1H, 13C, 15N Backbone Assignment of the Minimally Tied Trefoil Knot, MTTSA , a 23s rRNA SPOUT Methyltransferase

doi:10.21203/rs.3.rs-8320904/v1

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1H, 13C, 15N Backbone Assignment of the Minimally Tied Trefoil Knot, MTTSA , a 23s rRNA SPOUT Methyltransferase

https://doi.org/10.21203/rs.3.rs-8320904/v1

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Knotted Methyltransferases (MTase) from the SpoU-TrmD (SPOUT) family offer a unique opportunity to study a protein knot topology and enzymatic function. The knotted methyltransferase from Staphylococcus aureus, MTT_SA (PDB: 1vh0, 4fak), is an ɑ/β-knotted 23s rRNA MTase containing only the minimal scaffold among the SPOUT family members. This dimeric enzyme is a minimalist model to study the deep + 3₁ knot. Here, we report the non-proline backbone assignments with 98.8% completion using TROSY-based NMR experiments on uniformly labeled ²H/¹³C/¹⁵N-labeled protein. Complementary HBHA(CO)NH experiments on ¹³C/¹⁵N-labeled protein were completed for Hα/Hβ sidechain assignments. Both backbone and sidechain assignments were deposited in the Biological Magnetic Resonance Bank (BMRB ID: 53391). Of 164 assigned residues, TALOS-N predictions provided 88.4% unambiguous assignments based on the H_N, H_α, C_α, C_β and N chemical shifts. In addition, the predicted secondary structure based on torsion angels and the ΔδC_α-ΔδC_β profile are in agreement with the crystal structures.

Methyltransferase

Knotted protein

SPOUT

Protein NMR

MTT (minimally tied trefoil)

Knotted polypeptide chains represent a rare and complex topological feature among protein folds. With an increasing number of knotted proteins being identified across diverse structural families, understanding their unique topology provides insight into folding, stability, and enzymatic function of the knotted region as these proteins have become increasingly important (Capraro and Jennings. 2016). The folding mechanisms of knotted SPOUT MTase has been investigated through computational modeling and characterized with energy landscape theory (Andrews et al. 2017; Burban et al. 2015; Burban and Jennings, 2017; Capraro et al. 2020; Dahlstrom et al. 2022; Tkaczuk et al. 2007). Importantly, the correct chirality of the crossing loop is required to overcome the first energy barrier, forming the intermediate slipknot or plug topology. Subsequentially, the C-terminus threads through the loop to establish the native fold of a trefoil knot, which overcomes the second energy barrier (Strassler et al. 2022; Tkaczuk et al. 2007). Furthermore, matrix map analyses indicate that the active sites of SPOUT enzymes typically reside within the knotted region, where increased intra-chain contacts enhance structural stability and shield bound ligands from solvent accessibility (Dabrowski-Tumanski et al. 2016). Despite these computational and theoretical insights, experimental data at the residue level resolution of rRNA MTases remain limited (Andrews et al. 2017; Burban et al. 2015; Burban and Jennings, 2017; Capraro et al. 2020). Solution NMR spectroscopy has provided powerful insight, addressing this gap in knowledge (Burban and Jennings. 2017; Capraro et al. 2020). The residue-specific information has generated details regarding backbone conformation, dynamics, and ligand interactions that cannot be fully captured by modeling and crystallography alone (Burban et al. 2015; Burban and Jennings, 2017; Capraro et al. 2020; Zhong et al. 2019).

In this paper, we focus on a member of the SPOUT methyltransferase superfamily exhibiting a characteristic deep trefoil (+ 3₁) knot that contributes to both structural integrity and enzymatic activity. Staphylococcus aureus, MTT_SA (PDB code: 1vh0, 4fak), is a dimeric 23s rRNA methyltransferase and represents another member of the minimalist model within this knotted protein family (Andrews et al. 2013; Boundy et al. 2013; Burban et al. 2015; Burban and Jennings. 2017; Capraro et al. 2020; Capraro and Jennings. 2016; Strassler et al. 2002; Tkaczuk et al. 2007). Different from other SPOUT members such as TrmD and RlmB, which incorporate additional secondary structure elements around the knotted core, MTT_SA retains only the minimal catalytic architecture, making it a simplified model for investigating the structural and functional role of knots within proteins (Tkaczuk et al. 2007). MTT_SA adopts a highly conserved α/β knotted fold characteristic of SPOUT family, with five β-strands surrounded by five α-helices. The + 3₁ trefoil knot region is near the C-terminus and is defined by the crossing (T74-L85), wing (D104-L127) and threading loops (S128-F134).

NMR studies of MTT_SA enable direct characterization of how knot topology shapes folding, stability, and enzymatic function. Backbone and sidechain resonances assignments serve as an essential foundation for further analyses on many unresolved questions including if the trefoil knot can be fully untied and what addition role the trefoil knot may serve in enzymatic activities. Here, we report the non-proline backbone assignments and H_α H_β sidechain assignments for the 39kDa native homodimeric SPOUT MTase, MTT_SA. TROSY NMR was applied to acquire double and triple resonance NMR spectra on ²H/¹³C/¹⁵N labeled sample to enhance signals for backbone assignment, and the HBHA(CO)NH spectra on ¹³C/¹⁵N labeled sample were acquired for the sidechain assignment (Fernandez. 2003; Foster et al. 2007; Gardner and Kay. 1998; Pervushin et al. 1998; Xu and Matthews. 2011).

Protein Expression and Purification

The gene encoding MTT_SA was cloned into a pET-24d vector and expressed in Escherichia coli BL21(DE3) competent cells. Uniformly ²H/¹³C/¹⁵N and ¹³C/¹⁵N labeled samples were prepared in D₂O-M9 minimal medium. For ²H/¹³C/¹⁵N labeled sample, we used deuterated D-Glucose (U-¹³C₆, 1,2,3,4,5,6,6-D₇). MTT_SA expression with 50 mg/mL kanamycin as antibiotic was grown at 37°C until OD₆₀₀ reached 0.6 and was induced with 1mM Isopropyl-β-D-thiogalactopyranoside (IPTG) at 25°C for 16–18 hours. The cell culture was lysed by sonication in 50mM NaPO₄, 300mM NaCl, pH 8.0, and the protein was isolated using Ni-NTA (Qiagen) resin with increasing concentration of imidazole. The protein of interest was eluted using 50mM NaPO₄, 300mM NaCl, 150mM imidazole, pH 8.0. The protein was further purified by size-exclusion chromatography on Superdex 200 in 75mM NaOAc, pH 5.6, 1% glycerol.

NMR Sample Preparation

For backbone assignments, purified ²H, ¹³C, ¹⁵N labeled MTT_SA was exchanged into 75mM NaOAc-d3, pH 5.6, 1% Glycerol, and 10% D₂O. TROSY based triple resonance NMR spectra were acquired at 305K. For sidechain assignments, purified ¹³C, ¹⁵N labeled MTT_SA was exchanged into the same buffer conditions, and the HBHA(CO)NH spectra was acquired at 305K.

NMR Experiments

All NMR experiments for MTT_SA backbone assignments were performed on a Bruker Avance 800 MHz spectrometer equipped with a TROSY-optimized probe. The following TROSY double and triple resonance experiments were conducted to obtain resonance information for the backbone assignments: TROSY-HSQC, TROSY-HNCACB, TROSY-HNCO, TROSY-HNCACO (Fitzkee et al. 2010). Additionally, the HBHA(CO)NH experiment was performed on a Bruker Avance Neo600 MHz spectrometer in order to obtain side chain proton resonance, enhancing the completeness of MTT_SA backbone assignments.

All NMR datasets were acquired through Bruker TopSpin^™ NMR Software (Version 4.1.4). Raw spectral data was processed using NMRfx Analyst (Norris et al. 2016). Resonance assignments were carried out using Sparky (Lee et al. 2015). The backbone assignments were determined based on triple-resonance correlations (Toshio et al. 1994).

Secondary Structure Analysis

The secondary structure was evaluated experimentally using the ΔCα-ΔCβ profile (Wishart and Sykes. 1994). In α-helical regions, ΔCα is positive and ΔCβ is negative; in β-strands ΔCα is negative and ΔCβ is positive; value near zero indicates coil. ΔCα-ΔCβ values were calculated from backbone chemical shifts using random coil index as reference (δCα_observed - δCα_RCI) − (δCβ_Observed - δCβ_RCI) (Berjanskii and Wishart. 2005). This profile shows regions of positive values consistent with α-helices and negative values consistent with β-strands and interprets contiguous region of ≥ 3 residues that exceed ± 1ppm. Glycine without Cβ is only assessed with Δ δCα.

Extent of Assignment and Data Deposition

Here, we report the ¹H, ¹³C, ¹⁵N backbone assignment of MTT_SA, a 39kDa dimeric SPOUT MTase. Chemical shift assignments were deposited in the BMRB, ID 53391. Figure 1 represents the assigned ¹H-¹⁵N TROSY HSQC spectrum. NMR data is visualized and assigned as one protomer. All backbone and sidechain chemical shifts were determined through a series of 3D NMR experiments (see methods) by tracing the previous residue chemical shifts.

Assignments for H_α and H_β were obtained from the HBHA(CO)NH experiments to further complement and complete the backbone details. All chemical shifts assignments including H_N, H_α, C_α, C_β and N, were submitted to the TALOS-N server (https://spin.niddk.nih.gov/bax/software/TALOS-N/) for secondary structure prediction based on the submitted backbone φ/ψ torsion angles and sidechain χ1 torsion angles (Fig. 2a) (Shen and Bax. 2013). Of the 164 assigned residues, TALOS-N classified 81.7% as Good (Strong), and 6.7% as Good (Generous), yielding 88.4% unambiguously assigned coverage. The predicted secondary structure pattern agrees well with the crystal structure (PDB: 1vh0), especially all α-helices with some minor variations of the β-strands. In our ΔCα-ΔCβ profile (Fig. 2b), β2, β3 and β5 show negative values where each align with the corresponding β-strand in the crystal structure. The boundaries from the chemical shifts are in close agreement with the crystal structure. Interestingly, we observed several differences in the length determination of the β-strands. Our solution-NMR data indicated that β2 and β3 are shorter, and the 3-residue strand (β5) is not detected (Fig. 2c). The discrepancies between the NMR and crystal structure are due in part to the short β strands unraveling in the solution dynamics compared with static crystalline model (Eyal et al. 2005; Hinsen. 2008). Furthermore, β2, β3 and β5 lie near the ligand-binding pocket (Fig. 2c), a dynamic region where stabilization as a result of ligand-binding is observed Boundy et al. 2013; Wallin et al. 2007). Taken together, the results presented provide the basis for future evaluations of the folding, stability, ligand binding stabilization and enzyme function of MTT_SA.

Data Availability

Chemical shift assignments for MTT_SA reported in this manuscript are deposited in the Biological Magnetic Resonance Data Bank (BMRB), access ID: 53391. All other data sets are available upon request.

Authors disclose no financial nor non-financial interests that are directly or indirectly related to the work submitted for publication.

Funding Information

We would like to acknowledge National Science Foundation PHY2210636

Author Contribution

Tiange Tao performed the experiments, processed the data, prepared figures and wrote the manuscript. Dominique T. Capraro initiated the research project, generated preliminary research data, contributed experimental design, troubleshooting, and editing manuscripts. Patricia A. Jennings conceived and initiated the research project, contributed to experimental design and editing manuscript.

Acknowledgement

We would like to thank Dr. Lalit Deshmukh for his assistance with the TROSY-NMR experiment. Dr. Xuemei Huang for her guidance and maintenance at UCSD NMR facility.

Data Availability

Chemical shift assignments for MTT SA reported in this manuscript are deposited in the Biological Magnetic Resonance Data Bank (BMRB), access ID: 53391. All other data sets are available upon request.

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You are reading this latest preprint version

1H, 13C, 15N Backbone Assignment of the Minimally Tied Trefoil Knot, MTTSA , a 23s rRNA SPOUT Methyltransferase

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Version 1

Abstract

Figures

Biological Context

Methods

Protein Expression and Purification

NMR Sample Preparation

NMR Experiments

Secondary Structure Analysis

Extent of Assignment and Data Deposition

Data Availability

Declarations

Funding Information

Author Contribution

Acknowledgement

Data Availability

References

Additional Declarations

Supplementary Files

Status:

Version 1