Short Arginine Motifs Drive Protein Stickiness in the Escherichia coli

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Short Arginine Motifs Drive Protein Stickiness in the Escherichia coli Cytoplasm Ciara Kyne, and Peter B. Crowley Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.7b00731 • Publication Date (Web): 23 Aug 2017 Downloaded from http://pubs.acs.org on August 24, 2017

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Short Arginine Motifs Drive Protein Stickiness in the Escherichia coli Cytoplasm Ciara Kyne,*,† Peter B. Crowley*

School of Chemistry, National University of Ireland, Galway, University Road, Galway, Ireland -†

Present address: Astbury Centre for Structural Molecular Biology, School of Molecular and

Cellular Biology, University of Leeds, Leeds LS2 9JT, UK.

*Correspondence to; [email protected], +44 11 33 43 31 27 [email protected], + 353 91 49 24 80

Keywords: charge-charge interactions; GB1; in-cell NMR; quinary structure; supramolecular

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Abstract Although essential to numerous biotech applications, knowledge of molecular recognition by arginine-rich motifs in live cells remains limited. 1H, 15N HSQC and 19F NMR spectroscopy were used to investigate the effects of C-terminal -GRn (n = 1-5) motifs on GB1 interactions in Escherichia coli cells and cell extracts. While the ‘biologically inert’ GB1 yields highquality in-cell spectra, the -GRn fusions with n = 4 or 5 were undetectable. This result suggests that a tetra-arginine motif is sufficient to drive interactions between a test protein and macromolecules in the E. coli cytoplasm. The inclusion of a 12 residue flexible linker between GB1 and the -GR5 motif did not improve detection of the ‘inert’ domain. In contrast, all of the constructs were detectable in cell lysates and extracts, suggesting that the argininemediated complexes were weak. Together these data reveal the significance of weak, interactions between short arginine-rich motifs and the E. coli cytoplasm, and demonstrate the potential of such motifs to modify protein interactions in living cells. These interactions must be considered in the design of (in vivo) nano-scale assemblies that rely on arginine-rich sequences.


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Protein interactions mediate all cellular phenomena and the ability to modify protein interactions is of immense interest in chemical biology and drug discovery.1-6 Small molecules that stabilize protein interactions are attractive tools to elicit new assemblies and functions.6,7 An alternative and powerful strategy is to use peptides for guided assembly.1,3 This approach has been employed extensively in the development of bionanomaterials7-9 and coacervate droplets /protocell mimics.10-12 Several design rules are routinely applied in peptide-based assemblies. Complementary charge-charge interactions are favoured due to their long range nature.13-15 Interface ‘hot spot’ residues are frequently employed. Arginine, with its propensity to simultaneously engage in salt-bridges, hydrogen bonds, cation-π bonds and hydrophobic interactions is of particular interest.16-18 For example, arginine-rich motifs have been used to drive peptide assembly,19 protein encapsulation14 and liquid-liquid phase separation.11,20 Arginine-rich motifs also feature prominently in cell permeation and nuclear localization.2,3,21 Remarkably intricate systems emerge from the assembly of simple arginine-rich peptides. For instance, complexation of the S(SR)4 peptide with an anionic block copolymer produced wormlike micelles or spheres depending on the mixing ratio and sample conditions.19 Collagen mimetic peptides (CMPs) bearing N-terminal “supercharged” motifs composed of R, K, D or E can fold into triple helices.13 However, only the arginine-rich CMPs assembled into higher order structures upon interaction with anionic CMPs. Argininerich poly-dipeptides such as (PR)x and (GR)x, implicated in disease models,20 bind to disordered, low complexity domains of phase-separating proteins to impair the assembly and dynamic properties of membrane-less organelles. While bottom up investigations of peptide assembly remain important,11-13,14 the development of chemical biology tools will benefit from improved models of peptide interactions in the complex, heterogeneous cytoplasm. Therefore, we sought to explore the “stickiness” of short, arginine-rich motifs, -GRn, (n = 1-5) in the Escherichia coli cytoplasm. C-terminal fusion proteins of GB122,23 and the -GRn motifs (denoted GB1-GRn) were studied by using in-cell NMR spectroscopy. GB1 was chosen as a scaffold because it is stable,23 lacks arginine and is ‘biologically inert’ which facilitates rapid tumbling and high-quality incell NMR spectra.24-30 Precedent for this work comes from studies showing that a fusion of GB1 and the arginine-rich motif of HIV-1 Tat is sticky and undetectable by in-cell NMR.17,25 Here, we have identified the minimal arginine motif that renders GB1 sticky toward macromolecules in the E. coli cytoplasm. The pervasive, albeit weak, nature of -GRn mediated interactions was also demonstrated. The effect of separating GB1 from the -GR5 3 ACS Paragon Plus Environment

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motif by a disordered, 12 residue linker was investigated, with surprising results. The findings extend our understanding of arginine-rich motifs to facilitate their use in peptide therapeutics, nano-devices or supramolecular systems directed toward in vivo applications. Furthermore, we propose the GB1-GRn series (and related constructs) as probes of quinary structure.31-32


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Materials and Methods Mutagenesis. pET3a vectors encoding the gene for GB1-QDD22 with the C-terminal extensions -GRn (where n = 1–5), -GRGRGR and -AAPSAPAPSPAAGR5 (-linker-GR5) were synthesized and sequence-verified by GenScript.

Protein Expression and Purification. Unlabelled and

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N-labelled proteins were over-

expressed in E. coli BL21 (DE3) according to previously described methods.22,26 Fluorotryptophan labelled proteins were produced as described,33 with 60 mg/L 5fluoroindole in the minimal medium.

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N-arginine labelled protein was produced from

minimal medium that contained 100 mg/L 15N-arginine and all other unlabelled amino acids. Cells were harvested two hours post-induction by centrifugation at 4750 × g for 25 minutes at 4 °C. The cell pellet was resuspended in lysis buffer (10 mM Tris-HCl, 1 mM EDTA, pH 7.5) and frozen at -20 °C. Lysis was completed by thawing the cell suspension and sonicating on ice with repeated pulse cycles until the sample became partially clear and less viscous. 10 µg/mL DNase I was added and the cell debris was removed by centrifugation at 28,000 × g for 10 minutes at 4 °C. As GB1 is stable at low pH,22 acid precipitation (20 % acetic acid added dropwise to pH 4.5) was used to remove E. coli proteins from the extract. The suspension was cleared by centrifugation (28,000 × g for 10 minutes) and the pH was increased to 7.0 before loading onto a DEAE column equilibrated in 20 mM TRIS-HCl at pH 7.0. GB1 and GB1-GR1 bound to the column and were eluted using a linear NaCl gradient. Although GB1-GR2-5, GB1-GRGRGR and GB1-linker-GR5 eluted in the flow-through, sufficient purification was achieved by removal of anionic extract components. Purification was completed by size exclusion chromatography (Superdex G75) in 20 mM KH2PO4, 50 mM NaCl, pH 6.0. Protein purity and integrity was assessed by SDS-PAGE and mass spectrometry.

Mass spectrometry. The masses of all of the proteins were determined using a Waters LCT Premier XE Time of Flight mass spectrometer run in positive ion mode equipped with a Waters 2795 HPLC and MassLynx Software.

Native Gel Electrophoresis. 20 µL samples of 0.2 mM GB1-GRn were analysed in 2 % agarose gels prepared in 20 mM KH2PO4, 50 mM NaNO3 at pH 6.0.34 To minimize the effects of resistive heating, the gels were equilibrated for 30 min at 4 °C prior to sample 5 ACS Paragon Plus Environment

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loading. Electrophoresis was performed at a constant voltage (100 V) for 30 min at 4 °C. The gels were fixed directly after electrophoresis by incubating for 1 hour at 20 °C in a 45 % methanol, 10 % acetic acid solution, and stained overnight by incubating in a 50 % ethanol, 10 % acetic acid solution containing 0.005 % Coomassie brilliant blue R. Images of the gels were obtained using a flatbed scanner and processed in Adobe Photoshop.

NMR Sample Preparation. The typical sample conditions comprised 0.2-0.3 mM pure protein in 20 mM KH2PO4, 50 mM NaCl, 10 % D2O at pH 6.0. The sample pH was verified to be constant (± 0.05 pH units) before and after NMR data acquisition.

In-Cell NMR Sample Preparation. Two hours post-induction, 50 mL cultures expressing 15

N or

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F labelled protein were harvested by centrifugation at 530 × g at 20 °C for 10

minutes and the pellet was resuspended by gently mixing with 20 mM Na2HPO4, 40 % D2O. pH 7.0. Samples were prepared immediately prior to the NMR data collection and were in the magnet for 100.0


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Many globular proteins are ‘invisible’ or weakly detectable by in-cell NMR.24-27,29 This result is attributed to drastic resonance broadening caused by multifarious, attractive interactions between the test protein and cytoplasmic macromolecules which slows protein tumbling (and increases the rotational correlation time). Indeed, in-cell NMR studies support the significance of transient, non-specific or ‘quinary’ interactions that arise as a consequence of macromolecular crowding and confinement in cellulo.30-32 Several recent investigations have attempted to decipher the “physicochemical code” for quinary interactions in E. coli.17,26,27,30-32,34 Here, the quinary interactions of -GRn motifs were identified. The successive decrease in the in-cell spectral quality of GB1-GRn (n = 1-3) and the complete loss of signal at n = 4 or 5 likely arises from increased quinary interactions between the argininerich motifs and the E. coli cytoplasm. This result is consistent with the fact that the E. coli cytoplasm is rife with anionic macromolecules.43,44 Both charge-charge interactions, and the binding propensities of the guanidinium group16-19 are contributors to -GRn promiscuity. Cell Lysis Disrupts GB1-GRn Interactions. Interestingly, cell lysis by freeze/thaw was sufficient to render GB1-GR5 (Figure S4) and GB1-linker-GR5 (Figure S3) fully NMR detectable. These results suggest that the interactions between GB1-GR5 and cytoplasmic components were disrupted by gentle lysis procedures. Further NMR experiments were performed by spiking E. coli extracts with

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N-labelled test protein. Cell extracts are an

attractive, biologically-relevant medium for study as they are easy to handle and amenable to treatment with exogenous components.17,28-30,34,45-47 Recently, we quantified the total macromolecular concentration of E. coli extracts prepared from in-cell NMR samples to be ~2-fold lower than extracts prepared from saturated LB cultures.34 The latter, at macromolecular concentrations of ~90 mg/mL, can be considered moderately crowded. Figure S5 shows the HSQC spectra of each GB1-GRn mutant added to such extracts. High quality spectra were obtained in each case, suggesting that the interactions between E. coli macromolecules and GB1-GR4 or GB1-GR5 were diminished in extracts despite being appreciable in cellulo. 1HN line-widths were measured for 35 peaks in extracts containing GB1-GR1, GB1-GR3, or GB1-GR5 (Table S1). A comparison with the line-widths from the pure proteins revealed minor changes (~2 Hz on average) for GB1-GR1 and GB1-GR3. In the case of GB1-GR5, the average line-width increase was ~6 Hz suggesting that some weak, attractive interactions occur in the extract. To dissect further these interactions, extracts containing GB1-GR5 were loaded onto an SEC column26,34 equilibrated in 20 mM KH2PO4 50 mM NaCl pH 6.0. GB1-GR5 eluted at a volume (~80 mL) corresponding to the pure 13 ACS Paragon Plus Environment

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protein, indicating that GB1-GR5 interactions are too weak (i.e. Kd > 100 µM) to withstand the SEC column (Figure S6).


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Conclusions The precise control of protein assembly in physiological environments remains a major goal of chemical biology and supramolecular research. Arginine-rich motifs are versatile handles yet knowledge of their interaction propensities in cellulo is sparse. Here, 15N and

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F in-cell NMR methods revealed that fusion to a C-terminal -GRn (n = 1-5) motif

rendered the ‘biologically inert’ GB1 domain increasingly sticky relative to the E. coli cytosol. The GB1-GRn (n = 1-3) variants were detectable by in-cell NMR spectroscopy but the spectral quality decreased with increasing arginine content. GB1-GR4 and GB1-GR5 were undetectable suggesting that these proteins were sequestered into (non-specific) high molecular weight complexes. This promiscuity highlights the potential of short arginine motifs as mediators of macromolecular assembly. Although specificity is desirable in the design of protein-macromolecule interactions,1 the promiscuity of the -GR4 motif may be harnessed to modulate existing interactions. Other studies also support the significance of short arginine motif stickiness. For instance, a triarginine motif (R3W3) mediates RNA association on model protocell membranes,12 while a pentaarginine motif facilitates cellular uptake, endosomal escape and cytosolic localization.21 Although significant inside E. coli cells, the assemblies of GB1-GR4 or GB1-GR5 did not persist in cell extracts. The interactions were disrupted by gentle lysis procedures or in moderately crowded heterogeneous extracts. The transient nature of the GB1-GR4 or GB1GR5 interactions suggests a comparison with quinary interactions,30-32,45 which are usually destroyed by cell lysis and therefore difficult to study. Mounting evidence supports the significance of weak, electrostatic interactions between proximal macromolecular surfaces.31,32,34,45-47 The simple and physiologically-relevant48 -GRn series are useful models to investigate the physicochemical underpinnings of quinary structure mediated by arginine. We demonstrated the pervasive, attractive interactions between -GRn motifs and components of the E. coli cytoplasm. Given the abundance of charged molecules in E. coli,30,34,43,44 charge-based interactions (e.g. charge-charge, cation-π, salt bridges) likely govern -GRn quinary structure. These interactions must be considered when fusing short arginine motifs to protein surfaces in systems designed for in-cell applications. The -GRn motifs are relevant to disease biology. Short, disordered arginine-rich motifs are abundant in low complexity domains (LCDs) that mediate liquid-liquid phase separation through multivalent interactions.48-50 Additionally, simple arginine-containing poly-dipeptides (i.e. GR and PR) are toxic as they interact weakly but preferentially with the LCDs of RNA-binding proteins involved in membrane-less organelle formation.20 This 15 ACS Paragon Plus Environment

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phenomenon contributes to diseases such as amyotrophic lateral sclerosis and frontotemporal dementia. Although >50 dipeptide repeats are linked with pathogenicity, our data demonstrate the weak pervasive interactions of shorter motifs, akin to those expressed in healthy individuals.20 The systematic investigation of -GRn and (GR)n stickiness in physiological samples will help to delineate the role of charge clustering and multivalency in driving dipeptide repeat interactions with LCDs, liquid-liquid phase transitions and disease progression.


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Acknowledgements We thank M. C. Jürgens and M. Vignoles for technical assistance, and R. A. Doohan for maintaining the NMR facility.


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Supporting Information Line-widths of GB1-GRn 1HN resonances extracted from HSQC spectra acquired in buffer or in E. coli extract (Table S1). In-cell HSQC spectra of GB1-GR4, GB1-GR5, a plasmid-free E. coli slurry and supernatant (Figure S1). In-cell spectra of GB1-GR3 and GB1-GRGRGR (Figure S2).

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F NMR spectra of GB1-linker-GR5 in-cell and after freeze/thaw lysis (Figure

S3). Spectra of GB1-GR5 in-cell and in a lysate (Figure S4). HSQC spectra of 0.2 mM GB1GRn in concentrated E. coli extracts (Figure S5). SDS-PAGE analysis of the size exclusion chromatogram of an extract containing 0.2 mM GB1-GR5 (Figure S6).


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Funding Information This research was supported by NUI Galway and Science Foundation Ireland (grants 13/ERC/B2912 and 13/CDA/2168 to PBC).


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For Table of Contents Use Only Short Arginine Motifs Drive Protein Stickiness in the Escherichia coli Cytoplasm

Ciara Kyne, Peter B. Crowley


Short Arginine Motifs Drive Protein Stickiness in the Escherichia coli

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