Unveiling the activation dynamics of a fold-switch bacterial glycosyltransferase by 19F NMR
2020; Elsevier BV; Volume: 295; Issue: 29 Linguagem: Inglês
10.1074/jbc.ra120.014162
ISSN1083-351X
AutoresJobst Liebau, Montse Tersa, Beatriz Trastoy, Joan Patrick, Ane Rodrigo‐Unzueta, Francisco Corzana, Tobias Sparrman, Marcelo E. Guerin, Lena Mäler,
Tópico(s)Genomics and Phylogenetic Studies
ResumoFold-switch pathways remodel the secondary structure topology of proteins in response to the cellular environment. It is a major challenge to understand the dynamics of these folding processes. Here, we conducted an in-depth analysis of the α-helix–to–β-strand and β-strand–to–α-helix transitions and domain motions displayed by the essential mannosyltransferase PimA from mycobacteria. Using 19F NMR, we identified four functionally relevant states of PimA that coexist in dynamic equilibria on millisecond-to-second timescales in solution. We discovered that fold-switching is a slow process, on the order of seconds, whereas domain motions occur simultaneously but are substantially faster, on the order of milliseconds. Strikingly, the addition of substrate accelerated the fold-switching dynamics of PimA. We propose a model in which the fold-switching dynamics constitute a mechanism for PimA activation. Fold-switch pathways remodel the secondary structure topology of proteins in response to the cellular environment. It is a major challenge to understand the dynamics of these folding processes. Here, we conducted an in-depth analysis of the α-helix–to–β-strand and β-strand–to–α-helix transitions and domain motions displayed by the essential mannosyltransferase PimA from mycobacteria. Using 19F NMR, we identified four functionally relevant states of PimA that coexist in dynamic equilibria on millisecond-to-second timescales in solution. We discovered that fold-switching is a slow process, on the order of seconds, whereas domain motions occur simultaneously but are substantially faster, on the order of milliseconds. Strikingly, the addition of substrate accelerated the fold-switching dynamics of PimA. We propose a model in which the fold-switching dynamics constitute a mechanism for PimA activation. Proteins do not just occupy a single state but are in dynamic exchange between a set of conformations, and some of these states may at times be populated at very low levels, so-called invisible or dark states (1Wolynes P.G. Recent successes of the energy landscape theory of protein folding and function.Q. Rev. Biophys. 2005; 38 (16934172): 405-41010.1017/S0033583505004075Crossref PubMed Scopus (120) Google Scholar, 2Clore G.M. Visualizing lowly-populated regions of the free energy landscape of macromolecular complexes by paramagnetic relaxation enhancement.Mol. Biosyst. 2008; 4 (18931781): 1058-106910.1039/b810232eCrossref PubMed Scopus (59) Google Scholar, 3Sekhar A. Kay L.E. NMR paves the way for atomic level descriptions of sparsely populated, transiently formed biomolecular conformers.Proc. Natl. Acad. Sci. U S A. 2013; 110 (23868852): 12867-1287410.1073/pnas.1305688110Crossref PubMed Scopus (186) Google Scholar, 4Alderson T.R. Kay L.E. 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Lipids. 2017; 1862 (27826050): 1355-136710.1016/j.bbalip.2016.11.002Crossref PubMed Scopus (14) Google Scholar), both of which are virulence factors during tuberculosis (21Jankute M. Cox J.A. Harrison J. Besra G.S. Assembly of the mycobacterial cell wall.Annu. Rev. Microbiol. 2015; 69 (26488279): 405-42310.1146/annurev-micro-091014-104121Crossref PubMed Scopus (211) Google Scholar), one of the deadliest infections worldwide (22Horsburgh Jr, C.R. Barry III, C.E. Lange C. Treatment of tuberculosis.N. Engl. J. Med. 2015; 373 (26605929): 2149-216010.1056/NEJMra1413919Crossref PubMed Scopus (221) Google Scholar). Crystal structures were previously obtained of PimA in the unliganded state (23Giganti D. Albesa-Jové D. Urresti S. Rodrigo-Unzueta A. Martínez M.A. Comino N. Barilone N. Bellinzoni M. Chenal A. Guerin M.E. Alzari P.M. Secondary structure reshuffling modulates glycosyltransferase function at the membrane.Nat. Chem. 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Bellinzoni M. Chenal A. Guerin M.E. Alzari P.M. Secondary structure reshuffling modulates glycosyltransferase function at the membrane.Nat. Chem. Biol. 2015; 11 (25402770): 16-1810.1038/nchembio.1694Crossref PubMed Scopus (35) Google Scholar). Fold-switching activates PimA, since PimA mutants locked in the unliganded conformation are inactive, while mutants locked in the substrate-bound conformation are active (23Giganti D. Albesa-Jové D. Urresti S. Rodrigo-Unzueta A. Martínez M.A. Comino N. Barilone N. Bellinzoni M. Chenal A. Guerin M.E. Alzari P.M. Secondary structure reshuffling modulates glycosyltransferase function at the membrane.Nat. Chem. Biol. 2015; 11 (25402770): 16-1810.1038/nchembio.1694Crossref PubMed Scopus (35) Google Scholar). Finally, the occurrence of an open-to-closed motion between the N- and C-terminal Rossmann fold domains has been predicted and experimentally demonstrated to occur in PimA and other GT-B glycosyltransferases (24Giganti D. Alegre-Cebollada J. Urresti S. Albesa-Jové D. Rodrigo-Unzueta A. Comino N. Kachala M. López-Fernández S. Svergun D.I. Fernández J.M. Guerin M.E. Conformational plasticity of the essential membrane-associated mannosyltransferase PimA from mycobacteria.J. Biol. Chem. 2013; 288 (23963451): 29797-2980810.1074/jbc.M113.462705Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar, 25Guerin M.E. Schaeffer F. Chaffotte A. Gest P. Giganti D. Korduláková J. van der Woerd M. Jackson M. Alzari P.M. Substrate-induced conformational changes in the essential peripheral-membrane binding mannosyltransferase PimA from mycobacteria. Implications for catalysis.J. Biol. Chem. 2009; 284 (19520856): 21613-2162510.1074/jbc.M109.003947Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 26Albesa-Jové D. Giganti D. Jackson M. Alzari P.M. Guerin M.E. 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Our observations provide a basis to suggest an activation mechanism for PimA in which the addition of substrate accelerates dynamics between active and inactive conformations. 19F nuclei are NMR active with a gyromagnetic ratio that is 0.94 times that of protons, have 100% natural abundance, and display high sensitivity to their magnetic environment (29Gerig J. Fluorine NMR of proteins.Prog. Nucl. Magn. Res. Sp. 1994; 26: 293-37010.1016/0079-6565(94)80009-XAbstract Full Text PDF Scopus (239) Google Scholar, 30Kitevski-LeBlanc J.L. Prosser R.S. Current applications of 19F NMR to studies of protein structure and dynamics.Prog. Nucl. Magn. Res. Sp. 2012; 62 (22364614): 1-3310.1016/j.pnmrs.2011.06.003Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar, 31Didenko T. Liu J.J. Horst R. Stevens R.C. Wüthrich K. Fluorine-19NMR of integral membrane proteins illustrated with studies of GPCRs.Curr. Opin. Struct. 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Biol. 2013; 23 (23932201): 740-74710.1016/j.sbi.2013.07.011Crossref PubMed Scopus (68) Google Scholar). Based on analyses of the different crystallographic structures of PimA (Fig. 1A), we introduced a Cys mutation in position Arg144 (here termed PimAR144C) (Fig. 1A) and labeled it with 1,1,1-trifluoroacetone (TFA), giving rise to a 19F labeled mutant PimAR144C-TFA (see Experimental procedures). The introduced 19F label of the mutant PimAR144C-TFA is located in different chemical environments in the different crystallographic conformations of PimA, as highlighted in Fig. 1A. In the active state, the residue is buried inside the protein and, thus, not exposed to solvent, while it is solvent-exposed in the inactive states. 19F nuclei have been shown to be sensitive to such differences in the hydrophobicity of their immediate environment (32Manglik A. Kim T.H. Masureel M. Altenbach C. Yang Z. Hilger D. Lerch M.T. Kobilka T.S. Thian F.S. Hubbell W.L. Prosser R.S. Kobilka B.K. Structural insights into the dynamic process of β2-adrenergic receptor signaling.Cell. 2015; 161 (25981665): 1101-111110.1016/j.cell.2015.04.043Abstract Full Text Full Text PDF PubMed Scopus (437) Google Scholar, 33Evanics F. Kitevski J.L. Bezsonova I. Forman-Kay J. Prosser R.S. 19F NMR studies of solvent exposure and peptide binding to an SH3 domain.BBA-Gen. Subjects. 2007; 1770 (17182189): 221-23010.1016/j.bbagen.2006.10.017Crossref PubMed Scopus (42) Google Scholar). Thus, it can be expected that the 19F probe located at Cys144 can sense motions that we hypothesize to occur in the reshuffling region, which are 1) extended to compact domain motion and 2) fold-switching dynamics between the inactive and active states. Given that the probe experiences sufficiently distinct chemical environments, domain motions that are typically on the order of milliseconds can be probed by NMR relaxation dispersion experiments (34Carver J. Richards R. 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Ligand binding affinity determined by temperature-dependent circular dichroism: cyclin-dependent kinase 2 inhibitors.Anal. Biochem. 2005; 345 (16140252): 187-19710.1016/j.ab.2005.07.032Crossref PubMed Scopus (39) Google Scholar). We have previously described that the addition of GDP or GDP-Man to wild-type PimA increases the Tm of the enzyme, with the β-phosphate of the nucleotide group playing a prominent role (25Guerin M.E. Schaeffer F. Chaffotte A. Gest P. Giganti D. Korduláková J. van der Woerd M. Jackson M. Alzari P.M. Substrate-induced conformational changes in the essential peripheral-membrane binding mannosyltransferase PimA from mycobacteria. Implications for catalysis.J. Biol. Chem. 2009; 284 (19520856): 21613-2162510.1074/jbc.M109.003947Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). As depicted in Fig. S1B, thermal unfolding followed by the far-UV CD signal at 222 nm indicated slight differences in protein stability between unliganded WT PimA, PimAR144C, and PimAR144C-TFA. Interestingly, after the addition of GDP, wild-type PimA, PimAR144C, and PimAR144C-TFA displayed a clear increase in the Tm values (Table S1), supporting the notion that the mutation and labeling do not significantly perturb the enzymatic structure and function (Fig. S1). PimA contains two Cys residues buried inside the protein core that are not expected to be labeled during the labeling procedure (see Experimental procedures). To verify this, the labeling scheme was applied to WT PimA. Only resonances that are less than 5 Hz wide were observed in a 19F spectrum (Fig. S2), showing that they stem from small molecules. The resonances could be assigned to residual 3-bromo-1,1,1-trifluoroacetone (BTFA) label and 1,1,1-trifluoroacetone (TFA), the latter being the product of an incomplete labeling reaction. The resonance at −119.8 ppm stems from an impurity in the BTFA solution. Therefore, no labeling was observed for WT PimA. We conclude that PimAR144C-TFA is only labeled at the introduced Cys residue. Despite being singly labeled, two resonances are observed at −84.1 ppm and −82.7 ppm (Fig. 1B, bottom). As discussed below, these two peaks display saturation transfer, further corroborating that they stem from the same 19F label. To assign the resonances to conformations of PimA, we investigated the 19F spectrum of the triple mutant PimAT126C-V359C-R144C-TFA, previously shown to be locked in the inactive conformation. This variant cannot undergo reshuffling due to a disulfide bond formed between Cys126 and Cys359; these residues are in close spatial proximity in the inactive state but far apart in the active state (23Giganti D. Albesa-Jové D. Urresti S. Rodrigo-Unzueta A. Martínez M.A. Comino N. Barilone N. Bellinzoni M. Chenal A. Guerin M.E. Alzari P.M. Secondary structure reshuffling modulates glycosyltransferase function at the membrane.Nat. Chem. Biol. 2015; 11 (25402770): 16-1810.1038/nchembio.1694Crossref PubMed Scopus (35) Google Scholar). PimAT126C-V359C-R144C-TFA displays only a single resonance at −84.1 ppm (Fig. 1B, top), which therefore can be assigned to the inactive state. Consequently, the resonance at −82.7 ppm is assigned to the active state. This assignment is corroborated by the fact that the resonance at −84.1 ppm shifts 0.1 ppm upfield in 100% D2O, whereas the resonance at −82.7 ppm does not shift (Fig. 1B, center). For fully solvent-exposed 19F labels, upfield solvent isotope shifts of 0.2–0.3 ppm have been observed when comparing shifts in a 90% H2O/10% D2O to 100% D2O solvent (33Evanics F. Kitevski J.L. Bezsonova I. Forman-Kay J. Prosser R.S. 19F NMR studies of solvent exposure and peptide binding to an SH3 domain.BBA-Gen. Subjects. 2007; 1770 (17182189): 221-23010.1016/j.bbagen.2006.10.017Crossref PubMed Scopus (42) Google Scholar, 40Hull W.E. Sykes B.D. Fluorine-19 nuclear magnetic resonance study of fluorotyrosine alkaline phosphatase: the influence of zinc on protein structure and a conformational change induced by phosphate binding.Biochemistry. 1976; 15 (4091): 1535-154610.1021/bi00652a027Crossref PubMed Scopus (104) Google Scholar), indicating that the resonance at −84.1 ppm stems from partially exposed 19F label. In the crystal structure, Arg144 is shielded from solvent in the active state, whereas this is not the case for the inactive conformations (Fig. 1A), supporting the assignment. The assignment is further supported by extensive molecular dynamics (MD) simulations performed on the different states of PimA labeled with TFA. Based on these simulations, we calculated the solvent-accessible surface area (SASA) of the TFA label in the different states. A higher SASA value indicates that the label is more solvent-exposed. As shown in Fig. S3, the SASA of the TFA label in the active state is 19.3 Å2, which differs markedly from the values obtained for the protein in the inactive forms (96.7 Å2 and 60.1 Å2 for the extended and compact structures, respectively). Thus, MD simulations revealed that the TFA label in the active state is less surface exposed than that in the inactive state, in agreement with the NMR experiments. To test whether the two resonances in the 19F spectrum of PimAR144C-TFA are in slow exchange, we conducted saturation transfer experiments at increasing saturation times. Exchange rates were obtained from a fit to the model given in (Eq. 4), (Eq. 5), (Eq. 6), (Eq. 7). Experimental data to obtain R1 longitudinal relaxation rates that are required for the analysis are shown in Fig. S4 and Table S2. Figure 2 shows resonance intensity attenuation due to slow conformational exchange for the inactive and active state resonances upon saturation of the active or inactive state resonance, respectively. In the absence of GDP-Man, the exchange rate was determined to be 2.3 s−1. These dynamics correspond to fold-switching between the active and inactive states of the protein, as demonstrated by the assignments (Fig. 1B). The second timescale of the motion is in accordance with studies of the chemokine lymphotactin, for which fold-switching dynamics were shown to be on the order of seconds (14Volkman B.F. Liu T.Y. Peterson F.C. Lymphotactin structural dynamics.Methods Enzymol. 2009; 461 (19480914): 51-7010.1016/S0076-6879(09)05403-2Crossref PubMed Scopus (22) Google Scholar). In the absence of GDP-Man, the active and inactive states of PimA are nearly equally populated (Fig. 2C and Table 1).Table 1Parameters obtained from saturation transfer experiments for (A) the active state resonance and (B) for the inactive state resonance of PimAR144C-TFACondition and resonance stateChemical shift (ppm)kIAaI, the inactive state; A, the active state, i.e. kIA is the exchange rate from the inactive to the active state, and ΔGIA is the free energy difference between the inactive and active state and vice versa. (s−1)pIpAks (s−1)ΔGIA (kcal mol−1)Active stateApo−82.71.08 ± 0.050.47 ± 0.042.31 ± 0.09−0.070 ± 0.090GDP-Man−82.64.2 ± 0.20.34 ± 0.0413 ± 1−0.390 ± 0.180Inactive stateApo−84.11.23 ± 0.040.53 ± 0.042.31 ± 0.090.070 ± 0.090GDP-Man−84.38.3 ± 0.90.66 ± 0.1213 ± 10.390 ± 0.180a I, the inactive state; A, the active state, i.e. kIA is the exchange rate from the inactive to the active state, and ΔGIA is the free energy difference between the inactive and active state and vice versa. Open table in a new tab To test how GDP-Man affects these dynamics, we first conducted a titration experiment to corroborate that the sugar donor saturates the binding site. As shown in Fig. 1C, titration of GDP-Man to 100 μm PimAR144C-TFA leads to a gradual decrease in intensities of both resonances up to a nucleotide sugar concentration of 200 μm. At this concentration, the inactive state resonance shifts 0.2 ppm upfield, and further addition of GDP-Man does not alter the spectrum anymore. At the field strength employed in Fig. 1C (16.4 T), the active state resonance is too broad to clearly observe a resonance shift. Since the line broadening due to the field-dependent chemical shift anisotropy of 19F is reduced at lower field strength, we compared the spectra of PimAR144C-TFA in the presence and absence of GDP-Man at 14.1 T. At this lower field, a downfield shift of the active state resonance of 0.1 ppm was observab
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