Supplementary information.

MTP-PDI structure quality.

A protocol for the production of MTP in E.coli was established and the crystal structure of the full-

length heterodimeric protein complex was determined to 2.5 Å by molecular replacement using the

structure of lipovitellin (1) (PDB code: 1LSH) and full-length human PDI (2) (PDB code: 4EKZ).

Crystals of MTP belong to space group P1 and contain two copies of the MTPα-PDI heterodimer in

the asymmetric unit, with PDI annotated as chains A and B and the corresponding MTPα as chains

G and H. At this resolution, the electron density is generally of high quality with the majority of side

chains clearly visible. The electron density is not visible for the C-terminus of each PDI and MTPα

chain; chains A and B of PDI are missing 12 and 10 amino acid residues from the C-terminus,

respectively, while chains G and H of MTPα are missing the last 9 and 8 amino acids, respectively.

In each chain of MTPα, there is a short internal section for which the electron density is missing,

presumably corresponding to a flexible loop. This includes residues 717 – 721 in chain G and 717 –

722 in chain H. Excluding this minor difference, both copies of MTPα in the asymmetric unit are

similar, with a RMSD of 0.9 Å. In contrast, the calculated RMSD for the PDI subunits is 2.1 Å, with a

small shift in the position of the b domain relative to a and b domains and a moderate shift of the a

domain away from the rest of the PDI chain. This can be attributed to the overall flexibility of PDI

molecule. The interactions between MTPα and PDI chains are similar in both copies of the complex

in the asymmetric unit indicating that the interacting interfaces are not crystallization artifacts. Unless

stated, all analysis of the MTP complex was undertaken on chain B of PDI and chain H of MTPα, for

which the quality of electron density was judged to be marginally higher.

Small scale protein expression and purification.

Wild-type and mutant forms of MTP for biophysical analysis were expressed and purified as per the

protein for crystallization, with the following exceptions.

The media used was autoinduction terrific broth and prior to induction the temperature was lowered

to 15°C. Cells were harvested at 6,500 × g for 20 min and cell pellets were resuspended in lysis

buffer (50 mM NaP pH 7.4). Cell debris was cleared out by centrifugation at 30,600 × g for 40 min.

Instead of a HiTrap Chelating IMAC column, HisPur cobalt resin (Thermo Fisher Scientific) was used

for IMAC. The purification was performed in a cold room using gravity flow columns. The flow through

www.pnas.org/cgi/doi/10.1073/pnas.1903029116

was collected and column was additionally washed with 200 ml of 50 mM NaP pH 7.4, 500 mM NaCl,

15 mM Imidazole. The proteins of interest were eluted with 50 mM NaP pH 7.4, 300 mM Imidazole.

All fractions were analysed by SDS-PAGE and relevant fractions combined. Further purification (ABL

mutants and V664F and F813H ligand binding site mutants) was undertaken by concentrating IMAC

purified fractions to a final volume of 1.5 ml using Amicon® Ultra centrifugal filter with 30 kDa cut-off

membrane. Then size exclusion chromatography was performed using a HiLoad™ Superdex S200

16/60 pg column (GE Healthcare) previously equilibrated with 50 mM BisTris Propane pH 7.5, 200

mM NaCl. Eluted fractions were analysed on SDS-PAGE and fractions containing MTP were

combined.

 

Figure S1. Comparison of MTPα and lipovitellin structures. (A – B) Overlay of the N-terminal β-

barrel (A) and the α-helical domains (B) of MTPα and lipovitellin (grey). These domains maintain

similar architecture (RMSD of 2.16 and 3.04 Å, respectively). (C) Comparison of the MTPα structure

(depicted in the same colors as in Figure 1) to the structure of lipovitellin (grey) (1LSH). While the N-

terminal domains maintain the same domain architecture the C-terminal lipid binding domain differs

significantly between the two proteins reflecting their ability to bind and transfer different amounts of

lipids. Cysteine residues are shown in ball and stick representation and colored in red. Each view of

MTPα and lipovitellin are related by 180º rotation around a horizontal axis.

 

Figure S2. Lipid binding site of MTPα. (A) The electrostatic potential surface of the lipid binding

site of C- and A-sheets is colored based on charge distribution. The positive charge is shown in blue

and the negative charge is shown in red. PEG molecules are shown as stick models. (B) Electron

density maps for PEG molecule (chain G) before it is modelled. Molecules are shown in stick

representation; carbon atoms of the PEG molecule are shown in cyan, those of the residues forming

the lipid binding site of MTPα in grey. C-sheet is shown at the bottom of the figure, A-sheet is shown

on top of the figure. The BUSTER 2Fo-Fc map is shown at 0.9 σ level (blue), and the Fo-Fc map is

shown at 2.7 σ level (green) and -3 σ (red). (C) The REFMAC5 omit Fo-Fc map around the PEG

molecule site is shown in the same orientation as on Figure 3B at 2.4 σ level (green) and -3 σ level

(red). This map was calculated using combination of averaged phases from DMMULTI and phases

calculated from the model before addition of PEG molecules. To reduce the overestimation of phase

accuracy by density modification procedure a scale factor of 0.8 and B-factor of 25 Å2 were applied

to Hendrickson-Lattman coefficients representing DMMULTI phase probability distribution.

Unmodelled regions of the positive difference density indicate that the PEG molecule could be

present in several alternative conformations.

Figure S3. Analysis of mutations in lipid binding site of MTPα. (A) Coomassie stained SDS-

PAGE gel of IMAC purified MTP wild-type (lane 1) and L633F (lane 2), S662L (lane 3), V664F (lane

4), V778L (lane 5) and F813H (lane 6) mutants. (B – C) Relative lipid transfer activity of (B) IMAC

purified MTP mutants (n = 3; mean ± SD; concentration normalized based on densitometric analysis

of SDS-PAGE gel) and (C) IMAC and gel-filtration purified MTP mutants (n= 3; mean ± SD;

concentration determined by A280). Mutations cause both a significant increase (S662L, V664F,

V778L) and decrease (L633F and F813H) in lipid transfer activity (* P < 0.1; ** P < 0.05; *** P < 0.01;

t test with two tailed distribution and two-sample unequal variance). (D) The V664F (blue) and F813H

(red) mutations show the same near uv CD spectra as wild-type MTP (black). (E) Thermal stability

of wild-type MTP based on Thermofluor analysis. The insert panel shows the derivative of the change

in fluorescence signal and was used to determine the melting temperature (Tm). (F) Tm of the V664F

and F813H mutants is comparable with that of wild-type MTP. Note, the maximal change in volume

(48 Å3) resulting from any of these mutations is small compared with the volume of the lipid binding

cavity (2100 Å3).

Figure S4. Analysis of ABL causing mutations in MTPα. (A) Relative lipid transfer activity of the

S590I and N649S mutants (n = 3; mean ± SD). Both mutations result in a significant decrease in

lipid transfer activity (*** P < 0.01; t test with two tailed distribution and two-sample unequal variance).

(B) The S590I (blue) and N649S (red) mutations show the same near uv CD spectra as wild-type

MTP (black). (C) Melting temperature (Tm) of the N649S mutant is comparable with that of wild-type

MTP.

   

MILLAVLFLCFISSYSASVKGHTTGLSLNNDRLYKLTYSTEVLLDRGKGKLQDSVGYRISSNVDVALLWR 70 1 * 2 * * * ** 3 ** * ** * * * * * * *** 4 ********* * * * ** * * * * **** NPDGDDDQLIQITMKDVNVENVNQQRGEKSIFKGKSPSKIMGKENLEALQRPTLLHLIHGKVKEFYSYQN 140 1 * * 2 * * ** 3 * * * * * * ** *** 4 * *** * * ** ** * ** * * ** * *** EAVAIENIKRGLASLFQTQLSSGTTNEVDISGNCKVTYQAHQDKVIKIKALDSCKIARSGFTTPNQVLGV 210 1 * * * ** * * 2 *** * * * * * ** * * * * 3 * ******* * * ** * * ** * * * * * * 4 * ******* * ** *** * * ** * ** * * * * * * ** SSKATSVTTYKIEDSFVIAVLAEETHNFGLNFLQTIKGKIVSKQKLELKTTEAGPRLMSGKQAAAIIKAV 280 1 2 * * 3 * * *** * ** * * *** ** * 4 * * ***** ** * * * * * *** ** ** * DSKYTAIPIVGQVFQSHCKGCPSLSELWRSTRKYLQPDNLSKAEAVRNFLAFIQHLRTAKKEEILQILKM 350 1 2 * 3 * * * * * ** 4 * ** * * * * * * ** ** * ** ENKEVLPQLVDAVTSAQTSDSLEAILDFLDFKSDSSIILQERFLYACGFASHPNEELLRALISKFKGSIG 420 1 * * * * * * * 2 * * * * * * * * * 3 * * * *** * *** ******** ** * * * * * 4. ****** ***** * ****** ********* ** ** * ** * * * SSDIRETVMIITGTLVRKLCQNEGCKLKAVVEAKKLILGGLEKAEKKEDTRMYLLALKNALLPEGIPSLL 490 1 * 2 * * ** ** * 3 * * * ** * * ** *** * * * 4 * **** * * * * * ** * * ** * * *** **** **** * * ** KYAEAGEGPISHLATTALQRYDLPFITDEVKKTLNRIYHQNRKVHEKTVRTAAAAIILNNNPSYMDVKNI 560 1 * * * * 2 * ** * * * 3 * * ** * ** * *** **** ** ** * * * 4 * **** *** ** *** * *** ******* ** * * * ** LLSIGELPQEMNKYMLAIVQDILRFEMPASKIVRRVLKEMVAHNYDRFSRSGSSSAYTGYIERSPRSAST 630 1 * * ** * 2 * * ** * * * 3 ***** * ** *** ** * * * ** ** ** * ** ** * 4 ***** ** ** **** ** ** * ** *** ****** * ** *** *

YSLDILYSGSGILRRSNLNIFQYIGKAGLHGSQVVIEAQGLEALIAATPDEGEENLDSYAGMSAILFDVQ 700 1 * ** 2 * * * ** * ** **** * * * * * 3 * * *** ******** * *** **** * *** ** *** * * * * * * 4 * ** ************ * *** **** * ****** *** * * * * *** * LRPVTFFNGYSDLMSKMLSASGDPISVVKGLILLIDHSQELQLQSGLKANIEVQGGLAIDISGAMEFSLW 770 1 ** ** * ** * * * * *** 2 *** ** ** ** * ** * * * * * 3 ****** ** *** * * ** * * * * * *** * **** 4 ******* ** *** * * * *** * * **** **** * * *** * ***** YRESKTRVKNRVTVVITTDITVDSSFVKAGLETSTETEAGLEFISTVQFSQYPFLVCMQMDKDEAPFRQF 840 1 * * 2 *** * * * * * * 3 * **** ** * * * * ***** *** * ** * 4 ************ ** ** * * * ** ********** * *** * * EKKYERLSTGRGYVSQKRKESVLAGCEFPLHQENSEMCKVVFAPQPDSTSSGWF 1 * * * 2 * * * ** 3 * * * * * * * * * ** ** ** 4 * ***** ** * * * *** ** ** ** * ** Figure S5. Conservation of residues in MTP. Conservation in the full (1; 300 sequences),

vertebrate (2; 216 sequences), mammalian + bird (3; 156 sequences) and mammalian (4; 94

sequences) data subsets (conservation marked with *). For the full dataset where DNA sequencing

errors may be more prevalent, conservation is marked at 98%, for the other data sets it is marked at

100%.

   

Figure S6. Conservation of residues in MTP. A plot of conservation of a rolling 10 amino acid

window of MTPα from the 300 (98%) data set (A), vertebrate subset (B), mammalian + bird subset

(C), and mammalian subset (D). The dotted lines represent the mean + 2 standard deviations. Peaks

above this line are taken to show significant conservation of a linear stretch of amino acids.

   

PDI (MTP) DAPEEEDHVLVLRKSNFAEALAAHKYLLVEFYAPWCGHCKALAPEYAKAAGKLKAEGSEIRLAK PDI (4EL1) DAPEEEDHVLVLRKSNFAEALAAHKYLLVEFYAPWCGHCKALAPEYAKAAGKLKAEGSEIRLAK PDI (MTP) VDATEESDLAQQYGVRGYPTIKFFRNGDTASPKEYTAGREADDIVNWLKKRTGPAATTLPDGAA PDI (4EL1) VDATEESDLAQQYGVRGYPTIKFFRNGDTASPKEYTAGREADDIVNWLKKRTGPAATTLPDGAA PDI (MTP) AESLVESSEVAVIGFFKDVESDSAKQFLQAAEAIDDIPFGITSNSDVFSKYQLDKDGVVLFKKF PDI (4EL1) AESLVESSEVAVIGFFKDVESDSAKQFLQAAEAIDDIPFGITSNSDVFSKYQLDKDGVVLFKKF PDI (MTP) DEGRNNFEGEVTKENLLDFIKHNQLPLVIEFTEQTAPKIFGGEIKTHILLFLPKSVSDYDGKLS PDI (4EL1) DEGRNNFEGEVTKENLLDFIKHNQLPLVIEFTEQTAPKIFGGEIKTHILLFLPKSVSDYDGKLS PDI (MTP) NFKTAAESFKGKILFIFIDSDHTDNQRILEFFGLKKEECPAVRLITLEEEMTKYKPESEELTAE PDI (4EL1) NFKTAAESFKGKILFIFIDSDHTDNQRILEFFGLKKEECPAVRLITLEEEMTKYKPESEELTAE PDI (MTP) RITEFCHRFLEGKIKPHLMSQELPEDWDKQPVKVLVGKNFEDVAFDEKKNVFVEFYAPWCGHCK PDI (4EL1) RITEFCHRFLEGKIKPHLMSQELPEDWDKQPVKVLVGKNFEDVAFDEKKNVFVEFYAPWCGHCK PDI (MTP) QLAPIWDKLGETYKDHENIVIAKMDSTANEVEAVKVHSFPTLKFFPASADRTVIDYNGERTLDG PDI (4EL1) QLAPIWDKLGETYKDHENIVIAKMDSTANEVEAVKVHSFPTLKFFPASADRTVIDYNGERTLDG PDI (MTP) FKKFLE PDI (4EL1) FKKFLE Figure S7. Analysis of interaction sites in PDI. Sequence of mature human PDI (D17 – E471)

with yellow highlighted residues within 5 Å of MTPα (upper sequence) or the other PDI molecule in

the 4EL1 homodimer structure (lower sequence). Residues not visible in the 4ELI structure are

highlighted in blue. The contact sites in the a and a’ domains are similar while the potential contact

sites involving the b’ domain are absent in the structure, either due to missing density in the b’

domain or due to the N-terminal H6-SSGLEVLFQGPGS which is spatially juxtaposed to the b’

domain and D426 – Ala429 in the a’ domain being absent in the structure.

   

PDI (MTP) PLVIEFTEQTAPKIFGGEIKTHILLFLPKSVSDYDGKLSNFKTAAESFKGKILFIFID PDI (S1) PLVIEFTEQTAPKIFGGEIKTHILLFLPKSVSDYDGKLSNFKTAAESFKGKILFIFID PDI (S2) PLVIEFTEQTAPKIFGGEIKTHILLFLPKSVSDYDGKLSNFKTAAESFKGKILFIFID PDI (S3) PLVIEFTEQTAPKIFGGEIKTHILLFLPKSVSDYDGKLSNFKTAAESFKGKILFIFID PDI (MTP) SDHTDNQRILEFFGLKKEECPAVRLITLEEEMTKYKPESEELTAERITEFCHRFLEG PDI (S1) SDHTDNQRILEFFGLKKEECPAVRLITLEEEMTKYKPESEELTAERITEFCHRFLEG PDI (S2) SDHTDNQRILEFFGLKKEECPAVRLITLEEEMTKYKPESEELTAERITEFCHRFLEG PDI (S3) SDHTDNQRILEFFGLKKEECPAVRLITLEEEMTKYKPESEELTAERITEFCHRFLEG

Figure S8. Analysis of interaction sites in the b’ domain. Sequence of mature human PDI (P235

– G349) with yellow highlighted residues within 5 Å of MTPα (upper sequence) or ligand binding

sites reported previously and mapped by chemical shift mapping for KFWWFS (S1), ∆-somatostatin

(S2) (3) and unfolded RNase (S3) (4). The contact sites in the b’ domain are similar for both PDI

substrates and MTPα.

   

PDI M-LRRALLCLAVAALVRADAPEEEDHVLVLRKSNFAEALA---AHKYLLVEFYAPWCGHCKALAP : ::: : :: : : :: : :: :::: :::::::: ::: ERp57 MRLRRLALFPGVALLLAAARLAAASDVLELTDDNFESRISDTGSAGLMLVEFFAPWCGHCKRLAP PDI EYAKAAGKLKAEGSEIRLAKVDATEESDLAQQYGVRGYPTIKFFRNGDTASPKEYTAGREADDIV :: :: :: ::::: : ::: :::: : :: : : : : :: :: ERp57 EYEAAATRLKGI---VPLAKVDCTANTNTCNKYGVSGYPTLKIFRDGEEAG--AYDGPRTADGIV PDI NWLKKRTGPAATTLPDGAAAESLVESSEVAVIGFFKDVESDSAKQFLQAAEAI-DDIPFGITSNS ::: ::: : ::: : : :: :: : : : ERp57 SHLKKQAGPASVPLRTEEEFKKFISDKDASIVGFFDDSFSEAHSEFLKAASNLRDNYRFAHTNVE PDI DVFSKYQLDKDGVVLFK------KFDEGRNNF-EGEVTKENLLDFIKHNQLPLVIEFTEQTAPKI : : :: :: : : :: : :: : ERp57 SLVNEYDDNGEGIILFRPSHLTNKFEDKTVAYTEQKMTSGKIKKFIQENIFGICPHMTEDNKDLI PDI FGGEIKTHILLFLPKSVSDYDGKLSNFKTAAESFKGKILFI---FIDSDHTDNQRI--------- : :: : : : : ERp57 QGKDLL----------IAYYDVDYEKNAKGSNYWRNRVMMVAKKFLDAGHKLNFAVASRKTFSHE PDI LEFFGLKKE--ECPAVRLITLEEEMTKYKPESEELTAERITEFCHRFLEGKIKPHLMSQELPEDW : ::: : : : : : : : : : : : :: ERp57 LSDFGLESTAGEIPVVAIRTAKGEKFVMQEEFSR-DGKALERFLQDYFDGNLKRYLKSEPIPESN PDI DKQPVKVLVGKNFEDVAFDEKKNVFVEFYAPWCGHCKQLAPIWDKLGETYKDHENIVIAKMDSTA : :::: : :: : : : ::::::::::: : : ::: :::::::: :: ERp57 DG-PVKVVVAENFDEIVNNENKDVLIEFYAPWCGHCKNLEPKYKELGEKLSKDPNIVIAKMDATA PDI NEVEA-VKVHSFPTLKFFPASADRTVIDYNGERTLDGFKKFLE : : : ::: : :: : : : : : : ERp57 NDVPSPYEVRGFPTIYFSPANKKLNPKKYEGGRELSDFISYLQ

Figure S9. Alignment of human PDI (M1 – E471) and human ERp57 (M1 – Q481). Highlighted

residues are within 5 Å of MTPα (in PDI sequence) or tapasin (in ERp57 sequence) in the respective

heterodimeric crystal structures. The contact sites in the a and a’ domains are similar, while the b’

substrate binding site in ERp57 has become specialized for interacting with calnexin/calreticulin (5)

and does not interact with tapasin.

Supplementary references

1. Thompson JR, Banaszak LJ (2002) Lipid-protein interactions in lipovitellin. Biochemistry 41(30):9398–9409.

2. Wang C, et al. (2013) Structural insights into the redox-regulated dynamic conformations of human protein disulfide isomerase. Antioxid Redox Signal 19(1):36–45.

3. Byrne LJ, et al. (2009) Mapping of the ligand-binding site on the b’ domain of human PDI: interaction with peptide ligands and the x-linker region. Biochem J 423(2):209–217.

4. Denisov AY, et al. (2009) Solution structure of the bb’ domains of human protein disulfide isomerase. FEBS J 276(5):1440–1449.

5. Hatahet F, Ruddock LW (2009) Protein disulfide isomerase: a critical evaluation of its function in disulfide bond formation. Antioxid Redox Signal 11(11):2807–2850.