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Ligand binding reveals a role for heme in translationally-controlled tumor protein dimerization.

Lucas AT, Fu X, Liu J, Brannon MK, Yang J, Capelluto DG, Finkielstein CV - PLoS ONE (2014)

Bottom Line: Mutation in both His residues to Ala prevents hemin from binding and abrogates oligomerization, suggesting that the ligand site localizes at the interface of the oligomer.Unlike heme, binding of Ca2+ ligand to TCTP does not alter its monomeric state; although, Ca2+ is able to destabilize an existing TCTP dimer created by hemin addition.In agreement with TCTP's proposed buffer function, ligand binding occurs at high concentration, allowing the "buffer" condition to be dissociated from TCTP's role as a component of signal transduction mechanisms.

View Article: PubMed Central - PubMed

Affiliation: Integrated Cellular Responses Laboratory, Virginia Bioinformatics Institute, Department of Biological Sciences, Virginia Tech, Blacksburg, Virginia, United States of America; Protein Signaling Domains Laboratory, Virginia Bioinformatics Institute, Department of Biological Sciences, Virginia Tech, Blacksburg, Virginia, United States of America.

ABSTRACT
The translationally-controlled tumor protein (TCTP) is a highly conserved, ubiquitously expressed, abundant protein that is broadly distributed among eukaryotes. Its biological function spans numerous cellular processes ranging from regulation of the cell cycle and microtubule stabilization to cell growth, transformation, and death processes. In this work, we propose a new function for TCTP as a "buffer protein" controlling cellular homeostasis. We demonstrate that binding of hemin to TCTP is mediated by a conserved His-containing motif (His76His77) followed by dimerization, an event that involves ligand-mediated conformational changes and that is necessary to trigger TCTP's cytokine-like activity. Mutation in both His residues to Ala prevents hemin from binding and abrogates oligomerization, suggesting that the ligand site localizes at the interface of the oligomer. Unlike heme, binding of Ca2+ ligand to TCTP does not alter its monomeric state; although, Ca2+ is able to destabilize an existing TCTP dimer created by hemin addition. In agreement with TCTP's proposed buffer function, ligand binding occurs at high concentration, allowing the "buffer" condition to be dissociated from TCTP's role as a component of signal transduction mechanisms.

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Limited proteolysis defines structural regions important for heme binding in TCTP.A. Recombinant untagged TCTP was pre-incubated, or not (top panel), with either hemin (320 µM, middle panel) or Ca2+ (50 mM, bottom panel) before the addition of trypsin as described in “Materials and Methods”. Stability of TCTP under digestion conditions was evaluated at room temperature throughout the time course analyzed (second top down panel). Samples were collected at indicated times and fragments resolved by SDS-PAGE and visualized by Coomassie blue staining. Molecular mass markers (in kDa) are indicated on the left. B. Two views of the surface representation of TCTP (PDB access code: 1YZ1) where the trypsin resistant fragment generated after hemin binding is displayed in magenta. C. Elution profile of recombinant untagged-TCTP-HH resolved by gel filtration using a 16/60 Superdex 75 column as described in “Materials and Methods” (solid black line). In other experiments, untagged-TCTP-HH was loaded onto a 16/60 Superdex 75 column pre-equilibrated with 50 mM Tris-HCl, pH 7.8, 250 mM NaCl, and 1 mM hemin (solid red line). Experiments were performed as in Figure 1 with untagged-TCTP in the absence or presence (dashed red line) of 1 mM hemin.
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pone-0112823-g004: Limited proteolysis defines structural regions important for heme binding in TCTP.A. Recombinant untagged TCTP was pre-incubated, or not (top panel), with either hemin (320 µM, middle panel) or Ca2+ (50 mM, bottom panel) before the addition of trypsin as described in “Materials and Methods”. Stability of TCTP under digestion conditions was evaluated at room temperature throughout the time course analyzed (second top down panel). Samples were collected at indicated times and fragments resolved by SDS-PAGE and visualized by Coomassie blue staining. Molecular mass markers (in kDa) are indicated on the left. B. Two views of the surface representation of TCTP (PDB access code: 1YZ1) where the trypsin resistant fragment generated after hemin binding is displayed in magenta. C. Elution profile of recombinant untagged-TCTP-HH resolved by gel filtration using a 16/60 Superdex 75 column as described in “Materials and Methods” (solid black line). In other experiments, untagged-TCTP-HH was loaded onto a 16/60 Superdex 75 column pre-equilibrated with 50 mM Tris-HCl, pH 7.8, 250 mM NaCl, and 1 mM hemin (solid red line). Experiments were performed as in Figure 1 with untagged-TCTP in the absence or presence (dashed red line) of 1 mM hemin.

Mentions: We further analyzed conformational differences in TCTP as a result of either hemin or Ca2+ binding using a limited-proteolysis approach. A time course analysis of trypsin-treated TCTP showed that this protein displayed remarkable stability retaining both N- and C-terminus epitopes as identified by mass spectrometry sequencing of stained bands (Figure 4A, first and second panels from top). Remarkably, pre-incubation of TCTP with hemin at a concentration known to induce TCTP dimerization resulted in increased susceptibility to trypsin cleavage (Figure 4A, third panel from top). Thus, the lower band reflected the cleavage of the N-terminus domain, as outlined by mass spectroscopy, within a region (Gly40-Gly61) defined as highly mobile and disordered in the TCTP structure [49] and that comprises the TCTP1 motif important for interactions [50]. When analyzed in the context of TCTP's structure, residues 40 to 111 are located within the same interface comprising the predicted heme-binding site suggesting that this interface might mediate dimerization (Figure 4B). Remarkably, pre-incubation of TCTP with Ca2+ (up to 50 mM) did not result in changes associated with ligand binding where putative trypsin sites might be exposed (Figure 4A, bottom panel). Identification of approximate sites of proteolysis was carried out using mass spectrometry (Table S1). TCTP sequencing resulted in the generation of peptides that covered 32% of its complete amino acid sequence when digested with trypsin and up to 48% when using four different proteases. The most N-terminus residue identified in trypsin-digested samples was Asp6, while Asn131 was identified as the last C-terminus residue in TCTP. A large part of the C-terminus domain of TCTP (residues 133 to 172) did not contain closely spaced trypsin cleavage sites and, therefore, likely generated peptide fragments (>3.5–4.0 kDa) too large for detection by mass spectrometry. In other cases, two putative trypsin sites within the C-terminus were too close (residues 168 and 171) and, thus, the peptide resulting from the digest might be too small (<800 Da) for detection. Of note, we identified two peptides (63E.STVITGVDIVMNHHLQE.T81 and 102K.LEEQRPER.V111) by mass spectroscopy that had a high affinity for iron with one of them containing the putative bis-His axial ligand for heme binding, which was described as a potential binding site in the previous section (residues His76 and His77 underlined in the sequence). Overall, these results suggest that the increased proteolytic susceptibility is accompanied by TCTP reorganization upon hemin binding.


Ligand binding reveals a role for heme in translationally-controlled tumor protein dimerization.

Lucas AT, Fu X, Liu J, Brannon MK, Yang J, Capelluto DG, Finkielstein CV - PLoS ONE (2014)

Limited proteolysis defines structural regions important for heme binding in TCTP.A. Recombinant untagged TCTP was pre-incubated, or not (top panel), with either hemin (320 µM, middle panel) or Ca2+ (50 mM, bottom panel) before the addition of trypsin as described in “Materials and Methods”. Stability of TCTP under digestion conditions was evaluated at room temperature throughout the time course analyzed (second top down panel). Samples were collected at indicated times and fragments resolved by SDS-PAGE and visualized by Coomassie blue staining. Molecular mass markers (in kDa) are indicated on the left. B. Two views of the surface representation of TCTP (PDB access code: 1YZ1) where the trypsin resistant fragment generated after hemin binding is displayed in magenta. C. Elution profile of recombinant untagged-TCTP-HH resolved by gel filtration using a 16/60 Superdex 75 column as described in “Materials and Methods” (solid black line). In other experiments, untagged-TCTP-HH was loaded onto a 16/60 Superdex 75 column pre-equilibrated with 50 mM Tris-HCl, pH 7.8, 250 mM NaCl, and 1 mM hemin (solid red line). Experiments were performed as in Figure 1 with untagged-TCTP in the absence or presence (dashed red line) of 1 mM hemin.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4232476&req=5

pone-0112823-g004: Limited proteolysis defines structural regions important for heme binding in TCTP.A. Recombinant untagged TCTP was pre-incubated, or not (top panel), with either hemin (320 µM, middle panel) or Ca2+ (50 mM, bottom panel) before the addition of trypsin as described in “Materials and Methods”. Stability of TCTP under digestion conditions was evaluated at room temperature throughout the time course analyzed (second top down panel). Samples were collected at indicated times and fragments resolved by SDS-PAGE and visualized by Coomassie blue staining. Molecular mass markers (in kDa) are indicated on the left. B. Two views of the surface representation of TCTP (PDB access code: 1YZ1) where the trypsin resistant fragment generated after hemin binding is displayed in magenta. C. Elution profile of recombinant untagged-TCTP-HH resolved by gel filtration using a 16/60 Superdex 75 column as described in “Materials and Methods” (solid black line). In other experiments, untagged-TCTP-HH was loaded onto a 16/60 Superdex 75 column pre-equilibrated with 50 mM Tris-HCl, pH 7.8, 250 mM NaCl, and 1 mM hemin (solid red line). Experiments were performed as in Figure 1 with untagged-TCTP in the absence or presence (dashed red line) of 1 mM hemin.
Mentions: We further analyzed conformational differences in TCTP as a result of either hemin or Ca2+ binding using a limited-proteolysis approach. A time course analysis of trypsin-treated TCTP showed that this protein displayed remarkable stability retaining both N- and C-terminus epitopes as identified by mass spectrometry sequencing of stained bands (Figure 4A, first and second panels from top). Remarkably, pre-incubation of TCTP with hemin at a concentration known to induce TCTP dimerization resulted in increased susceptibility to trypsin cleavage (Figure 4A, third panel from top). Thus, the lower band reflected the cleavage of the N-terminus domain, as outlined by mass spectroscopy, within a region (Gly40-Gly61) defined as highly mobile and disordered in the TCTP structure [49] and that comprises the TCTP1 motif important for interactions [50]. When analyzed in the context of TCTP's structure, residues 40 to 111 are located within the same interface comprising the predicted heme-binding site suggesting that this interface might mediate dimerization (Figure 4B). Remarkably, pre-incubation of TCTP with Ca2+ (up to 50 mM) did not result in changes associated with ligand binding where putative trypsin sites might be exposed (Figure 4A, bottom panel). Identification of approximate sites of proteolysis was carried out using mass spectrometry (Table S1). TCTP sequencing resulted in the generation of peptides that covered 32% of its complete amino acid sequence when digested with trypsin and up to 48% when using four different proteases. The most N-terminus residue identified in trypsin-digested samples was Asp6, while Asn131 was identified as the last C-terminus residue in TCTP. A large part of the C-terminus domain of TCTP (residues 133 to 172) did not contain closely spaced trypsin cleavage sites and, therefore, likely generated peptide fragments (>3.5–4.0 kDa) too large for detection by mass spectrometry. In other cases, two putative trypsin sites within the C-terminus were too close (residues 168 and 171) and, thus, the peptide resulting from the digest might be too small (<800 Da) for detection. Of note, we identified two peptides (63E.STVITGVDIVMNHHLQE.T81 and 102K.LEEQRPER.V111) by mass spectroscopy that had a high affinity for iron with one of them containing the putative bis-His axial ligand for heme binding, which was described as a potential binding site in the previous section (residues His76 and His77 underlined in the sequence). Overall, these results suggest that the increased proteolytic susceptibility is accompanied by TCTP reorganization upon hemin binding.

Bottom Line: Mutation in both His residues to Ala prevents hemin from binding and abrogates oligomerization, suggesting that the ligand site localizes at the interface of the oligomer.Unlike heme, binding of Ca2+ ligand to TCTP does not alter its monomeric state; although, Ca2+ is able to destabilize an existing TCTP dimer created by hemin addition.In agreement with TCTP's proposed buffer function, ligand binding occurs at high concentration, allowing the "buffer" condition to be dissociated from TCTP's role as a component of signal transduction mechanisms.

View Article: PubMed Central - PubMed

Affiliation: Integrated Cellular Responses Laboratory, Virginia Bioinformatics Institute, Department of Biological Sciences, Virginia Tech, Blacksburg, Virginia, United States of America; Protein Signaling Domains Laboratory, Virginia Bioinformatics Institute, Department of Biological Sciences, Virginia Tech, Blacksburg, Virginia, United States of America.

ABSTRACT
The translationally-controlled tumor protein (TCTP) is a highly conserved, ubiquitously expressed, abundant protein that is broadly distributed among eukaryotes. Its biological function spans numerous cellular processes ranging from regulation of the cell cycle and microtubule stabilization to cell growth, transformation, and death processes. In this work, we propose a new function for TCTP as a "buffer protein" controlling cellular homeostasis. We demonstrate that binding of hemin to TCTP is mediated by a conserved His-containing motif (His76His77) followed by dimerization, an event that involves ligand-mediated conformational changes and that is necessary to trigger TCTP's cytokine-like activity. Mutation in both His residues to Ala prevents hemin from binding and abrogates oligomerization, suggesting that the ligand site localizes at the interface of the oligomer. Unlike heme, binding of Ca2+ ligand to TCTP does not alter its monomeric state; although, Ca2+ is able to destabilize an existing TCTP dimer created by hemin addition. In agreement with TCTP's proposed buffer function, ligand binding occurs at high concentration, allowing the "buffer" condition to be dissociated from TCTP's role as a component of signal transduction mechanisms.

Show MeSH
Related in: MedlinePlus