Probing protein quinary interactions by in-cell nuclear magnetic resonance spectroscopy

Abstract

Historically introduced by McConkey to explain the slow mutation rate of highly abundant proteins, weak protein (quinary) interactions are an emergent property of living cells. The protein complexes that result from quinary interactions are transient and thus difficult to study biochemically in vitro. Cross-correlated relaxation-induced polarization transfer-based in-cell nuclear magnetic resonance allows the characterization of protein quinary interactions with atomic resolution inside live prokaryotic and eukaryotic cells. We show that RNAs are an important component of protein quinary interactions. Protein quinary interactions are unique to the target protein and may affect physicochemical properties, protein activity, and interactions with drugs.

Figure 1

Quinary interactions of Trx in E. coli occlude the active site. (a) Overlay of the in-cell ¹H–¹⁵N CRINEPT–HMQC–TROSY spectrum of REDPRO-labeled Trx (blue) and that of the cellular lysate (red). The intensities of C33, C36, I39, and G98 peaks, residues involved in quinary interactions, are broadened out. The insets show overlays of the boxed regions of the in-cell spectrum (blue) and the corresponding regions of the ¹H–¹⁵N CRINEPT–HMQC–TROSY spectrum of lysate (red) and the ¹H–¹⁵N HSQC spectrum of purified Trx in 10 mM potassium phosphate buffer (pH 6.5) (black). (b) Residues involved in the quinary interactions (red) are mapped onto the molecular surface of Trx (PDB entry 1X0B); active site residues, C33 and G34, are shown in bold. (c) The relative volumes of the G52, G66, and G85 peaks in the in-cell ¹H–¹⁵N CRINEPT–HMQC–TROSY spectra of Trx are plotted vs CRINEPT transfer delay time. An endogenous tryptophan indole amide peak in the in-cell spectra is used as a reference. The optimal CRINEPT transfer delay for Trx is ~1.3 ms, which corresponds to an apparent molecular mass of ~1.1 MDa.

Figure 2

Multiple bound states of Trx inside bacterial cells. (a) Overlay of peaks from a ¹H–¹⁵N CRINEPT–HMQC–TROSY spectrum of REDPRO-labeled Trx in E. coli (blue) with peaks from the ¹H–¹⁵N HSQC spectrum of purified REDPRO-labeled Trx in 10 mM potassium phosphate buffer (pH 6.5) (black). G52, G66, and G85 exhibit broad in-cell peaks that are characteristic of multiple conformations of Trx in fast exchange on the NMR time scale. This implies that the quinary interactions are inherently transient and dynamic. (b) Bar plot showing the relative changes in in-cell ¹H–¹⁵N CRINEPT–HMQC–TROSY peak intensities of Trx residues due to quinary interactions. Residues E31, W32, C33, C36, M38, A40, A68, and Q99, annotated with asterisks, are also affected in total RNA-bound Trx. The horizontal line differentiates residues whose NMR peaks undergo significant broadening. (c) Overlays of selected regions of the in-cell spectrum (blue) and ¹H–¹⁵N HSQC spectrum of purified thioredoxin in 10 mM potassium phosphate buffer (pH 6.5) (black) and 10 mM deuterated sodium acetate buffer (pH 4.7) (red). The chemical shifts of thioredoxin peaks in the in-cell NMR spectrum are shifted downfield by ~0.1 ppm in the ¹H dimension compared to the in vitro spectrum of thioredoxin at pH 6.5. Specifically, the chemical shift of A68 resembles that observed at pH 4.7, whereas the indole amide of W29 resonates between pH 4.7 and 6.5.

Figure 3

Cisplatin perturbs the quinary interactions of Trx. (a) Overlay of the ¹H–¹⁵N CRINEPT–HMQC–TROSY spectra of REDPRO-labeled Trx in E. coli treated with (red) and without (blue) 10 μM cisplatin. The insets show peaks from active site residues F28, W32, G72, and L95, which are broadened (arrows) because of changes in the in-cell interaction of Trx induced by cisplatin. In addition, the indole amide of W32 is broadened. (b) The residues affected by quinary interactions (red) and cisplatin (cyan) are mapped onto the molecular surface of Trx (PDB entry 1X0B).

Figure 4

In-cell proteins exhibit megadalton apparent molecular masses. The curve, calculated using eq 1, shows the dependence of the apparent molecular mass at 700 MHz on the CRINEPT transfer delay that provides the optimal transfer efficiency, T_opt. To calibrate T_opt, 100 μM REDPRO-labeled Trx was dissolved in NMR buffer with 30, 65, 75, and 85% (w/w) d₅-glycerol and corresponding viscosities of 4, 34, 92, and 343 cP, respectively. T_opt was experimentally determined for each sample at 5 °C (red symbols). The in-cell apparent molecular masses of proteins used in this study and the in vitro apparent molecular masses of protein–RNA complexes are indicated. The in-cell apparent molecular mass of ADK (not shown) is greater than 1.2 MDa (Figure 10 of the Supporting Information).

Figure 5

Quinary interactions of ADK in E. coli maintain the enzyme in an open conformation. (a) Overlay of the in-cell ¹H–¹⁵N CRINEPT–HMQC–TROSY spectrum of REDPRO-labeled ADK (blue) and that of the cellular lysate (red). The insets show overlays of the boxed regions of the in-cell spectrum (blue) and the corresponding regions of the ¹H–¹⁵N CRINEPT–HMQC–TROSY spectrum of lysate (red). The intensities of I63, A66, I72, and A73, which are involved in quinary interactions, are broadened. (b) The residues involved in quinary interactions (red) are mapped onto the molecular surface of ADK (PDB entry 4AKE). The ATP and AMP binding regions are highlighted by circles. The residues involved in quinary interactions mostly lie in the CORE domain of ADK. (c) Bar plot showing the relative changes in in-cell ¹H–¹⁵N CRINEPT–HMQC–TROSY peak intensities of ADK residues due to quinary interactions. Residues that are affected by the interaction of ADK with total RNA are annotated with asterisks. The horizontal line differentiates residues whose NMR peaks undergo significant broadening.

Figure 6

Chloramphenicol induces binding of the adenine nucleotide to ADK in E. coli. (a) Overlay of the in-cell ¹H–¹⁵N CRINEPT–HMQC–TROSY spectrum of REDPRO-labeled ADK (purple) in chloramphenicol-treated cells and the ¹H–¹⁵N HSQC spectrum of purified ADK (blue). Chloramphenicol causes peak shift changes and broadening of the ADK residues due to chemical exchange between the different substrate-bound (AMP, ADP, and ATP) conformations of ADK. (b) Overlay of the in-cell ¹H–¹⁵N CRINEPT–HMQC–TROSY spectrum of REDPRO-labeled ADK in chloramphenicol-treated cells (magenta) and the ¹H–¹⁵N HSQC spectrum of purified 50 μM ADK with 3 mM ATP and 200 μM AMP (red). The trajectories of the in-cell peak changes after chloramphenicol treatment correlate with binding of ATP and AMP to ADK, consistent with a closed conformation of ADK. Specifically, F19, K23, G56, V111, E114, D118, A127, and A176 peaks in chloramphenicol-treated cells exhibit chemical shift changes consistent with ADK bound to 3 mM ATP and 200 μM AMP. These peaks are marked in both panels a and b. Double arrows denote F19 and A176 chemical shift changes. (c) The residues (magenta), whose peaks exhibit chemical shift changes and broadening, are mapped onto the cartoon representation of the AMP/ATP analogue, ANP, bound to ADK (PDB entry 1ANK). The ligands are shown as sticks (cyan). All residues indicated in the figure are part of either the ATP or AMP binding domains. The amino acids that form a quinary patch on the crystal structure (red) are located far from the ATP binding domain and are part of the CORE domain of ADK.

Figure 7

Ubiquitin quinary interactions block polyubiquitination sites, K27, K29, and K33. (a) Overlay of the in-cell ¹H–¹⁵N CRINEPT–HMQC–TROSY spectrum of purified REDPRO-labeled ubiquitin transfected into HeLa cells (blue) and the ¹H–¹⁵N HSQC spectrum of the cell lysate (red). NMR peaks corresponding to K29, K33, G35, and Q40 (insets) are broadened in the in-cell spectrum, suggesting that ubiquitin is involved in transient interactions with cellular components of the cytosol. (b) Residues (red), whose NMR peaks are broadened out (Figure 10 of the Supporting Information), form a contiguous interaction surface involved in ubiquitin quinary interactions (PDB entry 1D3Z). The seven lysines of ubiquitin, which are used for ubiquitylation, are colored purple. K27, K29, and K33 are a part of the interaction surface and are affected by the quinary interactions. The canonical I44 hydrophobic patch of ubiquitin (cyan) spanning L8, I44, and V70 is unperturbed by quinary interactions. The multiplet structure of the in-cell ubiquitin CRINEPT peaks suggests that there is a large population of free ubiquitin, which is in intermediate exchange on the NMR time scale with bound ubiquitin, in HeLa cells.

Figure 8

Total RNA–ubiquitin interactions and quinary interactions affect similar residues. (a) Overlay of in-cell ¹H–¹⁵N CRINEPT–HMQC–TROSY spectra of REDPRO-labeled ubiquitin treated with yeast total RNA (red) and untreated (blue). V5, K29, K33, G35, and Q40 peaks, indicated in the figure, are differentially broadened because of the interaction of ubiquitin with total RNA. (b) Bar plot showing ubiquitin residues whose peaks are differentially broadened because of quinary interactions in HeLa cells. Residues V5, I23, A28, K29, Q31, K33, G35, and Q40, annotated with asterisks, are also affected in total RNA-bound ubiquitin. The horizontal line differentiates residues whose peaks undergo significant broadening. The similarity between residues affected by both RNA binding and quinary interactions suggests that RNA is an important component of quinary interactions.

Figure 9

RNA is a key component of Trx quinary structure. (a) Overlay of in-cell ¹H–¹⁵N CRINEPT–HMQC–TROSY spectra of 15 μM REDPRO-labeled purified wild-type Trx with (red) and without (blue) 30 mg/mL total E. coli RNA. The indole NH of W29 exhibits a downfield shift in the RNA-bound and in-cell NMR spectra (Figures 1 and 2). The indole NH of W32, along with backbone amide peaks of E31, C33, C36, K37, I39, and A40 (boxed), is broadened due to the protein–RNA interaction that is similar to the quinary interaction observed in-cell. (b) The relative volumes of the G52, G66, and G85 peaks in the in vitro ¹H–¹⁵N CRINEPT–HMQC–TROSY spectrum of total RNA-bound wild-type Trx are plotted vs the CRINEPT transfer delay times. An endogenous tryptophan indole amide peak in the in vitro spectra is used as a reference. The optimal CRINEPT transfer delay is 3.5 ms, which corresponds to a molecular mass of ~0.3 MDa. (c) Overlay of in-cell ¹H–¹⁵N CRINEPT–HMQC–TROSY spectra of 15 μM REDPRO-labeled purified wild-type Trx treated with 15 mg/mL total E. coli RNA in the presence (black) and absence (red) of RNase A. The indole NH of W29 exhibits a downfield shift in the RNA-bound and RNase A-treated NMR spectra. The indole NH of W32, along with backbone amide peaks of E31, C33, C36, K37, I39, A40, and I42 (boxed), is broadened due to the protein–RNA interaction that is similar to the quinary interaction observed in-cell. (d) The relative volumes of the G52, G66, and G85 peaks in the in vitro ¹H–¹⁵N CRINEPT–HMQC–TROSY spectrum of RNase A-treated total RNA-bound wild-type Trx are plotted vs the CRINEPT transfer delay times. The optimal CRINEPT transfer delay is ~5.4 ms, which corresponds to a molecular mass of ~12 kDa.