Journal of Biomolecular NMR, 14: 195–196, 1999.KLUWER/ESCOM© 1999Kluwer Academic Publishers. Printed in the Netherlands.

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Letter to the Editor: Assignment of 1H, 13C and 15N resonances of thea′domain of protein disulfide isomerase

Klaas Dijkstraa, Päivi Karvonenb, Annamari Pirneskoskib, Peppi Koivunenb, Kari I. Kivirikko b,Nigel J. Darbyc,∗, Monique van Straatenc,d, Ruud M. Scheeka & Johan Kemminkc,d,∗∗aThe Groningen Biomolecular Science and Biotechnology Institute (GBB), University of Groningen, Nijenborgh 4,9747 AG Groningen, The Netherlands;bCollagen Research Unit, Biocenter and Department of Medical Biochem-istry, University of Oulu, Kajaanintie 52A, FIN-90220 Oulu, Finland;cEuropean Molecular Biology Laboratory,Meyerhofstrasse 1, D-69012 Heidelberg, Germany;dBiocenter and Department of Biochemistry, University ofOulu, P.O. Box 333, FIN-90571 Oulu, Finland

Received 1 February 1999; Accepted 13 April 1999

Key words:disulfide bond, prolyl-4-hydroxylase, protein disulfide isomerase, protein folding, thioredoxin

Biological context

Protein disulfide isomerase (PDI) is a 55 kDa mul-tifunctional protein mainly involved in the catalysisof disulfide bond formation, breakage and rearrange-ment. It also functions as a chaperone and a subunitin the enzymes prolyl-4-hydroxylase and the micro-somal triglyceride transfer protein (see Kivirikko andPihlajaniemi, 1998). The amino acid sequence of PDIindicated the presence of four structural domains (Ed-man et al., 1985). Two domains located at the N-and C-terminus of the PDI sequence, designatedaanda′, were clearly homologous to each other and tothe ubiquitous redox protein thioredoxin. NMR struc-tural studies of PDIa indeed confirmed the expectedthioredoxin-like structure (Kemmink et al., 1996). Themiddle section of the PDI sequence comprises twoother domains, termedb andb′, which are homologousto each other (Edman et al., 1985). Although there isno significant sequence similarity between these do-mains and either thea or a′ domain, structural studiesusing NMR spectroscopy revealed that theb domainof PDI also adopts a thioredoxin fold (Kemmink et al.,1997).

Both the individuala anda′ domains are good cat-alysts of disulfide bond formation. However, efficientcatalysis of rearrangement of disulfide bonds requiresthe presence of other domains. Theb′ domain of PDI

∗Present address: Astra Hässle AB, S-43183 Mölndal, Sweden.∗∗To whom correspondence should be addressed. E-mail:johan.kemmink@oulu.fi

appears to play an essential role in efficient rearrange-ment, but other domains are required depending onthe complexity of the peptide substrate (Darby et al.,1998). In particular thea′ domain plays an intriguingrole different from thea domain. Furthermore, thea′domain has been shown to be critical for the assemblyof the prolyl-4-hydroxylase tetramer (Koivunen et al.,1999). This prompted us to initiate NMR structuralstudies of thea′ domain of PDI.

Methods and results

The clone used for the expression of the PDIa′ do-main included residues D348-G462 of PDI clonedin pET12a (Novagen). The protein was expressed inE. coli strain BL21(DE3) pLysS (Novagen) and pu-rified as described by Darby and Creighton (1995)with some modifications. For preparation of the15N-labeled sample, 5.0 L of minimal medium contain-ing 1 g/L 15NH4Cl (Isotec) and supplemented with1xBME Vitamin Solution (Gibco), 1 mM CaCl2,2 mM MgSO4 plus 50 µg/mL carbenicillin and34 µg/mL chloramphenicol was inoculated from anovernight culture. Cells were grown until the ab-sorbance at 600 nm reached 0.4–0.5 and the geneexpression was induced by addition of IPTG to a con-centration of 0.5 mM. The cells were harvested after4–5 h by centrifugation and frozen. The cell pelletswere suspended in 20 mL of 50 mM Tris (pH 8),1 mM EDTA, 10 µM leupeptin and 0.1 mM PMSFand sonicated. The sonicated cells were centrifuged

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Figure 1. The 1H-15N HSQC spectrum of13C/15N-enriched PDIa′ domain in the dithiol form recorded at 300 K. The backbone NHcorrelations are indicated according to the type of amino acid andthe sequence numbering of intact PDI. The extra peaks are enclosedin boxes.

at 90000g for 60 min and DTT was added to a fi-nal concentration of 20 mM. The sample was appliedonto a 15× 1.5 cm Q-Sepharose High Performancecolumn (Pharmacia) equilibrated with 50 mM Tris(pH 8), 2 mM DTT (buffer A) and eluted by a lineargradient to 0.4 M NaCl in the same buffer. Fractionscontaining most of thea′ polypeptide were applied to aSephacryl S100 High Resolution gel filtration column(Pharmacia) equilibrated and eluted with buffer A. Fi-nally, thea′-containing fractions were dialyzed against50 mM Tris (pH 8), 5 mM DTT and concentrated. The13C/15N labeled sample was produced in a similar waystarting from a 2.5 L culture supplemented with 4 g/Lof D-glucose-13C6 (Isotec).

NMR experiments for resonance assignments ofPDI a′ domain were recorded on samples uniformlylabeled in either15N or 15N/13C at concentrationsof 1–2 mM in 93% H2O/7% D2O (v/v) containing10 mM DTT and 10 mM sodium phosphate buffer(pH 6.8). NMR spectra were recorded on a VarianUnity INOVA 600. Details about the employed NMRexperiments mentioned without any further referencecan be found in e.g. Cavanagh et al. (1996).

Extent of assignments and data deposition

Sequence specific assignment of the backbone1H,15N and 13C frequencies of reduced PDIa′ wasaccomplished using HNCA, HNCO, HN(CA)CO,

HNCACB, CBCA(CO)NH, HBHA(CBCACO)NHand COCAH (Dijkstra et al., 1995) experimentsrecorded at 300 K. The assignment process was com-plicated by the presence or appearance during thecourse of experiments of extra peaks. The temperaturestudies also solved a number of ambiguities present inthe spectra recorded at 300 K. The extra peaks can beclassified into two categories: (i) A limited number ofpeaks disappears after addition of DTT; these peaksare caused by the presence of PDIa′ in the oxidizedstate and (ii) the other ones most probably originatefrom irreversible degradation of the sample. More than98% of all backbone atoms that could possibly be as-signed using these experiments were assigned. Thebackbone1HN and15N assignments are summarizedin the HSQC spectrum shown in Figure 1. Assignmentof side chains was checked or extended by correlat-ing the Cα/β and Hα/β chemical shifts to the otheratoms using the HC(C)H-TOCSY and13C-NOESY-HSQC experiments. Aromatic side-chain assignmentswere obtained from (adapted) versions of the13C CT-HSQC, (HB)CB(CGCD)HD (Yamazaki et al., 1993),(HB)CB(CGCDCE)HE (Yamazaki et al., 1993) andHC(C)H-TOCSY experiments. More than 90% ofthe side-chain resonances that could possibly be as-signed using the experiments performed, have beenassigned. The assignments have been deposited withthe BioMagResBank, entry number 4302.

References

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