complex with EIN at the weakly charged interface. Our results indicate that different macromolecular crowders could influence transient EIN-HPr interaction through different mechanisms and provide new insights into protein-protein interaction regulation in native environments.

Figure 1

The averaged relative volume ratios of the amide cross-peaks of T9, G13, L14, V61, and T62 are plotted against the duration of the phosphoryl transfer in the dilute buffer (red dots), 10% Ficoll-70 (blue dots), and 10% BSA (black dots). The error bars show the standard deviations.

Figure 2

Global fitting of amide CSPs of EIN against a series of HPr concentrations. The ¹⁵N HSQC spectra of 200 μM ²H/¹⁵N EIN were recorded in dilute buffer (A), in 10% (w/v) Ficoll-70 (B), and in 10% (w/v) BSA (C), respectively. The K_D values calculated using the CSPs of L19, K30, F65, G110, and G134 are 2.82 ± 3.14 μM in a dilute environment, 2.45 ± 2.52 μM in a Ficoll-70 environment, and 3.02 ± 3.12 μM in a BSA environment.

Figure 3

Intramolecular PREs of E25C paramagnetically labeled HPr. The black, red, and blue cycles show the intensity ratio of the amide signals between the diamagnetic and the paramagnetic samples recorded in dilute buffer, Ficoll-70, and BSA environments, respectively.

Figure 4

Comparison between the PRE data in crowded environments (Y axis) and the PRE data in the dilute environment (X axis). The PRE data acquired with E5C, E25C, and E66C paramagnetically labeled HPr are shown as A/D, B/E, and C/F, respectively. The PREs caused by the specific interaction between EIN and HPr are shown using black cycles, and the PREs arising from the encounter complex are shown using red cycles.

Figure 5

Calculated ensemble structures of the EIN-HPr complex in dilute buffer (A), Ficoll-70 (B), and BSA (C). The specific complex of EIN (green) and HPr (cyan) are shown as cartoon, and the mass center of HPr molecules of the calculated EIN-HPr complex is shown as gray dots. According to the location on the EIN surface, HPr molecules are divided into five clusters marked using dashed cycles or noted as arrows.

Figure 6

SAXS analysis of the EIN-HPr complex in dilute buffer (A) and SANS analysis of this protein complex in the Ficoll-70 environment (B) and BSA environment (C). The top panels show the experimental scattering profiles (red dots) and the theoretical scattering profiles of the calculated EIN-HPr complexes in different environments (gray lines). The lower panels display the experimental paired-distance distribution function (PDDF) plots (red lines) and the theoretical PDDF plots of the calculated ensemble EIN-HPr structures in different environments (gray lines).