Báo cáo khoa học: Solution structure of the bb¢ domains of human protein disulfide isomerase docx

Here, we determined the solution structure of the sec-ond and third domains of human protein disulfide isomerase b and b¢, respectively by triple-resonance NMR spectroscopy and molecular

disulfide isomerase

Alexey Y Denisov*, Pekka Ma¨a¨tta¨nen*, Christian Dabrowski, Guennadi Kozlov, David Y Thomas and Kalle Gehring

Department of Biochemistry, McGill University, and Groupe de Recherche Axe´ sur la Structure des Prote´ines (GRASP), Montre´al, Canada

The endoplasmic reticulum (ER) is the cell

compart-ment where membrane and secretory proteins fold

The rate-limiting step for the folding of many proteins

is the formation of disulfide bonds As polypeptides

are synthesized, their cysteine thiols enter the oxidizing

environment of the ER and form covalent

intramolec-ular and intermolecintramolec-ular disulfide links Although this

oxidative folding process occurs spontaneously [1],

non-native disulfide-bonded intermediates often occur,

acting as kinetic traps along the folding pathway [2,3]

To avoid these, the ER contains a large family of

enzymes called protein disulfide isomerases (PDIs) that

catalyze both disulfide bond formation and the

rear-rangement of incorrect disulfide bonds [4–7]

PDI family members are loosely defined by

homology to thioredoxin and ER localization There

are at least 17 PDI family proteins in humans, 13 of which contain CXXC active-site motifs, and 9 have been shown to catalyze disulfide-exchange reactions [4,5] The best studied and most abundant member

of the family is PDI, a ubiquitous enzyme found at very high concentrations in the ER Its concentra-tion has been estimated to be 10 lm in dog pancre-atic microsomes [8], the highest of all ER resident proteins PDI has four thioredoxin-like domains, a-b-b¢-a¢, where the two a domains contain catalytic CGHC motifs, and the two b domains lack the conserved cysteine residues and are noncatalytic The linkers between the domains are generally short The longest is a stretch of 19 amino acids between the b¢ and a¢ domains, referred to as the x-linker [9]

Keywords

chaperone; endoplasmic reticulum; NMR

solution structure; protein disulfide

isomerase family

Correspondence

K Gehring, Department of Biochemistry,

McGill University, 3655 Promenade Sir

William Osler, Montreal, QC H3G 1Y6,

Canada

Fax: +1 (514) 398 7384

Tel: +1 (514) 398 7287

E-mail: kalle.gehring@mcgill.ca

*These authors contributed equally to this

work

(Received 16 November 2008, revised 30

December 2008, accepted 30 December

2008)

doi:10.1111/j.1742-4658.2009.06884.x

Protein disulfide isomerase is the most abundant and best studied of the disulfide isomerases that catalyze disulfide bond formation in the endoplas-mic reticulum, yet the specifics of how it binds substrate have been elusive Protein disulfide isomerase is composed of four thioredoxin-like domains (abb¢a¢) Cross-linking studies with radiolabeled peptides and unfolded pro-teins have shown that it binds incompletely folded propro-teins primarily via its third domain, b¢ Here, we determined the solution structure of the sec-ond and third domains of human protein disulfide isomerase (b and b¢, respectively) by triple-resonance NMR spectroscopy and molecular model-ing NMR titrations identified a large hydrophobic surface within the b¢ domain that binds unfolded ribonuclease A and the peptides mastoparan and somatostatin Protein disulfide isomerase-catalyzed refolding of reduced ribonuclease A in vitro was inhibited by these peptides at concen-trations equal to their affinity to the bb¢ fragment Our findings provide a structural basis for previous kinetic and cross-linking studies which have shown that protein disulfide isomerase exhibits a saturable, substrate-binding site

Abbreviations

ER, endoplasmic reticulum; GST, glutathione S-transferase; HSQC, heteronuclear single-quantum correlation; PDI, protein disulfide

isomerase; RDC, residual dipolar coupling; RNase A, ribonuclease A.

The structure of yeast PDI has been determined in

two crystal forms [10,11] In both structures, the

protein adopts a U shape with the catalytic a and a¢

domains on the same side of the protein

Compari-son of the two structures shows that considerable

flexibility exists in the interdomain linkers The

larg-est difference is a twist of over 120 in the relative

orientations of the a and b domains In one

struc-ture, the catalytic cysteines face each other; in the

other, the catalytic residues of the a domain face

away from the a¢ domain The crystal structures also

revealed the presence of a hydrophobic pocket

(which faces inwards at the base of the U) in the b¢

domain This b¢-domain pocket was postulated to be

the site for binding of incompletely folded proteins,

along with adjoining contiguous portions of the a, b

and a¢ domains [12] Cross-linking studies with

radio-labelled model peptides identified a homologous,

hydrophobic binding site on the b¢ domain of human

PDI [13]

Sequence identity between human and yeast PDI for

the b and b¢ domains is < 10% (Fig 1), making it

dif-ficult to compare the two proteins accurately The

structures of individual a [14], b [15], a¢ (Protein Data

Bank entry code 1X5C) and b¢ [16] domains of human

PDI have been solved by NMR or X-ray

crystallogra-phy The overall shape of full-length human PDI has

been investigated by small-angle X-ray scattering and

shown, at low resolution, to adopt a flat annular

arrangement [17]

Here, we reported the solution structure of the bb¢

fragment of human PDI and addressed the question of

how PDI recognizes unfolded proteins Using NMR

titrations, we mapped the region of PDI that binds

unfolded proteins and we showed that peptides which

bind to this region inhibit the PDI-catalyzed refolding

of ribonuclease A (RNase A)

Results

Spectra of the human PDI-bb¢ domains Dimerization of PDI fragments containing the hydro-phobic b¢ domain has complicated structural studies for more than a decade [11] NMR spectra of the isolated b¢ domain of human PDI showed broad lines and mul-tiple peaks as a result of the presence of a mixture of monomeric and dimeric forms (Fig S1) These forms can be separated by gel filtration but rapidly exchange

at 20–30C ( 20% of dimers in 2 h and more than 50% of dimers in 12 h, starting with pure 0.5 mm b¢ monomer at 30C) The 1H-15N heteronuclear single-quantum correlation (HSQC) spectrum of PDI-b¢ was significantly better upon the addition of hydrophobic compounds, such as peptide ligands or detergents (e.g 0.3% Triton X-100), that dissociate the dimer (Fig S1) Alternatively, the b domain moderates the tendency of the b¢ domain to dimerize and significantly slows inter-conversion The monomeric form of the PDI-bb¢ frag-ment converts into < 10% of dimers in 12 h and

< 25% of dimers in 3 days, starting with 0.5 mm of monomers at 30C Most of the1H-15N HSQC signals

of the dimeric form of PDI-bb¢ coincide with the signals

of the monomeric form or are weak as a result of the high molecular weight of the dimer The monomeric form gives good-quality spectra required for structural studies The 1H-15N spectrum of the 25 kDa PDI-bb¢ fragment shows signal dispersion typical for a well-folded protein and allows determination of the back-bone and side-chain NMR signal assignments [18]

Protein structure and comparison The solution structure of the human PDI-bb¢ fragment was calculated based on  2200 NMR-derived

con-Fig 1 Structure-based sequence alignment

of human and yeast PDI-bb¢ showing the

positions of the a-helices and the b-strands.

Color shading represents the size of the

amide chemical shift changes in human

PDI-bb¢ upon the binding of unfolded

RNase A (red, Dd > 0.10; yellow,

0.10 > Dd > 0.05 p.p.m.) Residues are

numbered from the initiator methionine

in the signal sequence.

straints, including 1H-15N residual dipolar couplings

(RDCs) (Fig S2) The set of best structures is

pre-sented in Fig 2A and the structural statistics are shown

in Table 1 The mean rmsd obtained from the average

structure was 0.7 A˚ for backbone atoms The greatest

uncertainties were in modeling helix a3 in the b domain

and the loop between b4¢ and b5¢ in the b¢ domain A

ribbon representation of the PDI-bb¢ structure is

pre-sented in Fig 2B The structures of both b and b¢

domains corresponded to a babababba

thioredoxin-like fold, where the central five-stranded b-sheet is

sur-rounded by a-helices on both sides Heteronuclear

15N{1H} NOEs were in the range of 0.6–0.9 (Fig S2),

indicating the absence of a flexible interdomain linker

Contacts between the b and b¢ domains could be

observed as long-range NOEs between protons

Ha(His231)⁄ Ha(Gly251), He1(His231)⁄ HN(Gly251),

Me(Val155)⁄ HN(Leu234) and He1(Phe209)⁄ HN(Leu236)

Analysis of RDCs for the two domains yielded the

same degree of alignment and rhombicity, which

fur-ther confirms the rigid structure of the bb¢ domain

frag-ment (Fig S2) It is interesting to note that the protein

surface and electrostatic potential is quite different for

the b and b¢ domains (Fig 2C)

The pairwise Ca-atomic coordinate rmsd between

human PDI-bb¢ and the crystal structure of yeast PDI

[10] was 3.5 A˚ for 198 structurally equivalent amino

acids (DALI Z-factor = 14.6) The principal

differ-ence between the protein fold of human and yeast PDI

bb¢ domains was an extra helix, a3, in the b domain of

the human protein and an extra a-helix in the b¢

domain from yeast (Figs 1 and 3B) In this sense, the

fold of human PDI-bb¢ is more similar to the fold of

human ERp57-bb¢ ERp57 is a disulfide isomerase that has the same domain architecture as PDI but shares very low sequence identity with PDI and is glycopro-tein specific via interaction with calnexin or calreticu-lin The rmsd between human PDI-bb¢ and the crystal structure of human ERp57-bb¢ [19] is 4.5 A˚ for 209 amino acids (Z = 14.9) A comparison of the b domain in our PDI-bb¢ structure with the reported solution structure of the isolated b domain [15] gave

an rmsd of 1.6 A˚ for 101 amino acids (Z = 16.1), showing that the structure is not changed significantly

by interaction with the b¢ domain

The pairwise Ca-atomic coordinate comparison of our solution structure of human PDI-bb¢ and the crys-tal structure of the I289A mutant of the human PDI-b¢x fragment [16] showed differences with an rmsd of 2.6 A˚ for 116 amino acids (DALI Z-factor = 12.1) Superimposition of these structures is shown in Fig 3C The differences could result from (a) an effect

of the second b domain in our PDI-bb¢ structure, (b) the I289A mutation, or (c) the presence of the x-linker

in the crystal structure In the b¢x structure, the hydro-phobic x-linker folds back and binds to the b¢ domain

in the region that we identified here as the hydropho-bic peptide-binding pocket (vide infra)

Binding site for unfolded ligands Analysis of the PDI-bb¢ electrostatic surface revealed a highly hydrophobic region within the b¢ domain (Fig 2C) Previous work has demonstrated that the amphipathic peptides mastoparan and D-somatostatin can bind directly to PDI, and that this interaction is

Tri-A

B C

Fig 2 The human PDI-bb¢ fold Stereoview

of the backbone superposition for 10 low-energy structures (A); ribbon representation

of the solution structure of PDI-bb¢ (B); and color-coded surface of PDI-bb¢, with red indicating negative electrostatic potential and blue indicating positive potential (C).

ton X-100 sensitive [13] This finding was confirmed by

NMR titrations of PDI-bb¢ by mastoparan and

somato-statin peptides and unfolded RNase A protein

Compar-ison of1H-15N HSQC spectra of PDI-bb¢ in the absence

or presence of unfolded ligands (Fig S1) was indicative

of strong shifts of the NMR signals for residues in heli-ces a1¢, a3¢ and all five b-strands of the b¢ domain The most strongly shifted HSQC signals were Thr241, Ala245, Phe249, Gly250, His256, Asp297, Glu322, Met324 when titrated with mastoparan (Fig 4A), Thr241, Gln243, Ile248, Gly250, Asp297, Arg300, Ile318, Thr325 when titrated with somatostatin and Thr241, Gly251, His256, Ile318, Thr319 and Glu321 when titrated with unfolded RNase A (Figs 1 and 4C) The chemical shift changes were plotted throughout the PDI-bb¢ sequence, and affected residues were mapped onto the protein backbone trace (Fig 4B,D) A close-up view of the hydrophobic pocket in the b¢ domain is shown in Fig 3A The binding pocket is large and could accommodate multiple hydrophobic residues The sig-nals identified by NMR belong to hydrophobic residues

of the binding pocket or neighboring residues, which could be influenced by steric contacts with the side chains of the hydrophobic residues and small changes in the conformation of the b¢ domain From the NMR titrations, the dissociation constant (Kd) was 130 ±

30 lm for mastoparan and 35 ± 15 lm for both somatostatin and unfolded RNase A (Fig S2) The higher affinity of somatostatin and unfolded RNase A compared with mastoparan is probably a result of the larger number of hydrophobic residues with aromatic side chains In control NMR titrations, folded RNase A showed essentially no binding to PDI-bb¢ (Kd > 2 mm),

as hydrophobic patches of RNase A are not exposed to solvent in the folded state

The structure of the bb¢ domains of the glycopro-tein-specific PDI homolog, ERp57, did not reveal a similar hydrophobic-binding pocket [19] ERp57 instead relies on substrate recruitment by the lectin-like chaperones calnexin and calreticulin, which bind the ERp57 b¢ domain on the surface opposite to the corre-sponding hydrophobic surface in PDI [5] Nonetheless, many of the hydrophobic residues in the PDI-b¢ pocket (shown in Fig 3A) are similar in other PDI family

Table 1 Structural statistics for PDI-bb¢.

Restraints for structure calculations

Final energies (kcalÆmol)1)

rmsd from idealized geometry

rmsd for experimental restraints

RDCs

Coordinate rmsd from the average structure (A ˚ ) a

Ramachandran analysis (%)

Residues in most favored regions 84.0 ± 2.2

Residues in additional allowed regions 12.8 ± 3.0

Residues in generously allowed regions 3.2 ± 1.2

a For residues 137–350.

A B C

Fig 3 (A) View of the peptide-binding hydrophobic pocket in the human PDI-b¢ domain with the residues displayed in stick representation (B) Superimposition of the solution structure of the human PDI-b¢ (blue) with the crystal structure of yeast PDI-b¢ (red, Protein Data Bank entry code 2B5E) (C) Superimposition of the solution structure of human PDI-b¢ (blue) with the crystal structure of the human PDI-b¢x I289A mutant (green, Protein Data Bank entry code 3BJ5) The x-linker tail of PDI-b¢x is shown in red.

members (Fig S3) It is likely that PDIp, PDILT,

ERp27 and ERp44 share features with PDI concerning

how they bind unfolded proteins [20–22]

Residue-specific interactions

The importance of individual amino acids in human

PDI for the binding of D-somatostatin was previously

investigated [23], but many of the reported mutations

were not in the region of the PDI-b¢ binding pocket

In that study it was reported that the mutation of

resi-due Ile289 (numbered as Ile272 without the PDI signal

sequence), which is located at the bottom of the b¢

hydrophobic pocket (Fig 3A), significantly reduced

cross-linking with D-somatostatin To explore the role

of individual amino acids in the b¢ domain, we

prepared two mutants (I289A and F240E) in both the

bb¢ fragment and the full-length protein Surprisingly,

NMR titration experiments of the binding of

somato-statin and mastoparan to the PDI-bb¢ I289A mutant

did not show a significant effect in comparison with

wild-type bb¢ domains (data not shown) We also

investigated the effect of the I289A mutant on the

PDI-catalyzed refolding of RNase A using a continu-ous spectroscopic assay of 2¢3¢ cCMP hydrolysis (Fig 5A) In agreement with the NMR titration, the I289A mutant did not diminish the foldase activity of the PDI By contrast, the mutation of F240E strongly decreased PDI-catalyzed refolding This mutation destablized the b¢ domain (most 1H-15N HSQC signals

of the b¢-domain of the PDI-bb¢ F240E mutant were shifted in comparison with wild-type protein and strongly broadened) and prevented peptide binding in the context of the bb¢ fragment

To identify peptide residues involved in binding to PDI, we carried out reciprocal NMR titrations by observing changes in the signals for mastoparan following the addition of PDI-bb¢ protein (Fig 6) At the lowest concentration of PDI-bb¢, at least half of the 14 mastoparan signals were significantly shifted by binding to the b¢ domain of PDI No strong selectivity

in residue binding was found At a protein⁄ mastoparan ratio of 1 : 15, practically all of the mas-toparan signals (except for those of the terminal amino acids) were strongly broadened as a result of binding

to the PDI-bb¢ protein Further work is necessary to

D

C

Fig 4 Mapping residues involved in ligand binding Magnitude of amide chemical shift changes in the primary sequence of PDI-bb¢ and backbone trace of PDI-bb¢ colored according to the magnitude of the chemical shift changes upon binding mastoparan (A, B) and unfolded RNase A (C, D).

determine the precise roles of substrate residues and

residues in the b¢ domain, but our preliminary results

indicate that the binding reaction involves multiple

redundant interactions

Inhibition of PDI in RNase A refolding

In order to understand better the contribution of the b¢ binding site to PDI activity, the inhibitory influence

on the refolding of RNase A caused by the peptides binding b¢ was examined In the assay, incubation of unfolded RNase with PDI led to RNase activity, which was measured by the hydrolysis of tRNA In the absence of an inhibitor or in the presence of a highly charged peptide, RNase A was rapidly refolded

in 10 min, leading to the disappearance of tRNA (Fig 5B) Addition of the hydrophobic peptides, mas-toparan or D-somatostatin, inhibited RNase A refold-ing at concentrations similar to their affinity to the bb¢ fragment Mastoparan completely inhibited RNase A

A

B

Fig 5 PDI-catalyzed RNase-refolding assays (A) Mutagenesis of

the b¢ domain reduces the efficiency of PDI-catalyzed refolding of

RNase A in a simultaneous refolding and cCMP hydrolysis assay.

The refolding rate of the F240E mutant was 50% lower than that

of the wild-type PDI or the I289A mutant relative to spontaneous

refolding in the absence of PDI A small increase in absorbance

was observed in the absence of RNase A (B) Peptides that bind to

the b¢ domain inhibit PDI refolding of RNase A in a dose-dependent

manner Folding reactions were carried out with the indicated

con-centrations of a control peptide (KEKEKVKQIPKAPK), mastoparan,

or D-somatostatin, and the activity of RNase A was measured in a

gel assay of tRNA hydrolysis In the presence of the control

pep-tide, PDI rapidly refolded RNase A, leading to the complete

degra-dation of the substrate tRNA Both mastoparan and D-somatostatin

blocked refolding.

Fig 6 NMR titrations of 2 m M mastoparan by human PDI-bb¢ protein and a plot of the magnitude of changes in mastoparan proton chemical shifts at a protein ⁄ mastoparan ratio of 1 : 15.

refolding at 120 lm, whereas D-somatostatin blocked

refolding at concentrations between 30 and 60 lm

These are similar to the KI of 80 lm reported for the

inhibition of PDI glutathione-insulin transhydrogenase

activity by the peptide somatostatin [24] Control

experiments with di(o-aminobenzyl)-labeled oxidized

glutathione showed no inhibition of PDI

oxido-reduc-tase activity by D-somatostatin (data not shown)

A systematic study of the kinetics of PDI-mediated

RNase A refolding showed that the refolding rate of

RNase A is saturable with increasing concentrations of

unfolded RNase A [25] The Km measured, 7 lm, is

close to the affinity measured by NMR for unfolded

RNase A binding to the isolated bb¢ domains The

sec-ondary importance of other domains for the binding of

large protein substrates has been previously

demon-strated [13,26] Mutational analysis of PDI revealed that

loss of the two cysteines in the C-terminal a¢ domain

increased the Kmto 30 lm, and loss of an additional

cys-teine in the a domain resulted in an increase of the Kmto

50 lm [25,27] On the other hand, the role of the b

domain seems to be to act simply as a spacer to allow

room for the a and a¢ domains to interact with substrate

thiols By NMR, we detected no interactions between

unfolded RNase A and the b domain

Discussion

There are many examples of chaperone proteins that

bind unfolded protein segments via hydrophobic

patches The best known is cytosolic Hsp70, which

binds and releases, through cycles of ATP binding and

hydrolysis, short stretches of hydrophobic polypeptides

that are in an extended conformation [28] In

Escheri-chia coli, the ClpA–ClpP chaperones disaggregate and

unfold proteins in order to degrade them ClpA binds

to substrates with low affinity, but broad specificity,

via a hydrophobic surface formed by two helices in its

N-terminal domain [29] Multisubunit GroEL binds

in vivo to more than 10% of newly synthesized

poly-peptides [30] via a groove between two alpha helices

that is lined with hydrophobic residues [31]

Neverthe-less, hydrophobic binding is not a universal mechanism

of chaperone function, and other chaperones use

charged and polar residues for interactions between

the chaperone and the substrate [31,32]

The relatively weak binding of PDI-bb¢ to peptides

and unfolded RNase A, and the large size of the

binding pocket, is consistent with a low degree of

specificity for hydrophobic ligands High specificity is

not expected because PDI acts on many substrates

with different primary sequences It is also important

that substrate proteins are released from PDI after

disulfide bond formation and protein folding A large, multivalent hydrophobic binding site is an effective way to bind a variety of substrates when unfolded and to release them once they acquire their native conformation with fewer hydrophobic residues exposed

To conclude, structural analysis of the bb¢ fragment of PDI has revealed a large hydrophobic surface that inter-acts with peptides and unfolded RNase A This site appears to be responsible for the saturable kinetics observed for RNase A folding by PDI, and blocking the site strongly inhibits the activity of PDI Structural anal-ysis of the substrate-binding sites of other disulfide isomerases should shed more light on their substrate specificities and help to explain why such a large variety

of disulfide isomerases is found in the mammalian

ER [5]

Experimental procedures

Sample preparation PDI was cloned from cDNA derived from human bronchial epithelial cells The bb¢ (residues P135–S357) and b¢ (residues L236–S357) fragments were subcloned into pGEX-6P-1 (Amersham Pharmacia Biotech, Piscataway,

NJ, USA) and expressed in E coli BL21 (DE3) as glutathi-one S-transferase (GST) fusion proteins To provide iso-tope-labeled samples for NMR, cultures were grown at

37C on minimal M9 medium supplemented with 15N ammonium chloride and [13C]-glucose (Cambridge Isotopes Laboratory, Andover, MA, USA) to produce uniformly 15

N- or 15N,13C-labeled proteins The protein was purified

by GST-affinity chromatography on a Glutathione Sepha-rose 4B column (Amersham) PreScission protease (Amer-sham) was used to cleave the fusion protein from GST The resulting proteins contained five extraneous N-terminal resi-dues (GPLGS) Further purification was carried out using gel-filtration chromatography on a Superdex-75 column Mass spectral analysis confirmed the sequence composition

of human PDI-bb¢ The NMR samples contained 0.1-1 mm protein in 90% H2O⁄ 10% D2O, 25 mm sodium phosphate buffer (pH 7.0), 70 mm NaCl, 0.5 mm EDTA and 5 mm dithiothreitol

Unlabeled 14 amino acid mastoparan INLKALAALAK KIL, D-somatostatin AGSKNFFWKTFTSS and charged KEKEKVKQIPKAPK peptides were chemically synthe-sized at EZBiolab (Westfield, IN, USA) and additionally purified by reverse-phase HPLC Somatostatin AGCKN FFWKTFTSC (‡ 97% pure by HPLC) was purchased from Sigma (St Louis, MO, USA) Bovine pancreatic RNase A from Sigma was unfolded and reduced for 20 min

at room temperature in 0.1 m Tris⁄ HCl (pH 8.0) containing

6 m guanidine⁄ HCl and 20 mm dithiothreitol [33] Unfolded

RNase A was desalted in 0.1% formic acid on a NAP-5

column (Amersham) and lyophilized The maximum

solubility of the unfolded RNase A in the NMR phosphate

buffer was 0.2 mm

Mutagenesis of PDI

Point mutants of full-length PDI and the bb¢ domains were

prepared in the vectors used for the expression of the

wild-type proteins using Quickchange site-directed mutagenesis

(Stratagene, La Jolla, CA, USA) with mismatched primers

and were verified by DNA sequencing

NMR spectroscopy

NMR spectra were recorded at 30C on Bruker DRX

600 MHz and Varian Unity Inova 800 MHz spectrometers

equipped with triple-resonance cryoprobes and pulsed-field

gradients Proton homonuclear NOEs were obtained from

15

N-edited and 13C-edited NOESY spectra recorded at

800 MHz with a mixing time of 80 ms Amide heteronuclear

15N{1H} NOEs were measured to determine high-mobility

regions of protein [34].1H-15N RDCs with precision ± 1 Hz

were extracted from in-phase/anti-phase-HSQC experiments

[35] on an isotropic sample and on a sample containing

12 mgÆmL)1 of Pf1 phage NMR spectra were processed

using nmrpipe [36] and xwinnmr (Bruker Biospin, Milton,

Canada) software, and then analyzed using xeasy [37] and

nmrview[38] Detailed analysis of ligand binding to PDI-bb¢

was carried out by comparison of chemical shifts for

back-bone amide signals in1H-15N HSQC spectra HSQC spectra

were recorded at 1 : 2, 1 : 1, 2 : 1, 4 : 1 and 8 : 1 peptide to

protein ratios The magnitude of amide chemical shift

changes was calculated as [(D1H shift)2+ (D15N

shift· 0.2)2

]1⁄ 2, in p.p.m Values of dissociation constants

were obtained by monitoring the chemical shift changes as a

function on ligand concentration using a simple binding

model A least-squares search was performed by varying the

values of Kdand the chemical shift of fully saturated protein

Standard deviations were derived for each Kdvalue by

com-paring different cross-peaks in the HSQC spectra

Assignments of the amide proton signals of mastoparan

were determined using 2D NOESY with a mixing time of

200 ms and TOCSY experiments on a 2 mm sample at 10C

Structure calculations

Regions of a-helical or b-strand secondary structure were

determined based on Ca-chemical shifts [39] and the NOE

patterns [40] ARIA-assigned [41] and manually verified

NOEs were collected from 15N- and 13C-edited NOESY

spectra Backbone angles were estimated from the chemical

shifts using the TALOS database [42] The starting

struc-ture was generated with modeller [43] using the yeast PDI

crystal structure (Protein Data Bank entry code 2B5E) and was in agreement with manually assigned NOEs The pro-tein structure was refined using the standard protocol in CNS version 1.1 [44], and the structural statistics for the 10 best structures is shown in Table 1 The atomic coordinates have been deposited as the Protein Data Bank entry 2K18 The pairwise coordinate rmsd comparisons between differ-ent proteins were obtained using dali [45] module software [46] was used for comparison of the RDCs with their back-calculated values Structural figures were generated using py-mol [47] and molmol [48] protskin (C Deprez and

K Gehring; http://www.mcgnmr.mcgill.ca/ProtSkin) soft-ware was used for mapping chemical shift changes onto pro-tein backbone traces procheck-nmr software [49] was used

to check the protein stereochemical geometry (Table 1)

Refolding of bovine RNase A by PDI PDI-catalyzed refolding of RNase A was measured in two assays: a continuous spectroscopic assay of 2¢3¢ cCMP hydrolysis; and a gel-based assay of RNA degradation PDI and unfolded RNase A were prepared as described above The first assay monitored the absorbance change at

296 nm and was carried out as previously described [50] with the following modifications Refolding was carried out

in 25 mm Hepes, pH 8.0, containing 0.5 mm oxidized gluta-thione, 2 mm reduced glutagluta-thione, 0.75 mm CaCl2 and

100 mm NaCl The concentration of reduced RNase A in the refolding reaction was 4.2 lm, and the concentration of PDI was 0.6 lm In the second assay, 0.18 lm RNase A was refolded with 0.3 lm PDI in 25 mm Hepes, pH 8.0, containing 0.5 mm oxidized glutathione, 2 mm reduced glu-tathione, 0.75 mm CaCl2and 100 mm NaCl Samples were removed during folding and free thiols were blocked with

an equal volume of 0.5 m iodoacetamide The RNase A activity at each time-point was assayed by incubation with

10 lg of yeast tRNA (Sigma) for 15 min at 25C followed

by electrophoresis in a 1% agarose gel containing ethidium bromide for visualization Gels were exposed to UV light and photographed using an Alpha Innotech Alpha Imager Control experiments with di(o-aminobenzyl)-labeled oxi-dized glutathione (a gift of Bulent Mutus) were carried out

as described previously [51]

Acknowledgements

The authors are grateful to Lloyd Ruddock for sharing data and helpful discussions and to Tara Sprules for assistance in running experiments at the Quebec-East-ern Canada High Field NMR Facility This work was funded by operating grants to D T and K G from the Canadian Institutes of Health Research (CIHR)

P M was supported by a CIHR Canada Graduate Scholarships Doctoral Award

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Supporting information

The following supplementary material is available: Fig S1 Comparison of 1H-15N HSQC spectra of human PDI-b¢ (A) and PDI-bb¢ (B) in the absence (black) and presence (red) of mastoparan (at 8 : 1 pep-tide-protein ratio)

Fig S2 Values of the15N{1H} heteronuclear NOE for backbone amides in human PDI-bb¢, the correlation between the observed and back-calculated RDCs for solution structure of human PDI-bb¢, and changes of chemical shifts in PDI-bb¢ Asp297 versus peptide concentrations

Fig S3 Multiple sequence alignment of the b¢ domains for human PDI family

This supplementary material can be found in the online version of this article

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