Mechanistic Insight into Trimethylamine N-Oxide Recognition by the Marine Bacterium Ruegeria pomeroyi DSS-3

ABSTRACT

Trimethylamine N-oxide (TMAO) is an important nitrogen source for marine bacteria. TMAO can also be metabolized by marine bacteria into volatile methylated amines, the precursors of the greenhouse gas nitrous oxide. However, it was not known how TMAO is recognized and imported by bacteria. Ruegeria pomeroyi DSS-3, a marine Roseobacter, has an ATP-binding cassette transporter, TmoXWV, specific for TMAO. TmoX is the substrate-binding protein of the TmoXWV transporter. In this study, the substrate specificity of TmoX of R. pomeroyi DSS-3 was characterized. We further determined the structure of the TmoX/TMAO complex and studied the TMAO-binding mechanism of TmoX by biochemical, structural, and mutational analyses. A Ca²⁺ ion chelated by an extended loop in TmoX was shown to be important for maintaining the stability of TmoX. Molecular dynamics simulations indicate that TmoX can alternate between “open” and “closed” states for binding TMAO. In the substrate-binding pocket, four tryptophan residues interact with the quaternary amine of TMAO by cation-π interactions, and Glu131 forms a hydrogen bond with the polar oxygen atom of TMAO. The π-π stacking interactions between the side chains of Phe and Trp are also essential for TMAO binding. Sequence analysis suggests that the TMAO-binding mechanism of TmoX may have universal significance in marine bacteria, especially in the marine Roseobacter clade. This study sheds light on how marine microorganisms utilize TMAO.

IMPORTANCE Trimethylamine N-oxide (TMAO) is an important nitrogen source for marine bacteria. The products of TMAO metabolized by bacteria are part of the precursors of the greenhouse gas nitrous oxide. It is unclear how TMAO is recognized and imported by bacteria. TmoX is the substrate-binding protein of a TMAO-specific transporter. Here, the substrate specificity of TmoX of Ruegeria pomeroyi DSS-3 was characterized. The TMAO-binding mechanism of TmoX was studied by biochemical, structural, and mutational analyses. Moreover, our results suggest that the TMAO-binding mechanism may have universal significance in marine bacteria. This study sheds light on how marine microorganisms utilize TMAO and should lead to a better understanding of marine nitrogen cycling.

INTRODUCTION

It has been reported that one-half of global primary production is generated in the oceans, mainly by marine phytoplankton, and a large fraction becomes dissolved organic matter (DOM) by various mechanisms (1, 2). Trimethylamine N-oxide (TMAO), which is widespread in the ocean, is an important component of the DOM and an osmoprotectant (3, 4). TMAO participates in many important physiological processes (5). In marine surface waters, the concentration of TMAO can reach 79 nM (4, 6). In deep-sea organisms, TMAO plays a key role in counteracting the protein-denaturing effects of urea and acts as a potent protein stabilizer (7, 8). In marine bacteria, TMAO can be catabolized to small, volatile, methylated amines (MAs), which are precursors of the greenhouse gas nitrous oxide in the marine atmosphere and in marine aerosols (3, 9). Because of their alkalinity, MAs can neutralize atmospheric acidity caused by organic and inorganic acids (10). Additionally, MAs are significant sources of methane for a variety of marine systems, including marine snow (11–13). They are thought to facilitate the coexistence of methanogenesis and sulfate reduction in marine sediments (11).

Marine microorganisms have efficient permeases that transport various DOMs (2). The BCCT (betaine, carnitine, and choline transporter) family and the ABC (ATP-binding cassette) superfamily are two types of transporters that have been widely reported to import organic osmolytes, including TMAO and some other compounds with molecular similarity to TMAO, such as betaine, choline, and carnitine (Fig. 1) (14, 15). BCCT carriers are membrane proteins, most of which contain 12 predicted trans-membrane α-helices (14, 15). ABC transporters are one of the oldest and largest protein superfamilies and are ubiquitous from prokaryotes to eukaryotes (16–18). In bacteria, ABC transporters are primarily responsible for cellular uptake of nutrients and the export of harmful substances (19). They can transport a wide range of substrates, including ions, sterols, amino acids, and drugs (16). ABC importers usually consist of three subunits: a transmembrane domain containing the translocation pathway, a cytoplasmic ATP-binding domain to provide the necessary energy for transport, and a periplasmic substrate-binding protein (SBP) to perform the first step in the recognition and binding of substrate. One group of ABC importers, termed Cbc, specialize in the transport of quaternary ammonium compounds (QACs), including choline, betaine, and carnitine (20). The crystal structures of Cbc-associated SBPs are similar, with three or four aromatic residues in the binding pocket forming cation-π interactions with QACs (21–27). TMAO is a QAC. An ABC importer that is specific for TMAO was identified in Aminobacter aminovorans in the Alphaproteobacteria (28), but the genes encoding this transporter have been unknown. Recently, the gene cluster encoding the ABC importer specific for TMAO in Ruegeria pomeroyi DSS-3, which was also found in a variety of marine bacteria, was identified and was termed tmoXWV (6). Marker exchange mutagenesis and lacZ reporter assays suggest that tmoXWV is essential for TMAO metabolism (6). Metatranscriptomic and metaproteomic data reveal that the tmoX gene encoding the periplasmic SBP TmoX is highly transcribed and expressed in marine environments (29–31). Phylogenetic analysis indicates that TmoX proteins form a distinct subcluster that does not contain any previously characterized SBPs (6), suggesting that TmoX may have characteristics distinct from those of other Cbc-associated SBPs. Despite these findings, the molecular mechanisms by which TMAO is recognized and transported by TmoXWV are still unclear.

FIG 1.

Chemical structures of TMAO, betaine, carnitine, and choline.

TMAO serves as an important nutrient for ecologically important marine heterotrophic bacteria, particularly those in the marine Roseobacter clade (MRC) (32), which is abundant in marine environments and is an important participant in marine C, S, and N cycles (33, 34). Because some ecologically relevant representatives of the MRC are amenable to genetic manipulation, they have become model organisms to investigate bacterial ecophysiology in the marine environment (6). R. pomeroyi DSS-3 is a marine heterotrophic bacterium isolated from seawater of coastal Georgia (35) and is one of the best-characterized models among members of the MRC (36–39). In recent years, some genes from R. pomeroyi DSS-3 related to TMAO metabolism have been identified (6, 40), including the ABC transporter genes tmoXWV specific for TMAO (6). In this study, we characterized the substrate specificity of TmoX of R. pomeroyi DSS-3 and studied the TMAO-binding mechanism of TmoX by biochemical, structural, and mutational analyses. Moreover, the substrate-binding mechanism of TmoX was compared with those of related osmolyte-binding proteins and TMAO-binding proteins. The results revealed that a TMAO-binding mechanism may have universal significance in marine bacteria.

MATERIALS AND METHODS

Bacterial strains and growth conditions.

R. pomeroyi DSS-3 was purchased from the Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures and was cultured in 974 medium at 30°C for 2 days according to the protocol provided (http://www.dsmz.de/). The Escherichia coli strains DH5α and BL21(DE3) were grown in Luria-Bertani (LB) medium at 37°C.

Gene cloning, point mutation, and protein expression and purification.

The full-length tmoX gene was amplified from the genomic DNA of R. pomeroyi DSS-3 using PCR and was subcloned into the pET22b (Novagen) vector with a C-terminal His tag. All of the point mutations in tmoX were introduced using PCR-based methods and were verified by DNA sequencing. The wild-type (WT) TmoX protein and all of the mutants were expressed in E. coli strain BL21(DE3). The cells were cultured at 37°C in LB medium to an optical density at 600 nm (OD₆₀₀) of 0.8 to 1.0 and then induced at 20°C for 16 h with 0.5 mM isopropyl β-d-1-thiogalactopyranoside (IPTG). The proteins were purified first with Ni²⁺-nitrilotriacetic acid (NTA) resin (Qiagen) and then fractionated by anion-exchange chromatography on a Source 15Q column and gel filtration on a Superdex-75 column (GE Healthcare). Approximately 10 mg recombinant TmoX protein was obtained from 1 liter of culture. The selenomethionine derivative of TmoX was overexpressed in E. coli strain BL21(DE3) through 0.5 mM IPTG induction in the M9 minimal medium supplemented with selenomethionine, lysine, valine, threonine, leucine, isoleucine, and phenylalanine. The recombinant selenomethionine derivative was purified in a manner similar to that for native proteins.

Gel filtration analysis.

A Superdex-75 column was preequilibrated with 10 mM Tris-HCl (pH 8.0), 100 mM NaCl and calibrated with ovalbumin (43,000 Da) and carbonic anhydrase (13,700 Da) molecular mass standards from GE Healthcare. The samples were eluted at a flow rate of 0.5 ml/min.

Isothermal titration calorimetry measurements.

Isothermal titration calorimetry (ITC) measurements were performed at 25°C using a MicroCal iTC₂₀₀ system (GE Healthcare). The sample cell was loaded with 250 μl of protein sample (∼60 μM), and the reference cell contained distilled water. The syringe was filled with 40 μl of TMAO (500 μM). The proteins and TMAO were kept in the same buffer containing 10 mM Tris-HCl (pH 8.0) and 100 mM NaCl. Titrations were carried out by adding 0.4 μl of TMAO for the first injection and 1.5 μl for the following 20 injections, with stirring at 1,000 rpm.

Crystallization and data collection.

The purified TmoX protein was concentrated to approximately 5.7 mg/ml in 10 mM Tris-HCl (pH 8.0) and 100 mM NaCl. To obtain crystals of the TmoX/TMAO complex, TmoX protein was mixed with TMAO in a 1:3 molar ratio. Initial crystallization trials for the TmoX/TMAO complex were performed at 20°C using the sitting-drop vapor diffusion method. Diffraction quality crystals of the TmoX/TMAO complex were obtained in hanging drops containing 0.2 M magnesium chloride, 0.1 M Bis-Tris (pH 5.5), and 25% (wt/vol) polyethylene glycol 3350 at 20°C after 1 week of incubation. Crystals of the TmoX/TMAO complex selenomethionine derivative were obtained under the same conditions. X-ray diffraction data were collected on the BL17U1 beamline at the Shanghai Synchrotron Radiation Facility using detector ADSC Quantum 315r. The initial diffraction data sets were processed with the HKL2000 program (41).

Structure determination and refinement.

The crystals of the native TmoX/TMAO complex belong to the P2₁ space group. The structure of the TmoX/TMAO complex Se derivative was determined by single-wavelength anomalous dispersion (SAD) phasing. Two molecules are arranged as a dimer in the asymmetric unit of the TmoX/TMAO complex. One monomer from the resulting structure was subsequently used as the search model for molecular replacement to determine the structure of the native TmoX/TMAO complex, using the CCP4 program Phaser (42). The refinement of the TmoX/TMAO complex structure was performed using Coot (43) and Phenix (44). All of the structure figures were made using the program PyMOL (http://www.pymol.org/).

Molecular dynamics simulations.

The molecular dynamics (MD) simulations for TmoX were performed using the NAMD program suite (45). The model was placed in a rectangular box with a size of 53 by 50 by 45 Å under periodic boundary conditions. Sodium and chlorine ions were randomly spread and located at least 5 Å from the model and at least 5 Å from each other. The entire system was minimized during the following 1 ns. Then, the equilibrated system was subjected to a 50-ns MD simulation at 300 K. The MD simulation ran on 80 cores of the Inspur Tiansuo at the Supercomputing Center of Shandong University. The average wall clock time was approximately 3 h for a 1-ns simulation.

Detection of metal ions.

Inductively coupled plasma atomic emission spectrometry (ICP-AES) measurement was performed using an IRIS Intrepid II XSP (Thermo Electron). To detect the metal ions in TmoX, 3 ml of TmoX was mixed with 8 ml nitric acid to a final TmoX concentration of 30 μM. The sample was incubated at 120°C overnight until the digestion was complete. The sample was then diluted to 10 ml with distilled water and filtered through a 0.22-μm filter membrane before detection.

Fluorescence measurements.

Protein fluorescence spectra were monitored at 25°C on an FP-6500 spectrofluorometer (Jasco). The final concentration of TmoX and all of the mutants was approximately 3 μM in a buffer containing 10 mM Tris-HCl (pH 8.0) and 100 mM NaCl. The wavelength of maximum fluorescence emission was 340 nm following excitation at 280 nm. The wavelength of maximum fluorescence excitation was 286 nm, with emission at 340 nm. Thus, we used an excitation wavelength of 286 nm to measure the intrinsic fluorescence of TmoX. Fluorescence spectra were collected from 300 nm to 420 nm at a scan speed of 1,000 nm/min with a bandwidth of 3 nm. TMAO at a final concentration of 5 μM was added to the protein samples. To determine whether Ca²⁺ has an effect on the binding affinity of TmoX for TMAO, EDTA was added to the protein samples at a final concentration of 1 mM, 2 mM, 3 mM, or 5 mM before fluorescence measurements.

Circular-dichroism spectroscopic assays.

WT TmoX and all of the mutant proteins were subjected to circular-dichroism (CD) spectroscopic assays at 25°C on a J-810 spectropolarimeter (Jasco). CD spectra of the samples at a final concentration of approximately 12 μM were collected from 250 nm to 200 nm at a scan speed of 500 nm/min with a bandwidth of 2 nm. All of the samples were in a buffer containing 10 mM Tris-HCl (pH 8.0) and 100 mM NaCl. To determine if the Ca²⁺ ion in TmoX has an effect on maintaining the structure of TmoX, EDTA was added to the TmoX samples at a final concentration of 5 mM.

Differential scanning calorimetry measurements.

Differential scanning calorimetry (DSC) measurements were carried out using a MicroCal VP-DSC (GE Healthcare). The sample cell was loaded with 250 μl of TmoX (∼15 μM), and the reference cell contained distilled water. The temperature was raised from 25°C to 90°C at a constant heating rate of 1°C/min. To determine the effects of Ca²⁺ on the binding affinity of TmoX, EDTA was added to the protein samples at a final concentration of 5 mM.

Protein structure accession number.

The structure of the TmoX/TMAO complex has been deposited in the Protein Data Bank (PDB) under the accession code 4XZ6.

RESULTS AND DISCUSSION

Characterization of the substrate specificity of TmoX of R. pomeroyi DSS-3.

Although the tmoX-lacZ fusion confirmed that the expression of tmoX was induced exclusively by TMAO (6), the substrate specificity of TmoX has not been biochemically confirmed. In order to analyze the substrate specificity of TmoX, the full-length tmoX gene containing 1,002 nucleotides was amplified from R. pomeroyi DSS-3 and was expressed in E. coli BL21(DE3) cells. The first 42 residues of TmoX were predicted to be a signal peptide (http://www.cbs.dtu.dk/services/SignalP/) and were verified by N-terminal residue sequencing. The first 10 N-terminal residues of the recombinant TmoX were confirmed to be Asp43-Ser-Ser-Asp-Pro-Ile-Val-Ile-Pro-Ile. To analyze the substrate specificity of TmoX, we tested the affinities of recombinant TmoX for TMAO, betaine, choline, and carnitine by using ITC measurements. Among these osmolytes, the recombinant TmoX possessed a high binding affinity for TMAO, with a K_d (dissociation constant) of 1.6 ± 0.1 μM (Fig. 2 and Table 1), similar to those of other reported Cbc-type SBPs for their substrates (21, 22, 24, 25). Surprisingly, the recombinant TmoX also presented a high binding affinity for choline, with a K_d of 3.8 ± 0.1 μM (Fig. 2 and Table 1). However, while TMAO led to a significant increase in the expression of tmoX in R. pomeroyi DSS-3, choline did not induce the expression of tmoX (6). It was also reported that when tmoX was mutated, the growth rate of R. pomeroyi DSS-3 was unaffected when grown on choline as a sole N source (6). Although these findings seem to suggest that the choline-binding affinity presented by recombinant TmoX may not make physiological sense, further experiments are still needed to sort out the possible role of choline as a putative substrate for TmoX.

FIG 2.

ITC data for titrations of TMAO (A) and choline (B) into recombinant TmoX. ITC traces (top) and integrated binding isotherms (bottom) are shown.

TABLE 1.

Thermodynamic parameters determined by ITC measurements^a

Substrate	Kd (μM)	ΔH (kcal/mol)	−TΔS (kcal/mol)
TMAO	1.6 ± 0.1	−4.0 ± 0.1	−3.9 ± 0.1
Betaine	−	−	−
Choline	3.8 ± 0.1	−6.6 ± 0.5	−0.8 ± 0.5
Carnitine	−	−	−

^aΔH, change in enthalpy; T, temperature; ΔS, change in entropy. −, little substrate-binding affinity was detected under the experimental conditions.

Overall structure of TmoX.

In order to analyze the mechanism of binding of TmoX to TMAO, we tried to obtain crystals of TmoX with and without TMAO. While attempts to obtain diffraction quality crystals of apo-TmoX were not successful, the crystal structure of the TmoX/TMAO complex was determined to 2.2 Å by the SAD method using a selenomethionine derivative (Table 2). The crystals of the TmoX/TMAO complex belong to the P2₁ space group, with two molecules arranged as a dimer in the asymmetric unit. Gel filtration analysis showed that TmoX exists in a monomeric state in solution (Fig. 3A), indicating that the dimeric TmoX observed in the crystal is a result of crystal packing. The PISA server (http://www.ebi.ac.uk/pdbe/prot_int/pistart.html) analysis also implied that the dimer is a result of crystal packing. Therefore, we limit our discussion to the monomer of TmoX. The overall structure of TmoX exhibits the typical topology of an F-III cluster of the ABC transporter superfamily (46). Each molecule of TmoX contains two distinct domains connected by two hinge regions (Asp126 to Thr129 and Ala265 to Tyr268), and a TMAO molecule is observed in the substrate-binding pocket between the two domains (Fig. 3B). Domain I comprises residues Asp43 to Val128 and Gly267 to Gly333, and domain II comprises residues Thr129 to Thr266. Both of the domains are made up of central β-sheets flanked by α-helices. Domain I is more compact than domain II because domain II contains more loops (Fig. 3B).

TABLE 2.

Crystallographic data collection and refinement

Parameter	Value(s)a
	TmoX/TMAO complex Se derivative	TmoX/TMAO complex
Diffraction data
Space group	P212121	P21
Unit cell dimensions
a, b, c (Å)	62.6, 68.7, 125.2	29.6, 135.0, 58.4
α, β, γ (°)	90.0, 90.0, 90.0	90.0, 90.2, 90. 0
Resolution range (Å)	50.0–1.95 (2.02–1.95)	50.0–2.2 (2.28–2.20)
Redundancy	13.6 (14.3)	3.7 (3.7)
Completeness (%)	100.0 (100.0)	99.9 (99.8)
Rmergeb	0.1 (0.4)	0.1 (0.4)
I/σI	60.6 (15.7)	15.1 (2.8)
Refinement statistics
R factor		0.17
Free R factor		0.24
RMSD from ideal geometry
Bond length (Å)		0.007
Bond angle (°)		1.05
Ramachandran plot (%)
Favored		95.0
Allowed		5.0
Overall B factor (Å2)		31.2

^aNumbers in parentheses refer to data in the highest-resolution shell.

^bR_merge = ∑_hkl∑_i|I(hkl)_i − <I(hkl)>|/∑_hkl∑_i<I(hkl)_i>, where I is the observed intensity, <I(hkl)> represents the average intensity, and I(hkl)_i represents the observed intensity of each unique reflection..

FIG 3.

Overall structure of the TmoX/TMAO complex. (A) Gel filtration analysis of TmoX. Ovalbumin (mass = 43,000 Da; GE Healthcare) and carbonic anhydrase (mass = 13,700 Da; GE Healthcare) were used as markers. The predicted molecular mass of mature TmoX is approximately 32,000 Da. (B) Overall structure of the TmoX/TMAO complex. Domain I is colored light blue, and domain II is dark blue. The locations of TMAO and Ca²⁺ are indicated by arrows.

A search using the Dali server (47) to identify structural homologues of TmoX showed that all of them are Cbc-type SBPs for specific osmolytes, such as betaine, choline, and carnitine. The structures of SBPs specific for betaine and choline have been extensively studied, including ProX from Archaeoglobus fulgidus and E. coli (21, 23), OpuAC from Bacillus subtilis and Lactococcus lactis (24, 27), OpuBC and OpuCC from B. subtilis (25, 26), and ChoX from Sinorhizobium meliloti (22). ProX and OpuAC are specific for betaine, OpuBC and ChoX are specific for choline, and OpuCC can recognize a broad spectrum of compatible solutes, including carnitine, betaine, and choline. Phylogenetic analysis indicates that TmoX proteins form a distinct subcluster within the F-III cluster that does not contain any previously characterized SBPs (6). Although the sequence identities between TmoX and these SBPs are only 11% to 18%, the overall structures of TmoX and these SBPs are similar, with root mean square deviations (RMSDs) of 1.6 to 8.0 Å between TmoX and other structures (Fig. 4).

FIG 4.

Structural comparison of TmoX and other Cbc-associated SBPs. (A) Overall structures of TmoX and other reported Cbc-associated SBPs. Domain I is colored light blue, and domain II is dark blue. The extended loop comprising the metal ion binding site is colored orange. The PDB code of each structure is shown. (B) Superimposed structures of TmoX and other reported Cbc-associated SBPs. TmoX, green; 2REG, cyan; 3TMG, magenta; 3L6H, yellow; 1R9L, salmon; 3PPP, light blue; 3R6U, slate; 1SW2, orange.

Except for ProX from E. coli (PDB ID 1R9L), which is reported to contain an unidentified metal ion (21), the reported Cbc-associated SBPs do not contain metal ions (Fig. 4). The electron density map of TmoX indicates that there is a metal ion located in domain II, far from the binding pocket. ICP-AES measurements indicate that the metal ion is Ca²⁺, with occupancy of approximately 1.0 per monomer. Ni, Zn, and Fe were detected, but at 1 order of magnitude less than Ca; no Cr, Co, Cd, Cu, or Mn was detectable.

Analyses of the function of the calcium ion of TmoX.

The Ca²⁺ ion in the TmoX molecule is coordinated by the carbonyls of Pro249, Val251, Asn254, and Ala257; the side chain of Asp260; and a water molecule in an octahedral arrangement (Fig. 5A). The sequence of the Ca²⁺ binding site (Pro249 to Asp260) of TmoX constitutes an EF-hand motif, which is common in Ca²⁺ binding folds (48). Protein sequence alignment suggests that the Ca²⁺ binding site is common to TmoX proteins from the MRC but not those from the SAR11 clade or from Deltaproteobacteria or Gammaproteobacteria (Fig. 5B). Most of the residues coordinating Ca²⁺ in MRC TmoX proteins are highly conserved, except for Val251 and Ala257. Given that Val251 and Ala257 coordinate Ca²⁺ through their main-chain carbonyls (Fig. 5A), variations at these positions should not affect Ca²⁺ binding in TmoX proteins.

FIG 5.

Analyses of the function of the Ca²⁺ ion in TmoX. (A) Ca²⁺ binding site in TmoX. Coordination bonds are shown with dashed lines. The 2-F_oF_c density for Ca²⁺ is contoured in blue at 3.0 σ. Wat, water. (B) Sequence alignment of TmoX proteins from the MRC, the SAR11 clade, Deltaproteobacteria, and Gammaproteobacteria. Conserved residues are colored red. The numbers above the sequences refer to the amino acid numbers in the TmoX sequence from R. pomeroyi DSS-3. Sequences 1 to 5 are from the MRC: 1, R. pomeroyi DSS-3; 2, Roseovarius sp. strain 217; 3, Ruegeria conchae; 4, Roseibium sp. strain TrichSKD4; 5, Roseovarius nubinhibens. Sequence 6 is from “Candidatus Pelagibacter” sp. strain HIMB5 belonging to the SAR11 clade. Sequence 7 is from Deltaproteobacterium SCGC AAA003-F15. Sequence 8 is from Gammaproteobacterium SCGC AAA076-F14. (C) Effect of EDTA on the binding affinity of TmoX for TMAO. The error bars represent standard deviations. (D) CD spectra of TmoX with and without EDTA. (E) DSC heat flow curves for TmoX with and without EDTA.

To analyze whether Ca²⁺ has an effect on the binding affinity of TmoX, we used EDTA to chelate Ca²⁺ away from TmoX. The removal of Ca²⁺ had little effect on either the binding affinity (Fig. 5C) or the secondary structure (Fig. 5D) of TmoX. Then, we measured the melting temperature (T_m) of TmoX to determine whether Ca²⁺ stabilizes TmoX. DSC measurements demonstrated that the thermostability of TmoX was dramatically decreased when Ca²⁺ was removed by EDTA, with a 13°C decrease in the T_m (from 54.5 ± 1.6°C to 41.4 ± 0.1°C) (Fig. 5E), indicating an important role for Ca²⁺ in maintaining the stability of TmoX. Ca²⁺ may play an important role as a switch to modulate proteins between stable and unstable states according to biological needs (49). It is possible that Ca²⁺ also modulates the protein stability of TmoX in response to biological needs and environmental changes.

Conformational changes in TmoX upon binding TMAO.

The TMAO molecule in the crystal structure of the TmoX/TMAO complex could not be observed in the electrostatic surface representation (Fig. 6A). The cross-sectional view of the complex reveals that the binding site for TMAO is completely buried in the binding pocket of TmoX (Fig. 6B). It has been reported that SBPs consist of two structurally conserved domains and that one domain rotates as a rigid body upon binding substrates (46, 50). To test if TmoX undergoes a conformational change during the binding of TMAO, we performed an MD simulation of the structure of TmoX without the TMAO molecule. The structure of apo-TmoX was obtained after an MD simulation for 50 ns. By comparing the structures of the TmoX/TMAO complex and apo-TmoX, we could identify two distinct conformations of TmoX: an “open” form and a “closed” form, with domain II rotating ∼40° as the rigid body (Fig. 6C). The space between the two domains of TmoX tended to expand during the 50 ns of dynamic motion (see Video S1 in the supplemental material). The distances between the two pairs of loops of domain I and domain II increased from 5.87 Å and 5.23 Å in the closed form to 9.93 Å and 13.65 Å in the open form (Fig. 6D and E). This rigid-body rotation is similar to that of other SBPs upon binding substrates (46). In the open form, the substrate-binding site of TmoX is solvent accessible, making it possible for TMAO to enter. After the TMAO molecule enters the pocket, TmoX transforms to the closed state and then associates with the transmembrane domain (TmoW) to release the substrate. After that, it returns to the open state and prepares to bind a new ligand.

FIG 6.

Analysis of the conformational changes of TmoX during binding of TMAO. (A) Electrostatic surface of the TmoX/TMAO complex. Red denotes negative potential, and blue denotes positive potential. (B) Cross-sectional view of the TmoX/TMAO complex. The TMAO molecule is buried in the binding pocket. (C) Superimposed structures of the TmoX/TMAO complex (blue) and apo-TmoX (orange) after a 50-ns MD simulation. (D) Overall structure of the TmoX/TMAO complex, with the distances between two pairs of loops in domain I and domain II labeled. (E) Overall structure of apo-TmoX after a 50-ns MD simulation, with the distances between the two pairs of loops of domain I and domain II labeled.

Roles of key residues in the substrate-binding pocket of TmoX.

Structural analysis showed that the binding pocket of TmoX is composed of Trp55, Trp102, and Phe106 from domain I and Glu 131, Trp177, Phe220, and Trp222 from domain II (Fig. 7A). The side chains of the four tryptophan residues form a rectangular aromatic box. Together with the side chains of Phe106 and Phe220, this box forms a hydrophobic cage to accommodate the quaternary amine of TMAO. Trp55 is located in the bottom of the cage, Glu131 is in the top of the cage, and the hydrophobic residues are scattered around the TMAO molecule (Fig. 7A). Glu131 forms a hydrogen bond with the oxygen atom of TMAO. Mutation of Glu131 to alanine nearly completely abolishes the binding affinity of TmoX, indicating an important role of Glu131 in binding TMAO (Fig. 7B). The positive charge of the quaternary amine is not localized in the nitrogen atom but delocalized over the three methyl groups of TMAO; thus, the quaternary amine is stabilized mainly via cation-π interactions with the side chains of Trp55, Trp102, Trp177, and Trp222 (Fig. 7A). To examine the roles of all four tryptophan residues, we generated TmoX mutants by replacing each of the tryptophan residues with an alanine or a phenylalanine. Mutations of tryptophan residues to alanine led to nearly complete abrogation of the TMAO-binding affinity of TmoX (Fig. 7B). Even though tryptophan and phenylalanine both contain aromatic rings, mutations of tryptophan residues to phenylalanine also resulted in significant decreases in the TMAO-binding affinity (Fig. 7B).

FIG 7.

Analyses of the roles of residues in the binding pocket of TmoX. (A) TMAO and the residues composing the binding pocket. The residues from domain I are shown in light blue, and the residues from domain II are shown in blue. The 2-F_oF_c density for TMAO is contoured in blue at 1.5 σ. (B) Binding affinities of the TmoX mutants for TMAO. The binding affinity of WT TmoX was defined as 100%. The error bars represent standard deviations. (C) Conformational architecture of π-π stacking interactions composed of aromatic rings. (D) CD spectra of WT TmoX and its mutants.

In addition to cation-π interactions, π-π stacking interactions between the side chains of phenylalanine and tryptophan are also observed in the structure. The benzene ring of Phe106 is approximately parallel to the indole ring of Trp55, and the benzene ring of Phe220 is located between the indole rings of Trp177 and Trp222 (Fig. 7C). The architecture of these aromatic rings forms a parallel-displaced configuration, which is one of the most stable conformations and can reduce repulsive quadrupole-quadrupole interactions and stabilize the binding pocket. The important roles of Phe106 and Phe220 have also been confirmed by mutational analysis (Fig. 7B).

CD spectroscopy assays showed that the secondary structures of the mutants are very similar to that of WT TmoX (Fig. 7D). This indicates that the decrease in the binding affinities of the mutants is a result of residue replacement rather than structural changes. Altogether, the mutational analyses are consistent with the structural observations.

Universality of the TMAO-binding mode of TmoX in marine bacteria.

The tmoX gene is widespread in a number of divergent marine bacteria, such as the MRC, SAR11 clade Alphaproteobacteria, and SAR324 clade Deltaproteobacteria, as well as many uncultivated bacteria (6). In addition, tmoX is highly transcribed and expressed in marine environments, as revealed by metatranscriptomic and metaproteomic data (29–31). To investigate the universality of the TMAO-binding mode of TmoX that we had revealed in R. pomeroyi DSS-3, we performed sequence alignments (51) of TmoX proteins. As shown in Fig. 8, the residues comprising the binding pocket of TmoX, including Trp55, Trp102, Phe106, Glu131, Trp177, Phe220, and Trp222, are all highly conserved in the MRC, the SAR11 clade, Deltaproteobacteria, and Gammaproteobacteria. This result suggests that the binding mechanism of TmoX in these marine bacteria may be similar to that in R. pomeroyi DSS-3.

FIG 8.

Sequence alignment of TmoX proteins from the MRC, the SAR11 clade, Deltaproteobacteria, and Gammaproteobacteria. The large dots indicate residues composing the binding pocket of TmoX proteins. R. pomeroyi DSS-3, Roseovarius sp. 217, R. conchae, Roseibium sp. TrichSKD4, and R. nubinhibens belong to the MRC. “Candidatus Pelagibacter” sp. strain HIMB5 belongs to the SAR11 clade. The alignment was performed with ESPript (51).

Comparison of the substrate-binding modes of TmoX and other Cbc-associated SBPs.

The substrate-binding modes of other reported Cbc-associated SBPs can be summarized as a rectangular aromatic box formed by three or four aromatic residues (Trp or Tyr) that form cation-π interactions with a quaternary amine, as well as two or three hydrophilic residues that interact with the polar oxygen atoms of QACs (21–27). The binding pockets of ProX from E. coli and OpuAC from B. subtilis and L. lactis are composed of 3 tryptophan residues forming cation-π interactions and 3 residues forming hydrogen bonds with betaine (Fig. 9A) (21, 24, 27). The binding pockets of ProX from A. fulgidus and OpuBC and OpuCC from B. subtilis are composed of 4 tyrosine residues forming cation-π interactions and 3 residues forming interactions with the ligand (Fig. 9B) (23, 25, 26). The binding pocket of ChoX from S. meliloti is composed of 3 tryptophan residues and 1 tyrosine residue that form cation-π interactions with choline and 2 residues that interact with the polar oxygen atom of choline (Fig. 9C) (22).

FIG 9.

Substrate-binding pockets of other reported Cbc-associated SBPs. The substrates and the residues composing the binding pocket are shown and labeled. The possible hydrogen bonds are shown with dashed lines. (A) Substrate-binding pocket of ProX from E. coli (PDB ID 1R9L). (B) Substrate-binding pocket of ProX from A. fulgidus (PDB ID 1SW2). (C) Substrate-binding pocket of ChoX from S. meliloti (PDB ID 2REG).

In general, the TMAO-binding mode of TmoX is similar to the substrate-binding mode of other Cbc-associated SBPs. Compared to other Cbc-associated SBPs, TmoX has more hydrophobic residues comprising the substrate-binding pocket. In addition to the four Trp residues, two Phe residues (Phe106 and Phe220) also participate in TMAO binding through π-π stacking interactions (Fig. 7C), which are also essential for TMAO binding to TmoX (Fig. 7B). These essential π-π stacking interactions have not been observed in any other characterized Cbc-associated SBPs (Fig. 9). In addition, there is only 1 residue (Glu131) to form a hydrogen bond with the polar oxygen atom of TMAO in TmoX (Fig. 7A), whereas other related SBPs usually have 2 or 3 residues interacting with the polar oxygen atoms of QACs (Fig. 9).

Comparison of the TMAO-binding modes of TmoX and TorT.

Besides the structural homologues of TmoX described above, we also found a TMAO-binding protein in a nonrelated protein family, TorT from Vibrio parahaemolyticus (52). TorT is the periplasmic binding protein of a histidine kinase receptor system that is involved in the TMAO reductase pathway (52). There is no significant similarity in either the sequences or the tertiary structures between TorT and TmoX. The overall structure of TorT is similar to that of ribose-binding proteins (52). However, we found that the overall architectures of the TMAO-binding pockets and the TMAO-binding modes of TorT and TmoX are generally similar. The TMAO-binding pocket of TorT is composed of three aromatic residues (Tyr44, Trp140, and Tyr252) to form an aromatic cage to interact with the quaternary amine of TMAO and 3 residues (Trp45, Tyr71, and Asp42) to form hydrogen bonds to the oxygen atom of TMAO (Fig. 10). Although the tertiary structures of TorT and TmoX are quite different, the overall architectures of their TMAO-binding pockets are similar, and they have similar TMAO-binding modes. This suggests that the TMAO-binding proteins with different sequences and structures may share similar TMAO-binding mechanisms.

FIG 10.

TMAO-binding pocket of TorT. TMAO and the residues composing the binding pocket of TorT (PDB ID 3O1H) are shown and labeled. The possible hydrogen bonds are shown with dashed lines.

Conclusion.

TMAO is a significant nutrient for marine bacteria. In this study, we characterized the substrate specificity of TmoX, the SBP of the ABC transporter genes tmoXWV specific for TMAO in R. pomeroyi DSS-3, and studied the TMAO-binding mode of TmoX in detail by biochemical, structural, and mutational analyses. We further compared the substrate-binding mechanism of TmoX with those of related osmolyte-binding proteins and TMAO-binding proteins. The results illustrated the TMAO-binding mechanism of TmoX, which may be shared by TMAO-binding proteins and may have universal significance in marine bacteria, such as the MRC and the SAR11 clade. This study provides insights into how marine bacteria absorb TMAO and promotes a better understanding of marine nitrogen cycling.