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. 2020 Apr 24;15(4):e0232015. doi: 10.1371/journal.pone.0232015

Intact ribosomes drive the formation of protein quinary structure

Leonard Breindel 1, Jianchao Yu 1, David S Burz 1, Alexander Shekhtman 1,*
Editor: Gideon Schreiber2
PMCID: PMC7182177  PMID: 32330166

Abstract

Transient, site-specific, or so-called quinary, interactions are omnipresent in live cells and modulate protein stability and activity. Quinary intreactions are readily detected by in-cell NMR spectroscopy as severe broadening of the NMR signals. Intact ribosome particles were shown to be necessary for the interactions that give rise to the NMR protein signal broadening observed in cell lysates and sufficient to mimic quinary interactions present in the crowded cytosol. Recovery of target protein NMR spectra that were broadened in lysates, in vitro and in the presence of purified ribosomes was achieved by RNase A digestion only after the structure of the ribosome was destabilized by removing magnesium ions from the system. Identifying intact ribosomal particles as the major protein-binding component of quinary interactions and consequent spectral peak broadening will facilitate quantitative characterization of macromolecular crowding effects in live cells and streamline models of metabolic activity.

Introduction

Broadening of target protein signals due to the formation of large slowly tumbling species that exhibit megadalton apparent molecular weights in-cell [1–3] during NMR spectroscopic experiments [4] and in concentrated cellular lysates is a common occurrence and regarded as evidence for specific transient complexes or protein quinary structure. [5–11] The source of quinary interactions has been investigated from the perspective of increased intracellular viscosity, [2, 12] volume exclusion, and the presence of neutral and charged polymeric and proteinaceous crowding agents. [12, 13] Although these non-specific physical phenomena do contribute to some degree to signal broadening, they fail to recapitulate the extent to which it is observed during in-cell NMR experiments except under non-physiological conditions. [14]

Recent work has advocated for ribosomes as the major target protein binding complement that give rise to quinary interactions and consequent spectral peak broadening [1, 15–17] and suggested that the ribosome may function as an electrostatic sponge that binds to a wide range of proteins and metabolites. In those studies in-cell NMR spectra of target proteins were compared to spectra obtained in vitro in the presence of total cellular RNA [16] and purified ribosomal preparations. [1, 15, 18] The work left open the question of whether intact ribosome particles per se, or unidentified proteins or free rRNA mediate these interactions.

The importance of quinary interactions is underscored by the observations that they may destabilize [19–21] or stabilize [22, 23] target proteins, alter ligand binding [24, 25] and catalytic activity [15, 26], thus adding another layer of complexity to the regulation of biological activity. The study of quinary interactions is complicated by the fact that they occur strictly inside cells and lysing the cells often destroys the quinary state. However the persistence of some NMR spectral broadening indicative of quinary-like interactions in concentrated lysates [8, 27] suggests that the interacting component is not absent but diluted beyond the physiological range. This assumption presents an opportunity to study quinary-like interactions in vitro.

The purpose of this study was twofold: First, to show that the intact ribosome particle is the viable binding complement that gives rise to quinary interactions for proteins that do not interact exclusively with mRNA. And second, to show that ribosome-protein interactions, RPIs, are a general property of ribosomes that gives rise to broadening of NMR spectral peaks and quinary structures.

Results

Protein quinary interactions are lost when the ribosome is destabilized

To demonstrate that intact ribosomes are a critical component of quinary interactions, the NMR spectrum of purified uniformly labeled [U- 15N] γD-crystallin was examined in the presence of stable and destabilized ribosomes in E. coli cell lysate. γD-crystallin is a small, 21 kDa, eukaryotic protein found in the eye lens of vertebrates. The protein was studied in E. coli lysate to provide an experimental environment that was devoid of specific binding interactions that could obscure the effects of RPIs. Since quinary interactions are transient, they are not expected to interfere with high affinity interactions involved in ribosomal function. Consequently, the effect of the binding interaction on the activity γD-crystallin or the ribosome was not considered in these experiments.

Uniformly labeled [U- 15N] γD-crystallin, yields a well-dispersed 15N isotope edited heteronuclear single quantum coherence, 1H-15N HSQC, NMR spectrum in vitro (Fig 1A), and that spectrum is extensively broadened in E. coli cells (Fig 1B). The loss of signal is attributed to a specific transient interaction between the target molecule and cellular constituents. To further explore the nature of signal broadening, [U- 15N] γD-crystallin was added to a clarified solution of E. coli cell lysate. To prevent premature degradation of ribosomes in the lysate by E. coli RNAses, NMR samples were supplemented with 10 units/mL of RNAse inhibitor, SUPERase In. Many of the [U- 15N] γD-crystallin spectral peaks were broadened, consistent with persistent quinary structural interactions between γD-crystallin and the cytosol (Fig 1C). Treating the lysate with 10 mM ethylenediaminetetraacetic acid, EDTA, which is a chelator of Mg2+, to destabilize the ribosome, and with 1 mM RNase A for 1 h to degrade rRNA, restored the NMR spectrum of [U- 15N] γD-crystallin (Fig 1D).

Fig 1. Protein quinary interactions are lost when the ribosome is destabilized.

Fig 1

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A) 1H-15N HSQC NMR spectrum of 10 μM purified [U- 15N] γD-crystallin in NMR buffer. B) [U- 15N] γD-crystallin overexpressed in E. coli cells. Note the extensive loss of signals. Most of the peaks are from 15N labeled metabolites. 1H-15N HSQC NMR spectra of 10 μM purified [U- 15N] γD-crystallin in C) E. coli cell lysate containing 10 mM EDTA. Peaks that broadened in the lysate are indicated by x; and D) E. coli lysate containing 10 mM EDTA treated with 1 mM RNase A for 1 h. The majority of previously broadened peaks, x, are recovered. All spectra are shown at the same contour level.

The results are consistent with what is known about the structural integrity of ribosomes in cell lysates. Treating cell lysates with RNase A in the presence of magnesium ions to disrupt the ribosome structure and liberate bound protein failed to recover sharp NMR signals. [8, 13] This result was not surprising because the ribosome structure is stabilized by magnesium ions, [28, 29] which are abundant in the cell and in lysates despite nuclease digestion. RNase A digestion of ribosomes yields RNA fragments averaging 30–40 nt [29] yet the ribosome remains intact due to the strength of the protein-protein and protein-RNA interactions that make up its structure. In addition, some rRNA remains protected from nuclease digestion even in partially unfolded ribosomes. [30]

The results of the NMR experiments were corroborated by native RNA gel electrophoresis. TRIzol extraction of RNA from intact cells showed intact 50S and 30S ribosomal subunits (Fig 2A lane 1). When EDTA and SUPERase In were added to the lysate used in the NMR experiments, the intact subunits were still evident (Fig 2A lane 2). Integral protein-rRNA contacts were sufficient to maintain the 50S and 30S structures despite the absence of Mg2+. Treating the lysate with RNase A, in the presence of EDTA resulted in the degradation of rRNA (Fig 2A lane 3). Thus complete disruption of the ribosome by nuclease digestion is possible only when the structure is destabilized by removing magnesium ions from the system.

Fig 2. Removal of magnesium ions and ribonuclease digestion are required to destabilize the ribosome particle.

Fig 2

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A) Native RNA gel: Left- RNA size markers; Lane 1- RNA extracted from E. coli shows intact ribosome 50S and 30S subunits; Lane 2- Lysate containing 10 mM EDTA and SUPERase In. The bright band at the bottom of Lanes 1 and 2 is digested RNA. The increased intensity of digested RNA in Lane 2 versus Lane 1 is due to the loss of SUPERase In activity after the 2 hour long NMR experiment. Lane 3- Ribosome preparation containing 10 mM EDTA treated with RNase A for 1 h. B) Denaturing protein gel: Left- Protein MW markers; Lane 1- Whole cell lysate; Lane 2- Lysate precipitate following treatment with RNase A for 2 h; Lane 3- Purified ribosome preparation.

Ribonuclease digestion of ribosomes results in the precipitation of ribosomal proteins, which in the absence of intact rRNA, are insoluble [31]. The presence of riboproteins in the precipitate was confirmed by using denaturing polyacrylamide gel electrophoresis, PAGE. Intact ribosomal proteins were evident in lysate containing EDTA (Fig 2B lane 1) and in the lysate precipitate resulting from RNase A digestion (Fig 2B lane 2). To confirm that the precipitate contains ribosomal proteins, purified ribosomes were electrophoresed (Fig 2B lane 3). Analysis of the protein content showed that the lysate precipitate consisted mostly of ribosomal proteins.

The interaction between γD-crystallin and the ribosome is electrostatic in nature

The observations made in-cell and with lysates were examined in vitro. Titrating [U- 15N] γD-crystallin with purified ribosomes from 0–6 μM resulted in broadening of the 1H-15N HSQC NMR spectrum (S1 Fig, S2 Fig, and S1 Table). At a ribosome concentration of 6 μM in vitro, in the presence of EDTA, the 1H-15N HSQC NMR spectrum of [U- 15N] γD-crystallin exhibited peak broadening comparable to that seen in cell lysates with an estimated ribosome concentration of 5 μM (S1 Fig, S2 Fig, S1 Table, and Fig 3A). This result is also consistent with the ribosome subunits maintaining their integrity in the absence of Mg2+. Treatment with RNase A in the presence of EDTA led to the complete destabilization of the ribosome, the disruption of binding interactions and recovery of the γD-crystallin NMR spectrum (Fig 3B).

Fig 3. The interaction between γD-crystallin and the ribosome is electrostatic in nature.

Fig 3

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1H-15N HSQC NMR spectra of purified [U- 15N] γD-crystallin: A) in the presence of 5 μM ribosomes containing 10 mM EDTA; and B) treated with 1 mM RNase A for 1 h in the presence of 5 μM ribosomes and 10 mM EDTA. Broadened peaks are indicated by x. C) Changes in peak intensities of purified γD-crystallin due to E. coli cell lysate (left) and 5 μM ribosomes (right). Unassigned residues are designated with an x. D) Surface residues involved in quinary interactions with E. coli lysate (pink), mapped onto the molecular surface of γD-crystallin (PDB entry 1HK0). The amide peak of R37 (cyan) is broadened in lysate (Fig 2B) but visible in the presence of purified ribosomes (panel B). E) Surface residues involved in quinary interactions with purified ribosomes. F) Electrostatic surface map γD-crystallin showing regions of positive, 1.25 kT/e (blue), and negative, −1.25 kT/e (red), potential. All spectra are shown at the same contour level.

Changes in peak intensities identified in lysates (Fig 3C left) and in the presence of purified ribosomes (Fig 3C right), were mapped onto the structure of γD-crystallin (Protein Data Bank, PDB entry 1HKO [32]) in Fig 3D and 3E, respectively. The residues perturbed in both cases are the same indicating a specific RPI when intact ribosome particles are present in solution. The interaction surface is located primarily on one face of the protein and consists mostly of charged residues (Fig 3F). Because of the preponderance of charged residues involved in the quinary interaction between γD-crystallin and ribosomes, the effect of salt on the quinary interactions between γD-crystallin and ribosomes was examined.

1H-15N HSQC NMR spectra of 10 μM [U- 15N] γD-crystallin were collected in the presence of increasing amounts of NaCl. The spectrum was broadened at 50 mM NaCl, the concentration used in all in vitro experiments. As the concentration of NaCl was increased to 200 mM and 500 mM, the spectrum was completely recovered (S3 Fig). The disruption of RPIs 200 and 500 mM NaCl is consistent with the concentrations used to disrupt protein-protein interactions [13] and is strong evidence for RPIs being mediated by electrostatic interactions.

Discussion

The broadening of protein crosspeaks observed in-cell during NMR experiments is considered a hallmark of quinary interactions. [5, 7, 33] Due to the extreme heterogeneity of in-cell NMR samples and high concentration of cellular ribosomes, ~20 μM, which completely broaden the protein NMR peaks (Fig 1A and 1B), direct quantitative comparison of in-cell and in vitro NMR spectra is not possible. Nevertheless, the observed in-cell peak broadening coincides with the presence of intact ribosomal particles in cell lysates and in vitro, and are consistent with intact ribosomes as the major interactor that gives rise to protein quinary interactions (Fig 3). In agreement with our observations, mass spectroscopic studies revealed that the ribo-interactome consists of about 430 proteins that remain bound to the ribosome after cell disruption and ribosome purification. [34] The results are also in agreement with the observation that translational diffusion of fluorescent proteins is reduced due to ribosome-protein interactions in E. coli, in the presence of purified ribosomes, [15, 17] and in NMR experiments performed in-cell and in vitro in the presence of total cellular RNA or purified ribosome preparations. [15, 16] The use of purified ribosomes to mimic the crowded cytosol provides an important means by which to investigate and characterize quinary-like interactions in vitro as a logical step towards a quantitative understanding of cellular biochemistry. [9]

The results further support the idea that intact ribosomes are primed to act as a chemical sponge. [15] The electrostatic surface of the ribosome exhibits extensive negative potential (Fig 4). Characterization of protein quinary interaction surfaces show that ribosome binding is mediated by highly charged surface residues involving a specific interaction surface that is unique to each protein [1, 15, 18]. The resulting quinary structure can give rise to biological activity that differs from that of the unbound state. [15] It is therefore critical to characterize the quinary state to properly analyze metabolic pathways.

Fig 4. Electrostatic surface of the ribosome.

Fig 4

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Electrostatic surface of the ribosome shows extensive negative potential consistent with the postulated function as an electrostatic sponge. Positive (blue, with potential > 10 kT/e) and negative (red, with potential < -10 kT/e) electrostatic isosurfaces of protonated E. coli ribosome (PDB code 4YBB [35]) were calculated by using Adaptive Poisson-Boltzmann Solver–Protein Data Bank to Protein Charge Radius, APBS-PDB2PQR, software. [36] White lines represent electric field lines at the surface of the ribosome imbedded in 0.3 M KCl. Electric field lines are calculated by using Visual Molecular Dynamics, VMD, software [37] with an magnitude gradient of 5 kT/(eÅ) and a maximum length of 29 Å, where k, T and e are the Boltzmann constant, temperature in Kelvin and electron charge, respectively. Ribosome subunits, 30S and 50S, as well as ribosomal protein L9 are indicated. The figure was rendered by using VMD.

Despite differences in the overall structure of eukaryotic and prokaryotic ribosomes, the ubiquity and abundance of ribosomes guarantees that these transient, site-specific quinary interactions are a generalized phenomenon. Because RPIs have dissociation constants in the micromolar range [1, 15], the inherently high, 1–20 μM [38], physiological concentration of ribosomes in cells provides the driving force for these interactions. Since most cellular proteins are present at concentrations ≤1 μM, at least half of the population of those that interact with ribosomes are expected to exist in the ribosome-bound or quinary state. Further regulation of quinary interactions occur because the concentration of ribosomes increases linearly with the growth rate in prokaryotes. [39] [40] This is especially important in prokaryotic cells where the ribosome concentration can be ten times greater than in eukaryotes. [38]

Materials and methods

RNA extraction

E. coli (∼7 × 107 cells) pellets were re-suspended in 10 mM potassium phosphate, 50 mM sodium chloride, and 10 mM EDTA at pH 7.0 and lysed by 6 cycles of freeze/thaw. The lysate was centrifuged at 14,500g for 30 min and the supernatant was incubated with either 10 U/mL SUPERase In (Invitrogen) or 1 mM RNAse A (Qiagen Inc.) and 10 U/mL SUPERase In. Total RNA was prepared as described previously. [41] The concentration of RNA was measured by the absorbance at 260 nm. The amount of total RNA loaded on a 1% agarose gel was 300 ng. SYBR Green II (Invitrogen) was used to stain the gel. The RiboRuler High Range RNA Ladder (Thermo Scientific) was used as a molecular size standard.

Ribosome preparation

Functionally active ribosomes were purified by using a published protocol [42] with slight modifications. E. coli strain MRE600, lacking RNAse A activity, was purchased from the American Tissue Culture Collection. MRE600 was grown in LB medium to an OD600 of 0.5 to 0.7 and the cell pellet (~10 g) was resuspended in lysis buffer, 10 mM tris(hydroxymethyl)aminomethane hydrochloride, Tris-HCl, pH 7.4, 200 mM ammonium chloride, 20 mM magnesium chloride, 0.1 mM EDTA, and 6 mM 2-mercaptoethanol, at a density of 1 g of cells per mL before sonicating with a Model 250 Digital Sonifier (Branson). The lysate was centrifuged at 30,000g for 45 min and the supernatant was centrifuged at 100,000g for 4.4 h at 4 °C in an Optima LE-90K Ultracentrifuge (Beckman Coulter) using a SW28 rotor. The pellet was resuspended in 10 mL of lysis buffer and 5.2 mL of the suspension was layered onto 5.2 mL of ribosome buffer, 10 mM Tris-HCl, pH 7.4, 500 mM ammonium chloride, 10 mM magnesium chloride, 0.1 mM EDTA, and 6 mM 2-mercaptoethanol, containing 30% sucrose prior to being centrifuged at 444,000g for 2 h at 4 °C in an Optima LE-90K Ultracentrifuge using a Type 90 Ti rotor. The ribosome pellet was washed four times with 3 mL of ribosome wash buffer, 20 mM Tris-HCl, pH 7.2, 1 M ammonium chloride, 10 mM magnesium chloride, 0.5 mM EDTA and 6 mM 2-mercaptoethanol, to remove residual ATPase activity. The clear ribosome pellet was resuspended in 0.3 mL of ribosome storage buffer, 10 mM potassium phosphate, pH 6.5, 10 mM magnesium acetate, and 1 mM dithiothreitol. Concentration was determined by absorbance at 260 nm, using an ε0.1% = 15 mL/(mg × cm). Ribosome solutions with a 260/280 nm ratio of 1.96 to 2.05 were used. For NMR experiments, ribosome was exchanged using Amicon Ultra centrifugal filters into 10 mM potassium phosphate, 50 mM sodium chloride, and 10 mM EDTA at pH 7.0 immediately prior to NMR experiments.

Protein gel analysis

E. coli (∼1 × 108 cells) were re-suspended in 10 mM potassium phosphate, 50 mM sodium chloride, and 10 mM EDTA at pH 7.0 before being sonicated with a Model 250 digital sonifier (Branson). The lysate was centrifuged at 14,500g for 30 min and the supernatant was incubated with 1 mM RNAse A and 10 U/mL SUPERase In. After incubation the lysate was centrifuged at 14,500g for 30 min and the supernatant was discarded. Proteins and purified ribosomes were electrophoresed on a 10% sodium dodecyl sulfate polyacrylamide gel. Coomassie blue G-250 was used to stain the gel. Precision Plus Protein (BioRad) was used as a molecular size standard.

Preparation of E. coli lysate

To prepare lysate, E. coli (∼30 × 109 cells) were re-suspended in a final volume of 2 mL of NMR buffer with 10 Units/mL of SUPERase In before being sonicated. The lysate was centrifuged at 14,500g for 30 min and the supernatant was removed for NMR experiments.

The concentration of ribosomes in the cell lysate was estimated as follows: 1 L of cells were grown to 0.5 OD600, which is equivalent to 2.5 × 108 cells/mL or 2.5 × 1011 total cells. Assuming an average cell volume of 2 μm3, or 2 × 10−12 mL/cell, [38] the total cell volume is therefore 2 × 10−12 mL/cell x 2.5 × 1011 cells = 5 × 10−1 mL. The ribosome concentration in fast growing cells is ~20 μM [38] and the cells were diluted 4-fold to 2 mL total lysate volume. This results in a final ribosome concentration of ~5 μM.

NMR experiments

10 μM [U- 15N] γD-crystallin (a gift from Dr. Pande, University at Albany) was dissolved in NMR buffer, 10 mM potassium phosphate, pH 7.0, 50 mM sodium chloride, and 10 mM EDTA in 90% H2O/10% D2O (v/v). To prepare 10 μM [U- 15N] γD-crystallin in the presence of E. coli lysate, 10 μL of 500 μM [U- 15N] γD-crystallin was dissolved in 490 μL of E. coli lysate. To prepare [U- 15N] γD-crystallin in the presence of ribosome, 5 μM ribosome was added to 10 μM [U- 15N] γD-crystallin in NMR buffer.

All NMR experiments were acquired at 298 K using a 700 MHz Bruker Avance II NMR spectrometer equipped with a TXI cryoprobe. 1H-15N HSQC spectra were acquired with 64 scans. The spectral widths in the 1H and 15N dimensions were 14 and 35 ppm, respectively and were digitized by 1024 and 128 points in the 1H and 15N dimensions, respectively.

Data analysis

Spectra were processed with Topspin 4.0.6 (Bruker) and analyzed by using CARA software (http://www.cara.nmr.ch). Changes in peak intensities were calculated by using ΔI = ((Ifree/Iref)-(Ibound/Iref))/ (Ifree/Iref), where Ifree/Iref is the scaled intensity of an individual peak in the spectrum of γD-crystallin in the absence of lysate or ribosome and Ibound/Iref is the scaled intensity of an individual peak in the spectrum of γD-crystallin in the presence of lysate or ribosome. Iref is the peak intensity of a glutamine side chain amide at 6.82 and 113.3 ppm in the proton and nitrogen dimensions respectively and does not shift in the presence of lysate or ribosome. The threshold to determine the residues involved in quinary interactions was set to ΔI > 0.5. Surface maps of γD-crystallin were constructed using Swiss PDB viewer. [43]

Supporting information

S1 Fig. Increasing concentration of ribosomes broadens NMR spectral crosspeaks.

(PDF)

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S2 Fig. Peak broadening of the 1H-15N HSQC spectrum of [U- 15N] γD-crystallin with diluted lysate is consistent with the presence of ~ 5 μM of ribosomes in the sample.

(PDF)

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S3 Fig. Increasing salt concentration restores NMR spectral crosspeaks.

(PDF)

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S1 Table. Normalized 1H-15N HSQC peak intensities of γD-crystallin titrated with ribosomes.

(PDF)

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S1 Raw images

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Data Availability

All relevant data are within the paper and its Supporting Information files.

Funding Statement

Funded studies. A.S.; United States National Institute of Health grant R01GM085006 The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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Decision Letter 0

Gideon Schreiber

30 Jan 2020

PONE-D-19-34774

Intact ribosomes are a major component of protein quinary structure.

PLOS ONE

Dear Prof. Shekhtman

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

In the current version, the manuscript suffers from major shortfalls as indicated by the reviewers. These include 1. relevance (gamma-D-crystalline is not a relevant protein to study soft interactions with the E.coli ribosomes). It is not clear why the authors chose to do the experiments using gamma-D-crystalline, and they don't give any rational explanation for this. 2. How do the authors reach the conclusion that non-specific electrostatic interactions are formed between gamma-D-crystalline and the ribosome. While they provide a number of references, they did not measure the salt effect on line broadening. Moreover, the E.coli cytoplasm has an ionic strength of ~200 mM. Would line broadening be observed in the E.coli cytoplasm? 3. Please answer in details all the additional points raised by the referees. 

We would appreciate receiving your revised manuscript. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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Gideon Schreiber

Academic Editor

PLOS ONE

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Reviewers' comments:

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Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: Partly

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: I Don't Know

Reviewer #2: N/A

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: This paper has the laudable goal of understanding the nature of RNA protein interactions in E. coli lysates. But in practice they focus on the interactions of intact ribosomes with gD-crystallin. The fact that it has electrostatic interactions with a highly charged ribosome is hardly surprising. The real question is why are these spurious interactions absent in the cell and /or managed so that they do not interfere with protein synthesis. This is not addressed in the slightest. Moreover, Gd crystallin is only enriched in human ovary cells, so it is not a particularly interesting protein. Overall, the impact of these experiments is minor and publication is not recommended.

Reviewer #2: The authors tried to identify the ribosome as the major component of the quinary structure. They prove their idea using a eukaryotic protein and prokaryotic ribosomes. The similar spectral broadening in the presence of purified ribosomes and lysate suggest the ribosome as the major source of broadening. Several major concerns needed to be addressed before publication:

1) The experimental data should include the HSQC spectrum in E.coli cells, it is critical to see if the degree of broadening in cells and in the purified ribosomes (the concentration is close to that in cells) are similar.

2) It will be fantastic if the authors can provide the quantitative data, such as the degree of resonances broadening as the function of the purified ribosomes concentration.

3) If the conclusion of the manuscript stands, the author should have more discussions on the difference between prokaryotic and eukaryotic cells and meaning for in-cell nmr filed.

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

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Reviewer #1: No

Reviewer #2: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org. Please note that Supporting Information files do not need this step.

PLoS One. 2020 Apr 24;15(4):e0232015. doi: 10.1371/journal.pone.0232015.r002

Author response to Decision Letter 0

12 Mar 2020

Response to Reviewers’

We thank our editor and reviewers for the comments. We addressed all of the concerns raised by the editor and reviewers. In addition, based on the comments, we revised the text throughout to emphasize the purpose of these experiments, i.e. to demonstrate that the ribosome must remain intact to affect quinary interactions, and that ribosome-protein interactions are a generalized phenomenon.

Editor:

In the current version, the manuscript suffers from major shortfalls as indicated by the reviewers. These include 1. relevance (gamma-D-crystalline is not a relevant protein to study soft interactions with the E.coli ribosomes). It is not clear why the authors chose to do the experiments using gamma-D-crystalline, and they don't give any rational explanation for this.

We included the rationale for studying γD-crystallin in E. coli in the first results section. “To demonstrate that intact ribosomes are a critical component of quinary interactions, the NMR spectrum of purified uniformly labeled [U- 15N] γD-crystallin was examined in the presence of stable and destabilized ribosomes in E. coli cell lysate. γD-crystallin is a small, 21 kDa, eukaryotic protein found in the eye lens of vertebrates. The protein was studied in E. coli lysate to provide an experimental environment that was devoid of specific binding interactions that could obscure the effects of RPIs. Since quinary interactions are transient, they are not expected to interfere with high affinity interactions involved in ribosomal function. Consequently, the effect of the binding interaction on the activity γD-crystallin or the ribosome was not considered in these experiments”.

2. How do the authors reach the conclusion that non-specific electrostatic interactions are formed between gamma-D-crystalline and the ribosome. While they provide a number of references, they did not measure the salt effect on line broadening. Moreover, the E.coli cytoplasm has an ionic strength of ~200 mM. Would line broadening be observed in the E.coli cytoplasm?

First, we added the in-cell NMR spectrum of γD-crystallin in E. coli (Fig 1B), which shows extensive line broadening. Second, we titrated γD-crystallin with NaCl in vitro to support our assertion and included Fig S2 in Supplementary Results. Based on these results, line broadening was evident at 200 mM NaCl.

Reviewer #1: This paper has the laudable goal of understanding the nature of RNA protein interactions in E. coli lysates. But in practice they focus on the interactions of intact ribosomes with gD-crystallin. The fact that it has electrostatic interactions with a highly charged ribosome is hardly surprising. The real question is why are these spurious interactions absent in the cell and /or managed so that they do not interfere with protein synthesis. This is not addressed in the slightest.

Quinary interactions are present in-cell as evidenced by the line broadening observed in the in-cell NMR spectrum of γD-crystallin (Fig 1B). Since quinary interactions are transient, they are not expected to interfere with high affinity interactions involved in ribosomal function. The goal of the paper is to show that it is the INTACT ribosome that mediates quinary interactions not a degraded form or “other” reactive species, and that quinary interactions are a generalized phenomenon associated with ribosomes. Consequently the effect of the interaction on activity was not considered. This is clarified in the introduction

Moreover, Gd crystallin is only enriched in human ovary cells, so it is not a particularly interesting protein. Overall, the impact of these experiments is minor and publication is not recommended.

We now included the rationale for studying γD-crystallin in E. coli in the first results section.

Reviewer #2: The authors tried to identify the ribosome as the major component of the quinary structure. They prove their idea using a eukaryotic protein and prokaryotic ribosomes. The similar spectral broadening in the presence of purified ribosomes and lysate suggest the ribosome as the major source of broadening. Several major concerns needed to be addressed before publication

1) The experimental data should include the HSQC spectrum in E.coli cells, it is critical to see if the degree of broadening in cells and in the purified ribosomes (the concentration is close to that in cells) are similar

The in-cell HSQC NMR spectrum of γD-crystallin in E. coli is now shown in Fig 1B.

2) It will be fantastic if the authors can provide the quantitative data, such as the degree of resonances broadening as the function of the purified ribosomes concentration.

The spectra of γD-crystallin with increasing amounts of ribosome are included in Fig S1. The intensities of γD-crystallin peaks as a function of ribosome concentration are included in a Table in Supplementary Results.

3) If the conclusion of the manuscript stands, the author should have more discussions on the difference between prokaryotic and eukaryotic cells and meaning for in-cell nmr field.

Based on our previous observations (ref. 1, 15-16) and our current work, we expect that in-cell NMR spectra of proteins are better resolved in eukaryotic then prokaryotic cells. It is a direct consequence of the fact that ribosome concentration in eukaryotes is about ten fold less than in prokaryotes. Because the emphasis is on the generic aspect of ribosome-protein interactions, we do not elaborate on specific differences between eukaryotic and prokaryotic ribosomes, except for their general role in regulation.

Attachment

Submitted filename: Response to Reviewers.pdf

Click here for additional data file. (539.8KB, pdf)

Decision Letter 1

Gideon Schreiber

25 Mar 2020

PONE-D-19-34774R1

Intact ribosomes drive the formation of protein quinary structure

PLOS ONE

Dear Dr. Shekhtman

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

We would appreciate receiving your revised manuscript by May 09 2020 11:59PM. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

Please include the following items when submitting your revised manuscript:

  • A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.

  • A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.

  • An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

We look forward to receiving your revised manuscript.

Kind regards,

Gideon Schreiber

Academic Editor

PLOS ONE

Additional Editor Comments (if provided):

For the manuscript to be accepted it has to comply with the request of the reviewer (which was given already in the original review)

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #2: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #2: Partly

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #2: N/A

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #2: The revised version still did not provide a quantitative analysis of resonance broadening contribution from ribosome vs other cellular components in in-cell NMR experiments, ie, at a physiological concentration of ribosome, the authors could estimate what percentage of broadening in living cells come from interacting with ribosomes for γD-crystallin since they have the in-cell and ribosome titration data. This analysis is important for the main conclusion of this manuscript "Intact ribosome particles were shown to be sufficient to mimic quinary interactions present in the crowded cytosol".

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #2: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org. Please note that Supporting Information files do not need this step.

PLoS One. 2020 Apr 24;15(4):e0232015. doi: 10.1371/journal.pone.0232015.r004

Author response to Decision Letter 1

30 Mar 2020

We thank our reviewer for the comments.

Reviewer#2

The revised version still did not provide a quantitative analysis of resonance broadening contribution from ribosome vs other cellular components in in-cell NMR experiments, ie, at a physiological concentration of ribosome, the authors could estimate what percentage of broadening in living cells come from interacting with ribosomes for γD-crystallin since they have the in-cell and ribosome titration data. This analysis is important for the main conclusion of this manuscript "Intact ribosome particles were shown to be sufficient to mimic quinary interactions present in the crowded cytosol".

We clarified our reasoning for using in vitro samples to analyze the role of ribosomes in quinary interactions: “Due to the extreme heterogeneity of in-cell NMR samples and high concentration of cellular ribosomes, ~20 μM, which completely broaden the protein NMR peaks (Fig 1A and Fig 1B), direct quantitative comparison of in-cell and in vitro NMR spectra is not possible. Nevertheless, the observed in-cell peak broadening coincides with the presence of intact ribosomal particles in cell lysates and in vitro, and is consistent with intact ribosomes as the major interactor that gives rise to protein quinary interactions (Fig 3).” (Lines 176-182). Furthermore, the quantitative comparison of peak broadening is made between in vitro samples of γD-crystallin with diluted lysate and with purified ribosomes (Lines 131-135 and Fig S2, Fig 3C, Fig 3D, and Fig 3E).

Attachment

Submitted filename: renamed_64b62.pdf

Click here for additional data file. (78.9KB, pdf)

Decision Letter 2

Gideon Schreiber

7 Apr 2020

Intact ribosomes drive the formation of protein quinary structure

PONE-D-19-34774R2

Dear Dr. Alexander Shekhtman

We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.

Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication.

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With kind regards,

Gideon Schreiber

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #2: N/A

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #2: (No Response)

**********

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Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #2: No

Acceptance letter

Gideon Schreiber

13 Apr 2020

PONE-D-19-34774R2

Intact ribosomes drive the formation of protein quinary structure

Dear Dr. Shekhtman:

I am pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

For any other questions or concerns, please email plosone@plos.org.

Thank you for submitting your work to PLOS ONE.

With kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Prof. Gideon Schreiber

Academic Editor

PLOS ONE

Associated Data

    This section collects any data citations, data availability statements, or supplementary materials included in this article.

    Supplementary Materials

    S1 Fig. Increasing concentration of ribosomes broadens NMR spectral crosspeaks.

    (PDF)

    Click here for additional data file. (793.4KB, pdf)
    S2 Fig. Peak broadening of the 1H-15N HSQC spectrum of [U- 15N] γD-crystallin with diluted lysate is consistent with the presence of ~ 5 μM of ribosomes in the sample.

    (PDF)

    Click here for additional data file. (512.8KB, pdf)
    S3 Fig. Increasing salt concentration restores NMR spectral crosspeaks.

    (PDF)

    Click here for additional data file. (776.5KB, pdf)
    S1 Table. Normalized 1H-15N HSQC peak intensities of γD-crystallin titrated with ribosomes.

    (PDF)

    Click here for additional data file. (114.2KB, pdf)
    S1 Raw images

    (PDF)

    Click here for additional data file. (3.4MB, pdf)
    Attachment

    Submitted filename: Response to Reviewers.pdf

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    Attachment

    Submitted filename: renamed_64b62.pdf

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    Data Availability Statement

    All relevant data are within the paper and its Supporting Information files.

    Articles from PLoS ONE are provided here courtesy of PLOS

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