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Crystal Structure of a Group I Energy Coupling Factor Vitamin Transporter S Component in Complex with Its Cognate Substrate

View Article: PubMed Central - PubMed

ABSTRACT

Energy coupling factor (ECF) transporters are responsible for the uptake of essential scarce nutrients in prokaryotes. This ATP-binding cassette transporter family comprises two subgroups that share a common architecture forming a tripartite membrane protein complex consisting of a translocation component and ATP hydrolyzing module and a substrate-capture (S) component. Here, we present the crystal structure of YkoE from Bacillus subtilis, the S component of the previously uncharacterized group I ECF transporter YkoEDC. Structural and biochemical analyses revealed the constituent residues of the thiamine-binding pocket as well as an unexpected mode of vitamin recognition. In addition, our experimental and bioinformatics data demonstrate major differences between YkoE and group II ECF transporters and indicate how group I vitamin transporter S components have diverged from other group I and group II ECF transporters.

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Binding of Thiamine to YkoE(A) Temperature-induced unfolding of YkoEM9 (open circles) and YkoETB (filled circles) monitored by CD spectroscopy. CD signal at 222 nm was recorded in 30 mM Tris, 200 mM NaCl (pH 7.1). The continuous line in each plot corresponds to a standard two-state unfolding model.(B) Fluorescence spectra of 200 nM YkoEM9 in the absence (dashed line) and presence of a saturating amount of thiamine (solid line; 800 nM).(C) Titration of 100 nM YkoEM9 with thiamine. Intrinsic protein fluorescence was measured with excitation wavelength of 280 nm and emission wavelength of 340 nm (filled circles) and 350 nm (open circles), respectively. The continuous line in each plot corresponds to a single-site binding model fit.(D) Table summarizing the binding affinities for various YkoE variants.
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fig6: Binding of Thiamine to YkoE(A) Temperature-induced unfolding of YkoEM9 (open circles) and YkoETB (filled circles) monitored by CD spectroscopy. CD signal at 222 nm was recorded in 30 mM Tris, 200 mM NaCl (pH 7.1). The continuous line in each plot corresponds to a standard two-state unfolding model.(B) Fluorescence spectra of 200 nM YkoEM9 in the absence (dashed line) and presence of a saturating amount of thiamine (solid line; 800 nM).(C) Titration of 100 nM YkoEM9 with thiamine. Intrinsic protein fluorescence was measured with excitation wavelength of 280 nm and emission wavelength of 340 nm (filled circles) and 350 nm (open circles), respectively. The continuous line in each plot corresponds to a single-site binding model fit.(D) Table summarizing the binding affinities for various YkoE variants.

Mentions: Escherichia coli is able to synthesize thiamine in its cytoplasm, therefore we decided to investigate whether YkoE co-purifies with its substrate in the pre-bound form as reported for several other S components (Erkens and Slotboom, 2010, Berntsson et al., 2012). We expressed the protein in standard terrific broth as well as M9 minimal media without the addition of thiamine as a co-factor. In both instances, thiamine could be detected using MALDI-TOF mass spectrometry from the denatured YkoE protein, confirming that, like for other S components, the affinity between YkoE and thiamine is very tight. To investigate whether there was a difference in the populations between the pre-bound versus apo-YkoE, we performed temperature melting circular dichroism (CD) experiments to assess the stability of proteins overexpressed under different conditions. YkoETB gave a Tm of 73°C whereas YkoEM9 had a Tm value of 68°C, which suggested that YkoE produced in M9 minimal media contained a substantial population of apo-YkoE molecules (Figure 6A). To confirm that the difference in Tm between the proteins is due to the presence of pre-bound thiamine, we added an excess of thiamine to YkoEM9 and repeated the CD melting experiments. The measured Tm of YkoEM9 supplemented with excess thiamine was 75°C, which confirmed that, when overexpressed in M9 minimal media, a substantial proportion of YkoE is in its apo form. We then proceeded to investigate the thiamine-YkoEM9 interactions using intrinsic Trp fluorescence measurements. The addition of excess thiamine led to the quenching of Trp fluorescence in YkoE and allowed us to determine an approximate dissociation constant (Kd) of 4.5 nM for YkoEM9-thiamine complex formation (Figures 6B and 6C). The YkoEW49A mutant did not exhibit any Trp quenching in response to the thiamine titration. Substitution of other thiamine coordinating residues with alanine (namely YkoEE77A, YkoED131A, YkoEQ95A, YkoEY46A) resulted in 2- to 5-fold weaker binding with the Q95A mutation showing the largest effect on affinity (Figure 6D). In addition, introduction of a bulky Trp side chain (YkoEQ95W mutant) abolished thiamine binding completely, possibly by causing a steric clash with the pyrimidine group at the bottom of the binding pocket. Altogether, the mutagenesis studies presented here corroborate with the observed orientation of thiamine in the substrate-binding site of YkoE in crystallo.


Crystal Structure of a Group I Energy Coupling Factor Vitamin Transporter S Component in Complex with Its Cognate Substrate
Binding of Thiamine to YkoE(A) Temperature-induced unfolding of YkoEM9 (open circles) and YkoETB (filled circles) monitored by CD spectroscopy. CD signal at 222 nm was recorded in 30 mM Tris, 200 mM NaCl (pH 7.1). The continuous line in each plot corresponds to a standard two-state unfolding model.(B) Fluorescence spectra of 200 nM YkoEM9 in the absence (dashed line) and presence of a saturating amount of thiamine (solid line; 800 nM).(C) Titration of 100 nM YkoEM9 with thiamine. Intrinsic protein fluorescence was measured with excitation wavelength of 280 nm and emission wavelength of 340 nm (filled circles) and 350 nm (open circles), respectively. The continuous line in each plot corresponds to a single-site binding model fit.(D) Table summarizing the binding affinities for various YkoE variants.
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fig6: Binding of Thiamine to YkoE(A) Temperature-induced unfolding of YkoEM9 (open circles) and YkoETB (filled circles) monitored by CD spectroscopy. CD signal at 222 nm was recorded in 30 mM Tris, 200 mM NaCl (pH 7.1). The continuous line in each plot corresponds to a standard two-state unfolding model.(B) Fluorescence spectra of 200 nM YkoEM9 in the absence (dashed line) and presence of a saturating amount of thiamine (solid line; 800 nM).(C) Titration of 100 nM YkoEM9 with thiamine. Intrinsic protein fluorescence was measured with excitation wavelength of 280 nm and emission wavelength of 340 nm (filled circles) and 350 nm (open circles), respectively. The continuous line in each plot corresponds to a single-site binding model fit.(D) Table summarizing the binding affinities for various YkoE variants.
Mentions: Escherichia coli is able to synthesize thiamine in its cytoplasm, therefore we decided to investigate whether YkoE co-purifies with its substrate in the pre-bound form as reported for several other S components (Erkens and Slotboom, 2010, Berntsson et al., 2012). We expressed the protein in standard terrific broth as well as M9 minimal media without the addition of thiamine as a co-factor. In both instances, thiamine could be detected using MALDI-TOF mass spectrometry from the denatured YkoE protein, confirming that, like for other S components, the affinity between YkoE and thiamine is very tight. To investigate whether there was a difference in the populations between the pre-bound versus apo-YkoE, we performed temperature melting circular dichroism (CD) experiments to assess the stability of proteins overexpressed under different conditions. YkoETB gave a Tm of 73°C whereas YkoEM9 had a Tm value of 68°C, which suggested that YkoE produced in M9 minimal media contained a substantial population of apo-YkoE molecules (Figure 6A). To confirm that the difference in Tm between the proteins is due to the presence of pre-bound thiamine, we added an excess of thiamine to YkoEM9 and repeated the CD melting experiments. The measured Tm of YkoEM9 supplemented with excess thiamine was 75°C, which confirmed that, when overexpressed in M9 minimal media, a substantial proportion of YkoE is in its apo form. We then proceeded to investigate the thiamine-YkoEM9 interactions using intrinsic Trp fluorescence measurements. The addition of excess thiamine led to the quenching of Trp fluorescence in YkoE and allowed us to determine an approximate dissociation constant (Kd) of 4.5 nM for YkoEM9-thiamine complex formation (Figures 6B and 6C). The YkoEW49A mutant did not exhibit any Trp quenching in response to the thiamine titration. Substitution of other thiamine coordinating residues with alanine (namely YkoEE77A, YkoED131A, YkoEQ95A, YkoEY46A) resulted in 2- to 5-fold weaker binding with the Q95A mutation showing the largest effect on affinity (Figure 6D). In addition, introduction of a bulky Trp side chain (YkoEQ95W mutant) abolished thiamine binding completely, possibly by causing a steric clash with the pyrimidine group at the bottom of the binding pocket. Altogether, the mutagenesis studies presented here corroborate with the observed orientation of thiamine in the substrate-binding site of YkoE in crystallo.

View Article: PubMed Central - PubMed

ABSTRACT

Energy coupling factor (ECF) transporters are responsible for the uptake of essential scarce nutrients in prokaryotes. This ATP-binding cassette transporter family comprises two subgroups that share a common architecture forming a tripartite membrane protein complex consisting of a translocation component and ATP hydrolyzing module and a substrate-capture (S) component. Here, we present the crystal structure of YkoE from Bacillus subtilis, the S component of the previously uncharacterized group I ECF transporter YkoEDC. Structural and biochemical analyses revealed the constituent residues of the thiamine-binding pocket as well as an unexpected mode of vitamin recognition. In addition, our experimental and bioinformatics data demonstrate major differences between YkoE and group II ECF transporters and indicate how group I vitamin transporter S components have diverged from other group I and group II ECF transporters.

No MeSH data available.


Related in: MedlinePlus