Hu, Kaifeng

Pervushin, Konstantin

Plückthun, Andreas

RERO DOC Digital Library

Journal of Biomolecular NMR 28: 405–406, 2004.KLUWER/ESCOM© 2004 Kluwer Academic Publishers. Printed in the Netherlands.405Letter to the Editor: Backbone HN, N, Cα, C′ and Cβ chemical shiftassignments and secondary structure of FkpA, a 245-residuepeptidyl-prolyl cis/trans isomerase with chaperone activityKaifeng Hua, Andreas Plückthunb & Konstantin Pervushina,∗aLaboratorium für Physikalische Chemie, Swiss Federal Institute of Technology, ETH- Hönggerberg, CH-8093,Zürich, Switzerland; bBiochemisches Institut, Universität Zürich, CH-8057, Zürich, SwitzerlandReceived 2 July 2003; Accepted 9 September 2003Key words: FkpA, Macrophage Infectivity Potentiator, MIP, molecular chaperone, NMR backbone assignments,peptidyl-prolyl cis/trans isomerase (PPIase), sFkpABiological contextFkpA is a 245-residue periplasmic peptidyl-prolylcis/trans isomerase (PPIase) from E. coli with chap-erone activity, which is induced by heat shock (Horneand Young, 1995; Missiakas et al., 1996). Previ-ous studies suggested that the chaperone activity ofFkpA is independent of its PPIase activity (Rammand Pluckthun, 2000, 2001) as it is also observedfor proteins devoid of cis-prolines. Overexpression ofFkpA suppresses the formation of inclusion bodiesfrom a defective folding variant of the maltose-bindingprotein and promotes the reactivation of denaturatedcitrate synthase (Arie et al., 2001). Coexpression ofFkpA can dramatically improve functional periplas-mic production of single-chain fragments of antibod-ies, even those not containing cis-prolines (Bothmannand Pluckthun, 2000). The folding-assisting functionof FkpA is hypothesized to be due to its interac-tion with early folding intermediates preventing theiraggregation, and its ability to reactivate inactive pro-teins, possibly by binding to partially unfolded species(Ramm and Pluckthun, 2000).Amino acid sequence alignment and homologymodelling indicated that the FkpA dimer is comprisedof two domains, the N-terminal dimerization domainand the C-terminal FK506-binding domain (FKBP).The high PPIase enzymatic activity towards proteins(among the highest in bacteria) and the chaperonefunction of the dimeric FkpA are suggested to bemechanistically related and confined to the FKBP do-main (Ramm and Pluckthun, 2001). Nevertheless,protein binding and the occupation of the active sitecan be independent, and thus these two functions arenot identical. The additional N-terminal domain ap-∗To whom correspondence should be addressed. E-mail:kope@phys.chem.ethz.chpears to be utilized to mediate the dimerization, whichplaces the two active sites of the FKBP domains in jux-taposition, such that they can simultaneously interactwith a protein substrate (Ramm and Pluckthun, 2001;Riboldi-Tunnicliffe et al., 2001). The amino acid se-quence of FkpA shows similarity to MIP (macrophageinfectivity potentiator) proteins produced by a numberof pathogenic bacteria (Rahfeld et al., 1996). Due tothe availability of the high resolution X-ray structureof the MIP, the side chain assignment of FkpA is notessential for characterization of interactions of FkpAwith polypeptide substrates in order to reveal mech-anistical details of its combined chaperone and PPIaseactivity.Experiments and methodsThe chemical shift assignments were obtained in turnfrom triple-resonance spectra of the mature full lengthFkpA protein (245 amino acid residues) and an engin-eered variant, the so-called ‘shortened FkpA’ (sFkpA)lacking the first presumably disordered 9 N-terminaland 18 C-terminal amino acid residues of the ma-ture FkpA, which are dispensable for its functionality(Ramm and Pluckthun, 2001).The mature full length FkpA protein was preparedas described in (Ramm and Pluckthun, 2000) usingM9 D2O minimal media supplemented with 15NH4Cl(99%, 1 g/L) and 13C6-glucose (99%, 4 g/L). Overpro-duction of the ‘shortened’ sFkpA (residues 10-227),also containing the C-terminal His6-tag, was carriedout in the E. coli strain BL21 at 25 ◦C. The final NMRsample conditions for both constructs were 0.6 mM ofprotein per monomer in 20 mM Mes buffer at pH 6.0with 50 mM NaCl.All NMR spectra of both constructs, FkpA(residues 1-245) and sFkpA (residues 10-227), wereacquired using TROSY (Pervushin et al., 1997) at406Figure 1. (a) 2D [15N,1HN]-TROSY spectrum of sFkpA (residues10-227) measured using the Avance 900 spectrometer at 37 ◦C. Thecross-peaks are annotated according to residue numbering of thefull length FkpA. The cross-peaks stemming from the guanidinumgroup of Arg side-chains are labeled with asterisks. The cross-peaksmarked with open triangles are degradation products which appearwith the aging of the protein samples (see Supplementary Mater-ial). (b) The polypeptide sequence and secondary structure of FkpA(open bars and arrows for α-helices and β-sheets) identified usingthe chemical shift index (CSI) based on the experimental 13Cαchemical shifts. The sequence of FkpA is aligned with the MIP(Macrophage Infectivity Potentiator, PDB code 1FD9), for whichthe secondary structure elements derived from the crystal structureare indicated with solid arrows and bars. Residues of FkpA withambiguously assigned backbone resonances are enclosed in boxes.25 ◦C and 37 ◦C on Bruker Avance 600 and 900 MHzspectrometers. The NMR experiments include 2D[1H,15N] TROSY, 3D TROSY-versions of HNCA,HNCACB, HNCO, HN(CA)CO and 3D 15N resolvedTROSY-NOESY (τmix = 100 ms).Extent of assignmentThe 1HN, 15N, 13C′, 13Cα and 13Cβ resonanceswere sequence-specifically assigned by identifica-tion of sequentially connected [1HN, 15N] fragmentsof the TROSY-HNCA, TROSY-HNCACB, TROSY-HNCO and TROSY-HN(CA)CO spectra supportedby TROSY-NOESY connectivities followed by globalfragment mapping with the program MAPPER (Gun-tert et al., 2000). With this approach ca. 60% of allresonances of the full length FkpA were sequence-specifically assigned at 25 ◦C. Genetical removal ofthe first 9 N-terminal and 18 C-terminal amino acidresidues in the so-called ‘shortened FkpA’ (sFkpA),together with the increase of temperature to 37 ◦Cand the use of the ultra high field NMR spectrometeroperating at 900 MHz permitted a largely completebackbone resonance assignement of sFkpA. The spec-tra of sFkpA were then aligned and compared to thecorresponding spectra of FkpA showing in most casesno significant perturbations of chemical shifts. Thispromoted the assignment of backbone resonances ofFkpA to 94%. Figure 1a shows the 2D [1HN, 15N]TROSY spectrum of sFkpA.In Figure 1b the polypeptide sequence of FkpAfrom E. coli is aligned with the sequence of the MIPfrom Legionella pneumophila, a PPIase for whichthe crystal structure was recently determined (PDBcode 1FD9) (Riboldi-Tunnicliffe et al., 2001). Despiteof the modest sequence identity of 34% the secondarystructure elements closely correspond to each otherin the aligned sequences of both proteins, indicatingpossible 3D structural similarity. The presented back-bone assignment should serve as a platform to invest-igate atomic resolution details of chaperone/proteinsubstrate interactions.AcknowledgementFinancial support was obtained from the Swiss Na-tional Science Foundation grant to K.P.Data deposition. The chemical shifts are available atBMRB database, accession code 5863.ReferencesArie, J.P., Sassoon, N. and Betton, J.M. (2001) Mol. Microbiol., 39,199–210.Bothmann, H. and Pluckthun, A. (2000) J. Biol. Chem., 275, 17100–17105.Güntert, P., Salzmann, M., Braun, D. and Wüthrich, K. (2000) J.Biomol. NMR, 18, 129–137.Horne, S.M. and Young, K.D. (1995) Arch. Microbiol., 163, 357–365.Missiakas, D., Betton, J.M. and Raina, S. (1996) Mol. Microbiol.,21, 871–884.Pervushin, K., Riek, R., Wider, G. and Wüthrich, K. (1997) Proc.Natl. Acad. Sci. USA, 94, 12366–12371.Rahfeld, J.U., Rucknagel, K.P., Stoller, G., Horne, S.M., Schier-horn, A., Young, K.D. and Fischer, G. (1996) J. Biol. Chem.,271, 22130–22138.Ramm, K. and Pluckthun, A. (2000) J. Biol. Chem., 275, 17106–17113.Ramm, K. and Pluckthun, A. (2001) J. Mol. Biol., 310, 485–498.Riboldi-Tunnicliffe, A., Konig, B., Jessen, S., Weiss, M.S., Rahfeld,J., Hacker, J., Fischer, G. and Hilgenfeld, R. (2001) Nat. Struct.Biol., 8, 779–783.

Letter to the Editor: Backbone HN, N, Cα, C′ and Cβ chemical shift assignments and secondary structure of FkpA, a 245-residue peptidyl-prolyl cis/trans isomerase with chaperone activity

http://doc.rero.ch/record/317693/files/10858_2004_Article_5252345.pdf

Letter to the Editor: Backbone HN, N, Cα, C′ and Cβ chemical shift assignments and secondary structure of FkpA, a 245-residue peptidyl-prolyl cis/trans isomerase with chaperone activity

Abstract

Similar works

Full text

Available Versions

RERO DOC Digital Library