Structural snapshots of CmoB in various states during wobble uridine modification of tRNA

https://doi.org/10.1016/j.bbrc.2020.11.033Get rights and content

Highlights

  • A novel open conformation of CmoB captured in an X-ray crystal structure.

  • An open state of the enzyme triggers the release of a cofactor.

  • Crystal structures of CmoB with CxSAM and SAH determined.

  • Correlation between the binding affinity and charge distribution within a cofactor.

Abstract

CmoB utilizes carboxy-S-adenosyl-l-methionine (CxSAM) to carry out unusual carboxymethyl transfer to form 5-carboxymethoxyuridine (cmo5U) of several tRNA species in Gram-negative bacteria. In this report, we present three X-ray crystal structures of CmoB from Vibrio vulnificus representing different states in the course of the reaction pathway; i.e., apo-, substrate-bound, and product-bound forms. Especially, the crystal structure of apo-CmoB unveils a novel open state of the enzyme, capturing unprecedented conformational dynamics around the substrate-binding site. The apo-structure demonstrates that the open conformation favors the release of CxSAM thus representing an inactive form. Our crystal structures of CmoB complexed with CxSAM and S-adenosyl-l-homocysteine (SAH) and combined binding assay results support the proposed mechanism underlying the cofactor selectivity, where CmoB preferentially senses negative charge around amino acid residues Lys-91, Tyr-200, and Arg-315.

Introduction

Crick proposed that the non-canonical base pairing can be formed between the 5′-nucleotide of anticodon and the 3′-nucleotide of a triplet codon in wobble theory [1]. For example, the uridine at the wobble position can read the G-ending codon in addition to the A-ending codon. Generally, a modification of wobble nucleoside within tRNA further confers a unique decoding property to anticodon [[2], [3], [4]], and numerous post-transcriptional modifications discovered since then added extra layers of complexity to the original model by Crick [[5], [6], [7], [8], [9]]. In particular, wobble uridines are frequently modified, for example, to 5-carboxymethoxyuridine (cmo5U) or 5-methoxyuridine (mo5U) in Gram-negative or Gram-positive bacteria, respectively. Biosynthetic pathways leading to both modifications are now fully identified (Fig. 1): TrhO or TrhP hydroxylates the C5 of the wobble uridine within the cognate tRNAs to produce 5-hydroxyuridine (ho5U) [10,11], which becomes the nucleophile in the following alkylation step by CmoB to form cmo5U in Gram-negatives [12,13], or by TrmR to form mo5U in Gram-positives [14]. Lastly, cmo5U in certain isoacceptors such as tRNAAla1, tRNASer1, tRNAPro3, and tRNAThr4 are targeted for further methylation by CmoM to produce its methyl ester, mcmo5U [8]. The (m)cmo5U/mo5U modification at the wobble position enables non-canonical base pairings with the 3′-nucleoside of a triplet codon, contributing to the expansion of codon degeneracy [15]. Selective tRNAs specific for Ala, Val, and Pro with wobble cmo5U or mcmo5U were demonstrated to recognize all four degenerate codons in the absence of other isoacceptors in Salmonella enterica [16,17].

CmoB belongs to the unique CxSAM-dependent carboxymethyl transferase family, which is best characterized in the biosynthesis of (m)cmo5U. The enzyme installs the carboxymethyl cap on the 5-hydroxyl group of ho5U within the hypomodified tRNAs using CxSAM as the two carbon donor, which is produced from SAM and prephenate by CmoA [12]. The CmoB-directed reaction results in the formation of cmo5U within tRNA anticodon stem-loop and SAH. The crystal structures of CmoB from E. coli complexed with CxSAM and of the cofactor-free form were reported [13]. Invariantly conserved amino acid residues Lys-91, Tyr-200, and Arg-315 participate in crucial polar interactions with the mobile carboxymethyl group of CxSAM in the complex structures. In the cofactor-free structure, a sulfate ion has been located in the active site, which is occupied by the carboxymethyl group of CxSAM in cofactor-bound structures. Collective structural data imply that an anion is preferably accommodated around this region in the CxSAM binding pocket. It was also shown that Lys-91 acts as an anti-determinant for SAM, which has an overall +1 charge at the physiological condition. Consequently, distinct electrostatic interactions with a cofactor provide the major principle for discriminating CxSAM vs. SAM.

Here we report X-ray crystal structures of ligand-free CmoB from V. vulnificus determined to 2.10 Å, disclosing an unprecedented open conformation of the enzyme around the substrate-binding pocket. Furthermore, we present crystal structures of CmoB complexed with its substrate, CxSAM, and the product, SAH, which have been determined to 2.90 Å and 2.30 Å, respectively. A series of structures of CmoB described herein offer snapshots of diverse catalytic states of the enzyme in the course of the wobble uridine modification. Lastly, we measured a dissociation constant of SAH by isothermal titration calorimetry and analyzed the implication in substrate selectivity.

Section snippets

Cloning and protein purification

The CmoB gene was amplified from the genomic DNA of Vibrio vulnificus MO6-24/O stain using Phusion DNA polymerase (Thermo Scientific) in a standard polymerase chain reaction (PCR) reaction. The description of primers used in PCR steps is included in Table S1. Their amplified PCR fragment was purified by gel extraction (Qiagen) and inserted into LIC-pLATE31 (Thermo Scientific) and verified by the DNA sequence analysis (Macrogen, Korea). Escherichia coli BL21 (DE3) cells were transformed with

Confirmation of in vitro activities of recombinant V. vulnificus CmoB

The activity of recombinant V. vulnificus CmoB was examined through in vitro assays using ho5U-containing tRNASer as a substrate. The hypomodified tRNASer was overexpressed from cmoB-deficient cells of E. coli, where the distinct size of the isoacceptor (94 nt including cloning sequence) allows the separation from other endogenous tRNA species (typically 76 nt) on a gel (Fig S1). This size-based purification method facilitates the preparation of an RNA sample, which precludes the need for a

Discussion

In this report we present three crystal structures of CmoB in different states; i.e., the ligand-free, substrate- and product-bound forms. The unprecedented ‘open’ conformation around the active site of CmoB has been captured in our ligand-free structure, in which a segment of approximately 16 amino acid residues forming a part of the active site wall swung out towards bulk solvent. The absence of electron densities corresponding to CxSAM in the ligand-binding pocket suggests a

Accession numbers

Coordinates and structure factors related to this work have been deposited in the PDB with accession numbers 7CT8, 7CT9, and 7CTA.

Funding

This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) (grant number: NRF-2020R1F1A1072849).

Acknowledgments

We thank Pohang Accelerator Laboratory and the beamlines 7A and 11C for their support with the X-ray diffraction data collection.

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