supplementary materials for...tetra detector (malvern instruments ltd), which monitors the...
TRANSCRIPT
www.sciencemag.org/cgi/content/full/339/6127/1604/DC1
Supplementary Materials for
The Structural Basis of ZMPSTE24-Dependent Laminopathies
Andrew Quigley, Yin Yao Dong, Ashley C. W. Pike, Liang Dong, Leela Shrestha,
Georgina Berridge, Phillip J. Stansfeld, Mark S. P. Sansom, Aled M. Edwards, Chas Bountra, Frank von Delft, Alex N. Bullock, Nicola A. Burgess-Brown,
Elisabeth P. Carpenter*
*To whom correspondence should be addressed. E-mail [email protected]
Published 29 March 2013, Science 339, 1604 (2013) DOI: 10.1126/science.1231513
This PDF file includes
Materials and Methods Supplementary Text Figs. S1 to S10 Tables S1 to S2 Full References
1
Supplementary Materials for
The structural basis of ZMPSTE24 dependentlaminopathies
Andrew Quigley1†, Yin Yao Dong1†, Ashley C.W. Pike1†, Liang Dong1, Leela Shrestha1,Georgina Berridge1, Phillip Stansfeld2, Mark S. P. Sansom2, Aled M. Edwards3, Chas
Bountra1, Frank von Delft1, Alex N. Bullock1, Nicola A. Burgess-Brown1, Elisabeth P.Carpenter1*.
Correspondence to: [email protected]
This PDF file includes:
Materials and MethodsSupplementary TextFigs. S1 to S10Tables S1 to S2References
2
Materials and Methods
Cloning and expressionThe gene for ZMPSTE24 was purchased from the Mammalian Gene Collection
IMAGE: 5269064. ZMPSTE24WT was expressed using a construct consisting of the fulllength wild type ZMPSTE24 gene with a C-terminal purification tag with a tobacco etchvirus (TEV) protease cleavage site, a 10xHis purification sequence and a FLAG tag in theexpression vector pFB-CT10HF-LIC (available from the SGC). A second construct,ZMPSTE24E305A,E306A,E307A, with surface entropy mutations, where residues E305EE weremutated to AAA, was produced using the same purification tag and vector. In addition,protein with reduced activity was produced by mutating the catalytic Glu336 to alanine.Baculoviruses were produced by transformation of DH10Bac cells. Spodopterafrugiperda (Sf9) insect cells in Sf-900 II SFM medium (Life Technologies) were infectedwith recombinant baculovirus and incubated for 72h at 27°C in shaker flasks.
Selection of suitable detergents for ZMPSTE24 purificationWe assessed the size and composition of the protein-detergent complex formed by
ZMPSTE24 in a range of detergents. We combined size exclusion chromatography (SEC)with a Shodex KW-803 column on a Dionex micro-HPLC system, with analysis with aTetra detector (Malvern Instruments Ltd), which monitors the refractive index, low (7˚)and right angle light scattering, viscosity and UV absorbance. Purifications wereperformed as described below with 1% detergent (w/v) +/- 0.1% cholesterylhemisuccinate (CHS) during protein solubilization and a concentration of 3x thedetergent critical micelle concentration (CMC, Affymetrix) +/- CHS (10:1detergent:CHS) in all other purification buffers. The mass of the PDC and its protein todetergent ratio were calculated as described in Slotboom et al., 2008 (33). Detergent (+/-CHS) standards in the SEC buffer (20mM HEPES, pH 7.5, 200mM NaCl with either0.03% DDM or 0.18% OGNG and 0.018% CHS) were analysed in triplicate on the samecolumn at concentrations of 5, 3.75, 2.85, 2.11, 1.58%. Detergent/lipid standard curveswere used to obtain the differential refractive index (dn/dc), and the differential UVabsorbance (dA/dc) of the detergent micelles. OGNG/CHS was found to give a smallerPDC than DDM and was subsequently used for purification and crystallization.
Production of ZMPSTE24 for crystallization and functional studiesCell pellets from 1 litre of insect cell culture were resuspended in 50ml of lysis
buffer (50mM HEPES, pH 7.5, 200mM NaCl, Roche protease inhibitor cocktail) andlysed by two passes through an EmulsiFlex-C5 homogenizer (Aventis). Protein wasextracted from cell membranes by incubation of the crude lysate with either 1% DDM or1% OGNG and 0.1% CHS for 1h at 4oC. Cell debris and unlysed cells were removed bycentrifugation at 40,000g for 1h. Detergent-solubilized protein was purified byimmobilized metal affinity chromatography by batch binding to Co2+ charged TALONresin (Clontech) at 4ºC for 1h. The resin was washed with 30 column volumes of washbuffer (50mM HEPES, pH 7.5, 200mM NaCl, 20mM imidazole with either 0.03% DDMor 0.18% OGNG and 0.018% CHS) and eluted with wash buffer supplemented with250mM imidazole. The protein was concentrated in a 100kDa cut-off Corningconcentrator and further purified by size exclusion chromatography (SEC) using a
3
Superdex 200 10/300GL column (GE Healthcare) equilibrated with SEC buffer (20mMHEPES, pH 7.5, 200mM NaCl with either 0.01% DDM or 0.12% OGNG and 0.012%CHS). The eluted protein was treated with 4:1 (w:w, protein:protease) TEV proteaseovernight at 4oC. The TEV protease-cleaved protein was separated from the 6xHis-taggedTEV protease and uncleaved ZMPSTE24 by incubation for 1h with TALON resin at 4oC.The resin was collected in a column and the flow-through and initial wash with SECbuffer were collected and concentrated to 10-20mg/ml using a 100kDa Sartoriusconcentrator for crystallization. The molecular weight of each purified ZMPSTE24construct was confirmed using an MSD-ToF electrospray ionization orthogonal time-of-flight mass spectrometer (Agilent Technologies inc. Palo Alto, CA, USA) (34).
Reconstitution of ZMPSTE24 in proteoliposomes for assaysA 3:1 stock of phosphatidyl-choline (PC) and phosphatidyl-ethanolamine (PE)
(Avanti), supplemented with 10% cholesterol (Affymetrix) was prepared. PurifiedZMPSTE24 was diluted 1 in 4000 with this stock and 2% Triton-X was added beforeincubation with Biobeads (Bio-rad) overnight. A further 1h incubation with freshBiobeads was followed by Biobead removal and centrifugation at 100,000g for 1h. Thepellet containing proteoliposomes was re-suspended in 1.2ml of SEC buffer (20mMHEPES pH7.5, 200mM NaCl). The centrifugation and resuspension steps were repeatedtwice more to ensure removal of all Triton-X.
CrystallizationProtein was concentrated to 20mg/ml, then diluted to 9–11mg/ml using SEC buffer
without added detergent. Crystals were grown at 20ºC from sitting drops (150-300nl) setup in 96-well format using a Mosquito crystallization robot (TTP Labtech) andprotein:reservoir ratios of 1.5:1 and 2:1. Two crystal forms were observed depending onthe purification detergent used. Monoclinic (P21) crystals were obtained with either wildtype ZMPSTE24 or the surface entropy mutant (ZMPSTE24E305A,E306A,E307A) purified in0.015% DDM and crystallized with a reservoir solution containing 30% (v/v) PEG 400,0.1M lithium sulfate, 0.1M sodium chloride, 0.1M MES, pH 6.5. Triclinic (P1) crystalswere obtained from protein with the wild type sequence, purified in 0.12%OGNG/0.012% CHS, and crystallized with a reservoir solution containing 30-42% (v/v)PEG 400, 0.1M HEPES pH 7.5, 0.1M calcium chloride. Crystals were cryocooled bytransfer into liquid nitrogen without the addition of a cryoprotectant. Diffraction of the P1crystal form was improved by transferring crystallization plates to 6ºC and equilibrationfor at least 24h prior to direct flash cooling of crystals.
Structure determination and refinementAll data were collected at 100K on beamlines I04 (P21 form) and I24 (P1 form) at
Diamond Light Source using helical / straight line scans with a beamsize of 10m x 10-30m (wxh) and processed with XDS (35) and AIMLESS (36). The final 3.4Å P1 datasetcomprises 545º degrees of data collected from two independent crystals. Diffraction fromthe monoclinic rods was anisotropic, extending to 3.4Å in the best direction and 5-6Å inthe worst direction. Data collection, phasing and refinement statistics are reported inSupplementary Table S2.
4
Phasing was initially performed with the P1 form using single isomorphousreplacement with anomalous scattering (SIRAS) from a single mercury derivative. Heavyatom derivative crystals were obtained by soaking crystals for 1-2 days with reservoirsolution containing OGNG/CHS and 1.2 mM ethylmercury thiosalicylate (EMTS). Theheavy atom sub-structure was determined using SHELXD (37). Twelve mercury siteswere identified initially, three per monomer, and phases were then calculated to 3.8Åusing SOLVE/RESOLVE (38). Initial electron density maps revealed a clear molecularboundary and could be moderately improved by four-fold averaging / phase extensionusing the 3.4Å native P1 data. An initial backbone model was built by manually fittingthe transmembrane helices into these maps and then transformed into the P21 asymmetricunit (AU) using PHASER (four copies per AU) (39). Subsequent eight-fold cross-crystalaveraging between the two crystal forms using DMMULTI (40) gave a substantialimprovement in the quality of the experimental phases and allowed tracing of both thetransmembrane and protease domains (Fig. S2). An initial auto-traced model produced byBUCCANEER (41) was manually rebuilt and extended to cover the entire proteasedomain using COOT (42). The electron density was continuous from TMH1-LH1,TMH3-2 strand in the protease domain and from MH3 to the C-terminus.
Validation of the TM helix assignment and sequence register was based onidentification of cysteine residues using the anomalous difference maps calculated fromdata collected from the mercury (EMTS)-soaked derivative crystals. Human ZMPSTE24contains five cysteines (Cys109, Cys176, Cys324, Cys359 and Cys406). Four anomalous peaks,as well the characteristic bulky electron density of sidechains of large hydrophobicresidues, were used to unambiguously assign TMH4, MH3, TMH6 and TMH7. Cys109
lies in a disordered region between LH1 and TMH3 and there was no appreciable peak inan EMTS anomalous difference map. MH3 assignment was also confirmed by theposition of the zinc ion, located using a strong anomalous difference peak calculated fromdata collected at the Zn edge (=1.282Å), as this helix contains the conserved HEXXHzinc-coordinating motif (Fig. S2). Sequence assignment for the kinked TMH3 helix isconsistent with the positioning of Pro140 (Fig. S2G). There was no density in the initialexperimental DMMULTI map or after model building for the N-terminus, the connectingloop region between LH1 and TMH3 or for 36 residues connecting MH2 and MH3,suggesting that these regions are disordered. In contrast, although electron density for theprotease’s -sheet domain (3-4 hairpin) was absent in the multi-crystal averaged mapsdue to local conformation differences in this region in the triclinic and monocliniccrystals, this region was clearly resolved in the averaged map using the 4-fold non-crystallographic symmetry (NCS) in the P1 crystal form.
Refinement was carried out with BUSTER (v. 2.11.2) (43) using all data to 3.4Åwith NCS and TLS restraints. Elongated tubes of persistent electron density, boundbetween TM5 and TM6 on the outer surface of the membrane-spanning region of thebarrel, were modeled as the alkyl chains of a single phospholipid moiety. Residualelectron density, located close to the charged patch around Glu86 within the barrel’s largeintramembrane chamber, could not be sensibly assigned and has been left un-modelled inthe final P1 structure.
5
The structure has been refined to acceptable R/Rfree values (24.6 / 26.4%) with goodmodel geometry (Table S2). The final P1 model comprises 4 copies of ZMPSTE24 witheach copy encompassing residues 10-107, 116-285, 322-472, a single zinc ion and twophospholipid alkyl chains. The region between MH2 and MH3, encompassing 36residues, could not be resolved in the electron density and no attempt was made to modelthis region. The N-terminus of chain A is more ordered and can be traced back to Gly2.The model has excellent geometry as assessed by the MOLPROBITY server (44) with97% of residues located in favored regions of the Ramachandran plot and no outliers. Thestructure of the monoclinic crystal form will be described elsewhere. The initial density-modified experimental phases, final BUSTER map coefficients and structure factors havebeen deposited in the PDB with codes 4AW6 for the P1 crystal form and 2YPT for thepeptide complex.
Initial P1 peptide co-crystals were obtained by mixing OGNG/CHS-purifiedZMPSTE24 E336A mutant with a 3-fold excess of farnesylated nonapeptide (Gln656-Met664) under similar conditions to the wild type protein. However, datasets collectedfrom such co-crystals showed that the peptide-binding site was empty. These ‘co-crystals’ were then successfully soaked with reservoir solution containing 5mM CSIMtetrapeptide and detergent for 24h at 4ºC. Data were collected to 3.8Å on beamline I24 atDiamond Light Source and processed with XDS (35) and AIMLESS (36). Thecoordinates of the native P1 structure were used as a starting model for refinement withBUSTER (43). Clear difference Fo-Fc electron density in each of the four molecules inthe asymmetric unit was interpreted based on the known binding mode of peptides andinhibitors to metalloproteases. The peptide (mis-)register, with Ser662 and Ile663
positioned in the S1 and S1′ subsites respectively, was determined based on both the length of the electron density, and the definition of the C-terminal methionine which wasreasonably well defined in two molecules (B&D; Fig. S7). The entire tetrapeptide wasmodeled in two cases (B&D) whereas only C661-I663 was modeled in molecules A & E. Inall cases, the quality of the electron density was not sufficient to resolve the CD1 atom ofIle663 and this atom was not included in the model. Refinement was performed withBUSTER with TLS, NCS and LSSR restraints to the native structure (SupplementaryTable S2). Structures were drawn using the open source version of the PyMOL MolecularGraphics System, (Version 1.5, http://www.pymol.org) and the UCSF Chimera package(Figs. 1 to 4, S2 to S5 and S8) (45).
Molecular dynamics simulationsAll MD simulations were performed using GROMACS v4.5.5 (46) at 323 K. Coarse
Grained (CG) MD simulations were run for 1μs to permit the assembly and equilibration of a POPC bilayer around the protease (47, 48). The systems were then converted toatomic detail using the CG2AT-align method described previously (49). The systemswere equilibrated for 1 ns with the protein restrained before 100 ns of unrestrained MD.
Peptide cleavage assayZMPSTE24WT or ZMPSTE24E336A were added to a final concentration of 0.9nM in
50mM HEPES (pH7.5), 200mM NaCl, DDM (0.25%) or OGNG/CHS (0.18%).Synthetic, prelamin A-based peptides (Eurogentec or Peptide Synthetics) were added to a
6
final concentration of 25uM, 50uM or 100uM in 30µL. The assay was performed intriplicate, using duplicate batches of purified ZMPSTE24, at 37oC for between 90min and48h. Reactions were stopped by heating at 95oC for 2min. Peptides were detected usingmass spectrometry. 5µL of each reaction was diluted, 1 in 10, with 1% formic acid.Reversed-phase chromatography was performed in-line prior to mass spectrometry usingan Agilent 1100 HPLC system (Agilent Technologies inc. Palo Alto, CA, USA). 20-40µlof each sample was injected onto a 1.0 mm x 75 mm Zorbax 5um 300SB-C18 column ora 200 mm long by 4.6 mm internal diameter Zorbax 300SB-C3 column in a column ovenat 60oC. The solvent system used consisted of 0.1% formic acid in ultra-high purity water(Millipore) (solvent A) and 0.1 % formic acid in methanol (LC-MS grade, Fluka)(solvent B). Chromatography was performed as follows: Initial conditions were 95% Aand 5% B and a flow rate of 0.5 ml/min. After 30 seconds at 5% B, a two-stage lineargradient from 10% B to 80% B was applied, over 1.5min and then from 80% B to 95% Bover 6 seconds. Elution proceeded isocratically at 95% B for 2min 24s followed byequilibration at initial conditions for a further 1.5min. Protein or peptide intact mass wasdetermined using an MSD-ToF electrospray ionisation orthogonal time-of-flight massspectrometer (Agilent Technologies Inc. Palo Alto, CA, USA). The instrument wasconfigured as described previously (34). Data analysis was performed using MassHunterQualitative Analysis Version B.04.00 Build 4.0.479.0 (Agilent) software. Individual LCscans were inspected and selected manually based on signal to noise. Varying numbers ofscans were combined depending on the width of the chromatographic peak. Once peptidepeaks had been identified, the extracted ion chromatogram (EIC) function was used tosearch for substrate and products in each assay sample.
Supplementary Text
Additional author notesAQ, YYD and ACWP contributed equally to this project. AQ, YYD and LD were
responsible for purification optimization and preparation of protein, growth andoptimization of crystals. YYD analysed the protein detergent complex and identifiedsuitable detergents for ZMPSTE24 purification. AQ performed functional studies. ACWPsupervised crystallisation, mounted and screened crystals, collected diffraction data andsolved and built the structures. NABB and LS performed cloning and expression tests.NABB and LD initiated purification trials and obtained the first crystals. GB performedmass spectrometry on protein samples and helped with activity assays. PS performed andMSPS supervised the MD simulations. FvD contributed to the crystallography. AE andCB initiated the project, helped in project management and helped prepare themanuscript. ANB and NABB were responsible for managing the pilot stages of thisproject. EPC is the project leader, collected data and contributed to the structure solution.AQ, YYD, ACWP and EPC analysed the data and prepared the manuscript.
Proteindetergent complex
Detergent micelles
A
B
RA
LS d
etec
tor r
eadi
ng (m
V)
RI d
etec
tor r
eadi
ng (m
V)
Abso
rban
ce a
t 280
nm (m
Au)
LALS
det
ecto
r rea
ding
(mV
)
4 6 8 10 12 14 16
-150
-100
0
100
200
300
400
500
-150-100
0
100
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300
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500
RI RALS LALS UV
SEC elution volume (ml)
-50-33
0
33
67
100
133
167
-150
-100
0
100
200
300
400
500
9.0 9.5 10.0 10.5 11.0
0
30
60
90
120
150
180
Abs
orba
nce
at 2
80nm
(mA
u)
SEC elution volume (ml)
DDM OGNG CHS
4 6 8 10 12 14 16
0
30
60
90
120
150
180
Fig. S1. Size exclusion chromatography-multi angle light scattering (SEC-MALS) analysis of the ZMPSTE24 protein detergent complex (PDC). (A) Overlay of the SEC 280nm UV absorbance profiles for ZMPSTE24 samples solubilized in DDM and OGNG/CHS, showing how the protein/detergent/lipid complexes eluted at different volumes. (B) Overlay of tetra detector analyses of a ZMPSTE24/OGNG/CHS sample’s refractive index (RI), low angle (7�, LALS) and right angle light scattering (RALS) and UV absorbance at 280nm. This data enabled the calculation of the molecular weight of the protein/detergent/lipid complex and the ratio of protein to detergent/lipid in the complex (see Table S1).
406
Zn
359
Lip Lip406
Zn
359
N
C C
N
A
B
C D
C324
C406
C359C176
N
C
Zn
TMH1TMH2TMH3TMH4TMH5TMH6TMH7
TMH1
TMH2
Nterm
TMH3
107
117
TMH4
TMH5TMH6
TMH7ALH2
LH1TMH1
TMH2
Zn
Loop 1-2
Cterm
TMH7B
C324
MH1
MH3
MH4
MH5
MH6
90˚90˚ 90˚
E
F20
F26
W33
R40F36
W28
Y31
F20
F26
W33
R40F36
W28
Y31
TMH1 E K R I F G A V L L F S W T V Y L W E T F L A Q R Q R R I Y K T. . .20 30 40
F
TMH2 S E T F E K S R L Y Q L D K S T F S F W S G L Y S E T E G T L I L L F. . .
Y70
F77F79
W80
Y84 W28
Y45
W141
Y144Y31
Y70
F77F79
W80
Y84 W28
Y45
W141
Y144Y31
70 80 90
G
TMH3 E Y E I T Q S L V F L L L A T L F S A L T G L P W S L Y N T F V I E E K.120 .130 .140 .150
P140
Y84
L101
F133
F126
F171
F163
W141
Y144
F147
P140
Y84
L101
F133
F126
F171
F163
W141
Y144
F147
H
TMH4 L G F F M K D A I K K F V V T Q C I L L P V S S L L L Y I I K I.160 .170 .180 .190
F162
F163
M164
F171
C176
F223
F203 F203
TMH5B
Y187
F162
F163
M164
F171
C176
F223
TMH5B
Y187
J
I
F421
F416
F414
C406
C359F361
F363 F363
F360
F357
F436
W340
F26
Y399F405
R413
F421
F416
F414
C406
C359F361
F360
F357
F436
W340
F26
Y399F405
R413
TMH6 H T V K N I I I S Q M N S F L C F F L F A V L.350
.400 .410 .420
.360
TMH7B S P Y N E V L S F C L T V L S R R F E F Q A D A F A K K
TMH5 F F I Y A W L F T L V V S L V L V T I Y A D Y.200 .210
A P L F.220
TMH5B
F203
P221
Lip
F223
W201F357
Y269
F198 F197
Y199
F203
P221
Lip
F223
W201F357
Y269
F198 F197
Y199
β3β2
β3β2
Fig. S2. Quality of experimental electron density for the native P1 structure. (A) Anomalous difference maps showing peaks corresponding to the locations of the four bound Hg atoms (magenta mesh) and the zinc ion (green mesh). The positions of four cysteine residues in human ZMPSTE24 are shown (the fifth cysteine (Cys109) lies in adisordered region). Two anomalous difference maps are superimposed on the samestructure, calculated at 6Å from data collected respectively from either the EMTS-soaked crystals or data from native crystals collected at the Zn-peak (λ=1.2816Å),contoured at either 4.5σ (Zn) or 3.5σ (Hg). (B) Stereoview of Hg-SIRAS phased experimental electron density maps after eight-fold cross-crystal averaging usingDMMULTI (40). Electron density (grey mesh) was averaged over the four moleculesin the P1 unit cell as well as a further four molecules in the monoclinic crystal formobtained in the presence of DDM. The Cα-trace of the final model is overlaid on maps. The zinc ion (magenta sphere) and lipid alkyl chains (pink) are also shown. The electron density map is contoured at 1σ. Anomalous difference peaks from the EMTS(magenta) and Zn-peak (green) datasets are also shown and contoured at 4σ. (C)View from the nucleoplasmic side showing connectivity in metalloprotease domain.The location of Cys324 at the N-terminal end of helix MH3 is indicated by ananomalous difference peak (magenta) in the EMTS dataset. (D) View of theexperimental density on the lumenal side of the barrel. (E-J) Stereoviews of theexperimental, DMMULTI-averaged map in the vicinity of the TM helices. Theelectron density map is contoured at 1σ and overlaid on the final model. Whererelevant, the 6Å anomalous difference map calculated from the EMTS-soaked data isshown and contoured at 3.5σ (in S2H) and 4.5σ (in S2J). The amino acid sequence ofeach TM helix is shown below each stereoview and bulky sidechains used in assigning the sequence are highlighted by green boxes. Cysteine residues which were modified by EMTS are indicated by pink boxes.
β1
β1β2
β3β4
β5β1β2
β2
β3 β3
MH1
MH2
MH3 TMH7
MH4
MH6
MH5
β4
[HEXXH] Glu helixGlu helixGlu helix
N
N
N
C
CC
65
7
[HEXXH][HEXXH]
1
5B
234
LH1
LH2
ZMPSTE24 M48 PEPTIDASE THERMOLYSIN(PDB: 3C37) (PDB: 3SSB)
HEXXH helixGlu helixZinc
A
B
H
H
H H
H H
EE
E E
E E
H
HZn
C
E
N
NP / CP
ER Lumen LH1LH2
MH1
MH4MH6MH3
β2
β1
β4 β3
5A
7A
11 2 3 4 5 6
proteasedomain
MH5
E
7
Fig. S3. Comparison of the structure of ZMPSTE24 with a soluble M48 family peptidase andthermolysin. (A) The overall fold of ZMPSTE24 exhibits a 7 TM alpha-helical barrelwith a zinc peptidase domain inserted between TM5 and TM6. Dali (50) searchesindicated that the closest structural homologues only showed similarity to the proteasedomain with a soluble M48 Zn-dependent peptidase from Geobacter sulfurreducens(PDB: 3C37; Dali Z-score 8.2). Lower structural similarity is observed with neprilysin (Z-score 4-4.5), several aminopeptidases and thermolysin (Dali Z-score 3.9). In ZMPSTE24, the core protease fold encompasses two contiguous regions ofsecondary structure. The first segment comprises a four-stranded β-sheet interruptedby an α-helix (metalloprotease helix 1 (MH1). The β-sheet is followed by a short α-helix and the HEXXH helix (MH3) containing the zinc-coordinating histidines (His335, His339) and the catalytic acid/base (Glu336). After a variable interveningregion, the second segment of the core protease fold comprises the glutamate helix(TMH7) which bears the third zinc-coordinating residue (Glu415) and a further 2-3 helices (MH4-MH6) which carry residues involved in defining the S1′ and S2′specificity pockets, a conserved histidine involved in catalysis (His459) and aconserved arginine (Arg465) that interacts with the P1′ residue. (B) Schematicrepresentation of the secondary structure of ZMPSTE24 with the protease domainhighlighted. Dotted lines indicate regions that were not resolved in the crystalstructures.
D E
Zinc
HH
A
B
C
TMH5 / TMH6TMH1 / TMH2TMH2 / TMH3TMH3 / TMH4Electronegative patch betweenTMH1 / TMH7Hydrophobic patch
bilayer accessible
NP accessible
}}
60˚150˚80˚
TMH1 TMH2 TMH3 TMH4 TMH5 TMH6 TMH7
-H
-
-
- -H H
H
Fig. S4. Fenestrations link the chamber of ZMPSTE24 to the nucleoplasm and theintramembrane space. Four views of the ZMPSTE24 structure viewed looking parallel to the lipid bilayer. (A) Ribbon representations. (B) Molecular surface view showing fenestrations in the exterior of the barrel wall. The four observed fenestrations aremarked with colored arrows. The surface has been backsliced so that the fenestrationsare clearly visible. (C-E) Sliced molecular surfaces highlight the properties of theinterior of the barrel. The molecular surfaces have been sliced perpendicular to theviewing axis. Molecules are viewed in the same orientation in panels A-C. (D) View looking onto the catalytic site from the ER side of the barrel. (E) View looking at thebase of the barrel from the nucleoplasmic side of the membrane. Molecular surfaceswere calculated from a molecule containing hydrogens and are colored byelectrostatic potential (contoured from -10 kT/e (red) to +10 kT/e (blue)).Electrostatics were calculated using the APBS plugin in PyMOL. The zinc ion wasnot included in the calculations. Prominent charged and hydrophobic patches on theinterior of the chamber are highlighted.
0
1
2
3
4
5
A B
E F
C
D
Residue number
Roo
t mea
n sq
aure
d flu
ctua
tion
(Å)
1 2 3 4 5 6 7
50 100 150 200 250 300 350 400 450
Fig. S5. ZMPSTE24 barrel architecture is stable over 100ns simulation in lipid bilayer. (A) Root mean square fluctuation (rmsf) of the protein during the simulation. (B & C) Comparison of the crystallographically-determined temperature factors (B) and rmsf values for the 100 ns simulation (C). A schematic worm representation of the crystalstructure is shown with the worm radius and color scaled to depict the range of B-factors (B) or rmsfs during the MD simulation (C). Red regions represent high temperature factors / rmsfs. (D) Snapshots of ZMPSTE24 structure over the timecourse of the 100ns simulation. Snapshots are shown at 1ns intervals colored from blue (0ns) to red (100ns) (E & F). The intramembrane chamber is maintainedduring simulation. The solvent-filled intramembrane chamber is shown for the crystalstructure (0ns) (E) and after 100ns simulation (F).
A
B
Counts vs. Acquisition Time (min)
+ESI Total Ion Chromatogram (T643-Q656)
8x10
0
0.2
0.4
0.6
0.8
1.0
1.2
4.0 4.5 5.0 6.5 7.0 7.5
4.9
5.1
5.1
5.1
7x10
0
0.8
1.6
2.4
3.2
4.0
4.8
16.3
16.3
16.3
16.1
16.1
13.1
13.1
+ ESI Total Ion Chromatogram (S636-C661)
11.5 12 12.5 13 13.5 14 14.5 15 15.5 16 16.5
Counts vs. Acquisition Time (min)
C
S636-C661
T643-Q656
C-terminusPrelamin A
Q656-M664(fCys)
S G D L S G Q SL L S S QF N V T R Y P R T P N CN
G G S F G D N L V T R S Y G P R T Q S P Q N C S I ML L N S S640 650 660
S G QL L S ST R Y P R TN
Q S QP N C S I M
Q S QP N C S I M
RCE1 / ZMPSTE24ZMPSTE2412
NH2
NH2
NH2
NH2
1257Da1806Da
1071Da958Da845Da
966Da879Da
875Da
Farnesyl GroupPrelamin A Peptide Substrate
Prelamin A Peptide Product
762Da
Q656-M664
5x10
0
0.8
1.6
2.4
3045.60
600 1400 2200 3000
3045.59
3045.63
+ESI Scan Substrate,16.3min
1578.90
200 600 1000 1400
6x10
0
0.4
0.8
1.2
1.6
2.0
2.41578.89
1578.89+ESI Scan Subtrate, 5.1min
Counts vs. Mass (Da)
6x10
0
0.4
0.8
1.2
1.6
2.0
2.4
1071.61
845.44958.52
1071.61
200 600 1000 1400
+ESI Scan products, 4.9min
Counts vs. Mass (Da)
1805.99
1806.00
0
1.0
0.2
0.4
0.6
0.8
1257.63
+ESI Scan Products, 13.1min & 16.1min
1257.64
600 1400 2200 3000
6x10
D
0.8
1.6
2.4
3.4
16 16.8 17.6 18.4
7x10
17.2
17.2
17.2
16.4
Counts vs. Acquisition Time (min)
8x10
0.4
1.2
2.0
2.8
3.6
6 7 8 9 10 11 12
11.9
11.9
11.9
7.3 7.6
966.48
0.4
0
0.8
1.21.6
6x10
200 400 600 800 1000 1200
762.26
1006.44
1006.48
874.29
0.2
0.6
1.0
6x10
200 400 600 800 1000
200 400 600 800 1000
0.2
0.6
1.0
6x10
200 400 600 800 1000
2.04.06.0
4x10
8.0
1210.56
0.4
0
0.8
1.21.6
6x10
200 400 600 800 1000 1200
1210.57
1210.57
E1006.42
16.6
0.20
0.40.60.8
5x10
200 400 600 800 1000 1200
879.43
* *+ESI Scan Product, 16.6min
Counts vs. Mass-to-Charge (m/z)
+ESI Scan Product, 16.4min
+ESI Scan Substrate, 17.2min
+ESI Scan Product, 7.6min
+ESI Scan Product, 7.3min
+ESI Scan Substrate, 11.9min
Counts vs. Mass-to-Charge (m/z)
Counts vs. Acquisition Time (min)
+ESI Total Ion Chromatogram (Q656-M664, fCys)
+ESI Total Ion Chromatogram (Q656-M664)
Fig. S6. ZMPSTE24 cleaves peptides derived from the C-terminus of prelamin A at the twoexpected sites and at additional sites. (A) Schematic illustrating the observed productsafter ZMPSTE24 cleavage of a range of Prelamin A based synthetic peptides. (B)Total ion chromatogram (TIC) and deconvoluted substrate and product spectra for Prelamin AS636-C661. Substrate (3045Da) has a retention time of 16.3min with products(12757Da, 1805Da) at 16.1min and 13.1min. ZMPSTE24WT produced 80-86% more product than the partial inactivation mutant, ZMPSTE24E336A. (C) TIC and deconvoluted product and substrate spectra for Prelamin AT643-Q656. Substrate(1578Da) has a retention time of 5.1min with products (1071Da, 958Da, 845Da) at4.9min. (D) TIC and deconvoluted product and substrate spectra for Prelamin AQ656-
M664 (fCys). Substrate (1211Da) has a retention time of 17.2min with products (966Da, 879Da) at 16.4min and 16.6min respectively. The substrate and products for thisreaction elute adjacent to detergents, so the TIC spectra have additional peaks. The
presence of peaks with the exact masses for the substrate and product peptides,however, clearly indicates the presence of the peptides. (E) TIC and deconvolutedproduct and substrate spectra for Prelamin AQ656-M664. Substrate (1006Da) has a retention time of 11.9min with products (762Da, 874Da) at 7.3min and 7.6minrespectively. Peak shifts are observed in the TICs on addition of ZMPSTE24WT (blue)or ZMPSTE24E336A (green) to substrate (red). Analysis of the associated mass-chargeratios for each peak and subsequent deconvolution enables identification of peptidesubstrate and products. Similar results are observed for all peptides when ZMPSTE24is purified in OGNG/CHS or when reconstituted into proteoliposomes. Additionallyall short peptides have gave similar results for ZMPSTE24 purified in DDM and when reconstituted into proteoliposomes, with exception of ZMPSTE24E336A which is not stable in DDM and was therefore only assayed after purification in andreconstitution from OGNG/CHS containing buffer.
O
HN
R
HN
O
NH
ROHN
R
Zn2+
R
O
N
Phe 268
O H
Glu 336
O OH
Asn 265
O NH2
H2N
H2N
HN
HN
His 459His 335
His 339 Glu 415
Zn2+
His 335
His 339 Glu 415
OHH
Glu 336
O O HN
HN
His 459
HN
OZn2+
His 335
His 339 Glu 415
O
H
HGlu 336
O
O
HN
HN
His 459
HN
O
Zn2+
His 335
His 339 Glu 415
O
H
HGlu 336
O
O
HN
HN
His 459
HN
O
HN
H
H
Zn2+
His 335
His 339 Glu 415
OGlu 336
O
O
HN
HN
His 459
O
Arg 465
+1
+2 -1-2
*
B
A
Fig. S7. Proposed catalytic mechanism for ZMPSTE24. (A) Reaction mechanism based on thegenerally accepted, two step mechanism of peptide hydrolysis by thermolysin (23,51). The scissile peptide bond of the substrate is schematically represented in blue.ZMPSTE24 differs from thermolysin in that there are no residues directly equivalentto Tyr157 and Asp226 (interacts with protonated histidine), which appear to beassociated with thermolysin-mediated peptide cleavage catalysis. (B) Schematicrepresentation of peptide binding to the active site of ZMPSTE24. Residuescoordinating the catalytic zinc and interacting with the substrate peptide are shown. Numbers in green circles refer to the standard peptide subsite positions relative to thescissile bond. In the initial CAAX cleavage of the prelamin A C-terminus, the farnesylated Cys661 lies in the P1 (+1) position with Ser662 and Ile663 lying in the P1′ (-1) and P2′ (-2) positions respectively. For the second cleavage, Leu647 and Leu648
occupy the P1′ and P2′ positions and Tyr646 occupies the P1 subsite. Figures drawn with ChemDraw (CambridgeSoft Ltd).
-1+1+2
+3
+4-2*
H231
R203
E166
H146
N165
N112E143
H142
A B
C D E
F
53-1+1+2
-2*
H459
H339E415
H335
N265
A336
CCS I
M
H344
661
662663
664
661 661661663
661663
664664
54
55
56
57
58
CHAIN ABprot: 171Å2
Bpep: 186Å2
CHAIN EBprot: 190Å2
Bpep: 230Å2
CHAIN DBprot: 168Å2
Bpep: 181Å2
CHAIN BBprot: 179Å2
Bpep: 217Å2
Fig. S8. Substrate peptide binding in ZMPSTE24 and comparison with thermolysin. (A) Structure of the 3.8Å ZMPSTE24E336A mutant in complex with a tetrapeptidecorresponding to the unfarnesylated C-terminus of prelamin A (Ser661-Met664).Interactions between the peptide (green carbons) and ZMPSTE24 are shown as greendotted lines. Unexpectedly, the peptide is positioned so that the peptide carbonylgroup of Ser662 interacts with the catalytic zinc ion so that the Ser662 (S1 subsite) -Ile663 (S1′ subsite) peptide bond is poised to be cleaved (bond to be cleaved indicatedby an asterisk). The hydrophobic sidechains of Ile663 and Met664 are directed towards alarge hydrophobic S1′ – S2′ subsite formed by residues from strand β3, MH3, MH4 and MH6. Asn265 on the β3 strand moves towards to the bound peptide and mayinteract directly with the peptide. (B) View of cleaved inhibitor bound to thermolysin (PDB: 3SSB, (26)). For clarity, only the part of the inhibitor interacting with the active site subsites (S4 to S2′) is shown (residues 53-58). The peptide bond betweenAsn56 and Ile57 has been cleaved. This structure was used as a template, in
combination with the experimental electron density for the CSIM peptide, to constructa model of the C-terminus of prelamin A bound to the active site of the nativeZMPSTE24 enzyme with the correct registration so that Cys661 is positioned in the S1position (Fig. 3B & 3C). (C-F) Omit Fo-Fc electron density for the CSIM-soaked crystals at 3.8Å. The catalytic site of each of the four molecules in the P1 asymmetricunit (A, B, D, E) is viewed as in (A). The omit electron density, calculated afterBUSTER refinement (43) using the coordinates of the native P1 structure alone, is shown as a green mesh and contoured at 3σ. The average temperature factors areindicated for each of the protein chains / peptides in the final model. Sufficientdensity was observed for two chains (B/D) to allow modeling of the entiretetrapeptide.
H. sapiensC. elegansD. melanogasterO. sativaS. cerevisiae
H. sapiensC. elegansD. melanogasterO. sativaS. cerevisiae
H. sapiensC. elegansD. melanogasterO. sativaS. cerevisiae
H. sapiensC. elegansD. melanogasterO. sativaS. cerevisiae
H. sapiensC. elegansD. melanogasterO. sativaS. cerevisiae
H. sapiensC. elegansD. melanogasterO. sativaS. cerevisiae
H. sapiensC. elegansD. melanogasterO. sativaS. cerevisiae
H. sapiensC. elegansD. melanogasterO. sativaS. cerevisiae
H. sapiensC. elegansD. melanogasterO. sativaS. cerevisiae
M G M W A S L W E M P A E K R I F G A L . V R I K T H V P E
G Q I S L Q S S S G E E G . G Y L G
Y A . . . G F G P E Y E I Q S T E G
K A K K I L D
K E V K D .
E E Y S V L N K D I Q E D S G M E P R N E E E G N S E E I K A K V K N K K Q G K .
K K N F V I G R K E A Y . D S . .
. . . . . . . . . . . L F E L R F A K
K Y S K S W T K Q
. . . . . . . S C L F K A A . L Y A K A K R N E
K E L E D K H G H S Q L L . G F A .
. . . . . . . . . . . . . H D T D G
V K K V N S T
D K Q A S .
. . . . . . . . . . . S G A E K E K V H E L Y V A A G E K I E E T E N D K K R M D
A I N A Y Y K W E A Q H . . D . .
. . . . . . . . . . . M F Q H S A E A
E I G V N S A Q A N K
M S S . . . . . . . D T V L L S F . E V V Q A K V A E
K S H E K G E G K A D A L I G V L D
K L . . . Q W D S K N E I V K I E R
W Q G Q R G I
A S D A K .
. . . . . L N K G K P D D S E L . . . . . . . . . S E E E K G K . . . . . . G T .
K K H L N F K Y P P V Q P G T . .
. . . . . . . . . . . L Y A L R F E F
E G Q V Y W R E K E K K L N
M A L P Y . . . . . . . . . . . . . C . M H A K P . L K P
V G V G A S S H H E I M D . R W K T
N A . . . G L N A E N E I H S T A W
R M G Q N F I
V K K A S .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Q Q S D
K Y L G L R N S K D E E . . D . .
. . . . . . . . . . . . H H V A F A K
P R A E T P D D S K E N
M K T I L D H P N I Q W K L S G Q Y K . E K L P V
E D E D N S A S G G L Q K . D K M N
A V L P V R F H M V S T V Q S H E Q
T M S D K F V
E S S D G P
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . N N D
Q N H S S Y R N T S N F L E K T G
S F V D P V I T K E F . D C I T H A K
Q C R I V P Y S E K K N
1 10 20 30 40 50
60 70 80 90 100 110
120 130 140 150 160
170 180 190 200 210
220 230 240 250 260 270
280 290 300 310 320
330 340 350 360 370 380
390 400 410 420
430 440 450 460 470
L V F S W T Y L W T F L A Q R Y T T
M D T E S L K T F W L Y S T T L I L L F G I L R S G R F C
T S L V L L A T L F A L T G W L N I K Q L F F
M F V V T Q C I L L V S S L L L Y I D Y F I A L T V S V L V A Y
D F P E K E M I T V Y E F F F K N
F L C N E V
L G G T V N I I I S M S C L A L L F A Q
T L L I I Q I F S Y N V L S C L T V F K K
A D I N D G F P V F M W H Y P L Q K M H
M L T N W A F L W D Q I T F K H N V V
I G D Y K A I N L F F W F N L T A Q L I G Y Y L Y T A S Y P
L V A V S I N S I I E T I I D W L S I I A I F Y
F M L V G F A L T M V Y G I E W I P Y F V I L V V V L L M F
D Y F P D K T L Y T E L Y N A M F W K N
Y L G N N V
V G W T L N L V I T E V L F S V G F L Y G Y T
P V M L I Q V L A L Y N L A S I G M V I F G N H
G N T G D S M P I W C T H T P V V V A V R F K
L I L V V I N A L I I S L K Y T L
M G D T H A L Q K I F V V M V L C M E L Y L I A L Q S V Q V V
I S C V V L I S N V L T F K G F I K L T A F F
A L F L V T Q V L M I T A A I I F V D N F I W L I T I S V L L L F
D Y E K R Q L T L F E F L W N S
F L C D E V
L G G V T N I I I M V L M V G V F Y A M R
I L V L I V T V L A Y N L M N A M T I Y K F
A Q I N D G F P V T W N H T L Q N K L E
L E V F M I L Y I F T L D I R L L T
I S K E R S L K K F I A V T L T T I L Y Y V L V K S G E L A
L T L A A G V M I W Q I T D F L S I K I L F
I I L L S I L L G P V A A I I I V P Y L A I L G M A L S V M M V
N F P E R E L K L F D T M F F K N
Y I C S E I
V S I A N T V S F V A V L M Q G Y T V L F S Q
V I L I I Q T I I V Q L L S C L N L F N Y
A Q V Q E S A M N W A Y H Y P V S E A K
F L I I F S I A F S F S L T Y Q L S T
I D T H S R A K K I F V Y N A L V F I K Y L F I H A V S L S
A S L C G L L S S L T L V D L Y S L K F L V L W
I L T L A Y A I G G L Y L F L K F F P T D L W I M V L V Q I L A M I F
M M N F E D K K R V D I F I D F T L F T S
F V S S T I
T A I K N I V M V I F S L T I L T I F Y T S
I I F M L N L L T L E A M Q V M S L Y Y K Y
K N I D Q K S T M N S Y H Y T A T D V K
D A L E R Q P L
E F K R Y D F P W
F L S L P Y F V E H G F N Q T
D I K P I G G F Y W F V L T I Y I
A P L F K T P L G L I E A S F P L K V V G S K R S S H S N A Y Y G
K R I V L D T L E
A V L H E L G H W L H Q F L F F F G F
P I G F P F S R E Q A D F A L G
L A L K L N L D L S S H P L E R L A L
D A L Y R Q P
E K R Y D F P W
F L L P Y F E H G F N K Q T
D I K P I I G G F Y W F V L T I Y P I
A P L F K P L G L I E L A S P L V V G S K R S H S N A Y Y G
K R I V L D T L E
A V L H E L G H W L H F F F F G
P I G F S R E A D F A L G
L A L K L N L D L Y S H P E R A
D A L E Y R Q P L
F K R Y D F W
F S L P Y F V E H G F N K Q T
D K P I I G G F W F V L T Y P I
A P L F K T P L G L I E L A S F P L K V V G S K R S S H S N A Y Y G
K R I V L D T L E
A V L H E L G H W L H Q F L F F G F
P G F P F S R E Q A D F A L G
L A L K L N L D L Y S S H P L R L L
A L E Y R Q P L
E F R Y D F P W
F L S L P Y F V E H G F N K Q T
D I K P I I G G Y W F L T I Y P I
A P L F K T P L G L I E L A S F P L K V V G S R S S H S N A Y Y G
K R I V L D T L E
V H E L G H W L H Q F L F F G F
P I G F P F S R E Q A D F A L G
L A L K L N L D Y S S H P L E R L A L
D E Y R Q P L
E F K R Y F P W
F L S L P Y F V E G F N K T
D I K P I I F Y F V T I P I
P F K T P L G L I E L A F P L K V G S K R S S H S N A Y G
K R I V L D T L E
A V L H E G H W H Q F L F F F G F
P I G F P F S R E Q A D A L G
L A L L N L D L Y S S H P L E R L A L
TMH1
TMH2
TMH3
TMH4 TMH5
TMH5B MH1
MH2
MH3 TMH6
TMH7B
MH4 MH5 MH6
TMH7A
LH2
LH1
β1 β2 β3
β4
Fig. S9. Alignment of ZMPSTE24 sequences. Residues that exhibit a high degree ofconservation are colored red. Secondary structure elements from the humanZMPSTE24 crystal structure are shown and labeled. The regions enclosed by dottedlines represent the metalloprotease domain. Residues involved in coordinating thezinc ion are highlighted by magenta circles. Other catalytically important residues arehighlighted by blue spheres. Black stars indicate residues that are mutated in HGPS /MAD / RD. Figure prepared using ALINE (52).
Zn
C
EH
H
N
11 2 3 4 5 6 7
5B
β1
β2 β4 β3
7ALH1
MH
4
MH
6MH
1
MH5
MH2
LH2
Q41trunc
E239trunc
E231trunc
L91del
Y195FfsP98Lfs
Y70Sfs
I19Yfs
W450trunc
Q417trunc
V402Sfs
E362FfsI198Yfs
T159del
K17Sfs
L94P
L438F
L462R
W340R
P248L
N265S
E16
F95
S61
T47
G97
L104E117
K152
L160
I191
F196
Y218
A220F223
L284
D281
F226
N326
L366
G270
E371
Q393T384
A375
D441
A428
W450W455
T471
L462
S244
K233
L279
S397
K424
NP/CP
ERLumen
V255
Fig. S10. ZMPSTE24 mutations mapped onto a schematic representation of the structure.Deleterious mutations (frameshifts (fs), truncations (trunc), deletions (del)) are indicated by white stars. Less severe mutations caused by single amino acid changes(L94P, P248L, N265S, W340R, L438F, L462R) that result in HPGS, MAD-B or RD are indicated by red stars. Mutations are shown in black fonts. Residues at thebeginning and end of secondary structure elements are shown in blue. Thenucleoplasmic (NP)/cytoplasmic (CP) and the ER lumen faces of the membrane areindicated on the left.
Supplementary Tables:
Table S1.
SEC-MALS data for ZMPSTE24 samples solubilized in two different detergent lipid
systems.
α n-dodecyl-β-D-maltopyranoside (DDM), octyl glucose neopentyl glycol (OGNG) +
cholesteryl hemisuccinate (CHS) β Protein detergent complex
γ Mass of detergent with the PDC
Using refractive index, right angle and low angle light scattering and UV data shown in
Fig. S1B, the molecular weight of the protein detergent complex (PDC), the amount of
detergent in the complex and the percentage of protein in the complex was calculated as
described in Slotboom et al. 2008 (33). The amount of detergent bound to the protein and
therefore the size of the PDC varied with the detergent used to solubilize the protein.
Detergentα PDC Mw (KDa)β Detergent Mwγ (KDa) % Protein
DDM 201.04 142.94 28.9
OGNG + CHS 127.13 69.03 45.7
Table S2. Data collection, phasing and refinement statistics
EMTS soak Monoclinic form Native CSIM soak
Detergent OGNG / CHS DDM OGNG / CHS OGNG / CHS
Data Collection
Space group P1 P21 P1 P1
Molecules / AU 4 4 4 4
Cell dimensions
a, b, c (Å)
, , (º)
60.44, 94.69, 130.1
77, 79.74, 76.85
152.66, 83.95, 154.77
90, 114.03, 90
60.99, 95.45, 131.1
76.73, 79.64, 72.61
61.32, 95.54, 132.03
76.26,79.67,72.30
Resolution [Å]1 3.75 (3.75-3.85)1 3.6 (3.6-3.69) 3.4 (3.4-3.49) 3.8 (3.8-3.9)
Resolution limits [Å]2 3.97, 4.15, 3.75 3.42, 3.42, 5.05 3.4, 3,4, 3.4 3.98, 3.95, 3.8
Rmerge 0.115 (1.446)1 0.162 (0.976) 0.109 (1.106) 0.053 (0.687)
Rmeas 0.136 (1.706)1 0.223 (1.348) 0.136 (1.390) 0.075 (0.971)
Rpim 0.072 (0.896)1 0.109 (0.641) 0.081 (0.832) 0.053 (0.687)
I / I 9.7 (1.3)1 6.2 (1.5) 10.7 (1.7) 10.8 (1.6)
Completeness [%] 99.4 (98.9)1 99.8 (99.9) 99.9 (99.8) 99.2 (99.4)
Redundancy 7.0 (7.1)1 4.0 (4.0) 5.3 (4.9) 3.3 (3.2)
Refinement
Resolution (Å) 38 – 3.40 36.05 – 3.80
No. reflections 37465 26935
Rwork / Rfree 24.57 / 26.42 26.04 / 27.92
No. atoms
Protein
Other
12866
112
12855
94
B-factors
Protein
Other
132
112
177
204
R.m.s. deviations
Bond lengths (Å)
Bond angles (°)
0.008
0.87
0.008
0.86 1 Values in parentheses are statistics for highest resolution shell
2 Anisotropic resolution limits as defined by AIMLESS based resolution at which half dataset
correlation (CC1/2) > 0.50.
References
33. D. J. Slotboom, R. H. Duurkens, K. Olieman, G. B. Erkens, Static light scattering to
characterize membrane proteins in detergent solution. Methods (Duluth) 46, 73-82
(2008).
34. G. Berridge et al., High-performance liquid chromatography separation and intact
mass analysis of detergent-solubilized integral membrane proteins. Anal Biochem,
(2010).
35. W. Kabsch, Xds. Acta Crystallogr. D 66, 125-132 (2010).
36. P. Evans, Scaling and assessment of data quality. Acta Crystallogr. D 62, 72-82
(2006).
37. G. M. Sheldrick, A short history of SHELX. Acta Crystallogr. A 64, 112-122
(2008).
38. T. Terwilliger, SOLVE and RESOLVE: automated structure solution, density
modification and model building. J Synchrotron Radiat 11, 49-52 (2004).
39. A. J. McCoy et al., Phaser crystallographic software. J. Appl. Cryst. 40, 658-674
(2007).
40. K. Cowtan, DMMULTI. Joint CCP4 and ESF-EACBM Newsletter on Protein
Crystallography, 31, 34-38 (1994).
41. K. Cowtan, The Buccaneer software for automated model building. 1. Tracing
protein chains. Acta Crystallogr D Biol Crystallogr 62, 1002-1011 (2006).
42. P. Emsley, B. Lohkamp, W. G. Scott, K. Cowtan, Features and development of
Coot. Acta Crystallogr. D 66, 486-501 (2010).
43. G. Bricogne et al. (Global Phasing Ltd, Cambridge, UK, 2011).
44. V. B. Chen et al., MolProbity: all-atom structure validation for macromolecular
crystallography. Acta Crystallogr. D 66, 12-21 (2010).
45. E. F. Pettersen et al., UCSF Chimera--a visualization system for exploratory
research and analysis. J Comput Chem 25, 1605-1612 (2004).
46. B. Hess, C. Kutzner, D. van der Spoel, E. Lindahl, GROMACS 4: Algorithms for
Highly Efficient, Load-Balanced, and Scalable Molecular Simulation. Journal of
Chemical Theory and Computation 4, 435-447 (2008).
47. L. Monticelli et al., The MARTINI Coarse-Grained Force Field: Extension to
Proteins. J Chem Theory Comput 4, 819–834 (2008).
48. K. A. Scott et al., Coarse-grained MD simulations of membrane protein-bilayer
self-assembly. Structure 16, 621-630 (2008).
49. P. J. Stansfeld, M. S. Sansom, Molecular simulation approaches to membrane
proteins. Structure 19, 1562-1572 (2011).
50. L. Holm, P. Rosenstrom, Dali server: conservation mapping in 3D. Nucleic Acids
Res 38, W545-549 (2010).
51. V. Pelmenschikov, M. R. Blomberg, P. E. Siegbahn, A theoretical study of the
mechanism for peptide hydrolysis by thermolysin. J Biol Inorg Chem 7, 284-298
(2002).
52. C. S. Bond, A. W. Schuttelkopf, ALINE: a WYSIWYG protein-sequence alignment
editor for publication-quality alignments. Acta Crystallogr. D 65, 510-512 (2009).
References 1. T. Dechat, S. A. Adam, P. Taimen, T. Shimi, R. D. Goldman, Nuclear lamins. Cold
Spring Harb. Perspect. Biol. 2, a000547 (2010).
2. T. A. Dittmer, T. Misteli, The lamin protein family. Genome Biol. 12, 222 (2011).
3. M. O. Bergo et al., Zmpste24 deficiency in mice causes spontaneous bone fractures, muscle weakness, and a prelamin A processing defect. Proc. Natl. Acad. Sci. U.S.A. 99, 13049 (2002).
4. C. L. Navarro et al., Loss of ZMPSTE24 (FACE-1) causes autosomal recessive restrictive dermopathy and accumulation of Lamin A precursors. Hum. Mol. Genet. 14, 1503 (2005).
5. J. Barrowman, S. Michaelis, ZMPSTE24, an integral membrane zinc metalloprotease with a connection to progeroid disorders. Biol. Chem. 390, 761 (2009).
6. V. L. Boyartchuk, M. N. Ashby, J. Rine, Modulation of Ras and a-factor function by carboxyl-terminal proteolysis. Science 275, 1796 (1997).
7. S. Michaelis, J. Barrowman, Biogenesis of the Saccharomyces cerevisiae pheromone a-factor, from yeast mating to human disease. Microbiol. Mol. Biol. Rev. 76, 626 (2012).
8. A. M. Pendás et al., Defective prelamin A processing and muscular and adipocyte alterations in Zmpste24 metalloproteinase-deficient mice. Nat. Genet. 31, 94 (2002).
9. J. Barrowman, P. A. Wiley, S. E. Hudon-Miller, C. A. Hrycyna, S. Michaelis, Human ZMPSTE24 disease mutations: Residual proteolytic activity correlates with disease severity. Hum. Mol. Genet. 21, 4084 (2012).
10. C. L. Navarro et al., Lamin A and ZMPSTE24 (FACE-1) defects cause nuclear disorganization and identify restrictive dermopathy as a lethal neonatal laminopathy. Hum. Mol. Genet. 13, 2493 (2004).
11. D. R. Witt et al., Restrictive dermopathy: A newly recognized autosomal recessive skin dysplasia. Am. J. Med. Genet. 24, 631 (1986).
12. C. L. Moulson et al., Homozygous and compound heterozygous mutations in ZMPSTE24 cause the laminopathy restrictive dermopathy. J. Invest. Dermatol. 125, 913 (2005).
13. J. Denecke et al., A homozygous ZMPSTE24 null mutation in combination with a heterozygous mutation in the LMNA gene causes Hutchinson-Gilford progeria syndrome (HGPS): Insights into the pathophysiology of HGPS. Hum. Mutat. 27, 524 (2006).
14. A. K. Agarwal, J. P. Fryns, R. J. Auchus, A. Garg, Zinc metalloproteinase, ZMPSTE24, is mutated in mandibuloacral dysplasia. Hum. Mol. Genet. 12, 1995 (2003).
15. A. Dutour et al., High prevalence of laminopathies among patients with metabolic syndrome. Hum. Mol. Genet. 20, 3779 (2011).
16. A. Garg, Lipodystrophies: Genetic and acquired body fat disorders. J. Clin. Endocrinol. Metab. 96, 3313 (2011).
17. R. Ben Yaou et al., Type B mandibuloacral dysplasia with congenital myopathy due to homozygous ZMPSTE24 missense mutation. Eur. J. Hum. Genet. 19, 647 (2011).
18. C. D. Ragnauth et al., Prelamin A acts to accelerate smooth muscle cell senescence and is a novel biomarker of human vascular aging. Circulation 121, 2200 (2010).
19. S. Rodríguez, M. Eriksson, Evidence for the involvement of lamins in aging. Curr Aging Sci 3, 81 (2010).
20. C. Coffinier et al., HIV protease inhibitors block the zinc metalloproteinase ZMPSTE24 and lead to an accumulation of prelamin A in cells. Proc. Natl. Acad. Sci. U.S.A. 104, 13432 (2007).
<foot>21. Materials and methods are available as supplementary materials on Science Online.</foot>
22. N. D. Rawlings, A. J. Barrett, A. Bateman, MEROPS: The database of proteolytic enzymes, their substrates and inhibitors. Nucleic Acids Res. 40(Database issue), D343 (2012).
23. B. W. Matthews, Structural basis of the action of thermolysin and related zinc peptidases. Acc. Chem. Res. 21, 333 (1988).
24. A. P. Kuzin et al., PDB entry 3c37, 10.2210/pdb3c37/pdb (2008).
25. J. Barrowman, C. Hamblet, M. S. Kane, S. Michaelis, Requirements for efficient proteolytic cleavage of prelamin A by ZMPSTE24. PLoS ONE 7, e32120 (2012).
26. J. L. Arolas, T. O. Botelho, A. Vilcinskas, F. X. Gomis-Rüth, Structural evidence for standard-mechanism inhibition in metallopeptidases from a complex poised to resynthesize a peptide bond. Angew. Chem. Int. Ed. Engl. 50, 10357 (2011).
27. A. K. Agarwal et al., Focal segmental glomerulosclerosis in patients with mandibuloacral dysplasia owing to ZMPSTE24 deficiency. J. Investig. Med. 54, 208 (2006).
28. V. J. Cunningham, M. R. D’Apice, N. Licata, G. Novelli, T. Cundy, Skeletal phenotype of mandibuloacral dysplasia associated with mutations in ZMPSTE24. Bone 47, 591 (2010).
29. S. Shackleton et al., Compound heterozygous ZMPSTE24 mutations reduce prelamin A processing and result in a severe progeroid phenotype. J. Med. Genet. 42, e36 (2005).
30. M. Thill et al., Restrictive dermopathy: A rare laminopathy. Arch. Gynecol. Obstet. 278, 201 (2008).
31. Y. Miyoshi et al., Severe mandibuloacral dysplasia caused by novel compound heterozygous ZMPSTE24 mutations in two Japanese siblings. Clin. Genet. 73, 535 (2008).
32. Z. Ahmad, E. Zackai, L. Medne, A. Garg, Early onset mandibuloacral dysplasia due to compound heterozygous mutations in ZMPSTE24. Am. J. Med. Genet. A. 152A, 2703 (2010).
33. D. J. Slotboom, R. H. Duurkens, K. Olieman, G. B. Erkens, Static light scattering to characterize membrane proteins in detergent solution. Methods 46, 73 (2008).
34. G. Berridge et al., High-performance liquid chromatography separation and intact mass analysis of detergent-solubilized integral membrane proteins. Anal. Biochem. 410, 272 (2011).
35. W. Kabsch, Xds. Acta Crystallogr. D Biol. Crystallogr. 66, 125 (2010).
36. P. Evans, Scaling and assessment of data quality. Acta Crystallogr. D Biol. Crystallogr. 62, 72 (2006).
37. G. M. Sheldrick, A short history of SHELX. Acta Crystallogr. A 64, 112 (2008).
38. T. Terwilliger, SOLVE and RESOLVE: Automated structure solution, density modification and model building. J. Synchrotron Radiat. 11, 49 (2004).
39. A. J. McCoy et al., Phaser crystallographic software. J. Appl. Cryst. 40, 658 (2007).
40. K. Cowtan, DMMULTI. Joint CCP4 and ESF-EACBM Newsl. Protein Crystallogr. 31, 34 (1994).
41. K. Cowtan, The Buccaneer software for automated model building. 1. Tracing protein chains. Acta Crystallogr. D Biol. Crystallogr. 62, 1002 (2006).
42. P. Emsley, B. Lohkamp, W. G. Scott, K. Cowtan, Features and development of Coot. Acta Crystallogr. D Biol. Crystallogr. 66, 486 (2010).
43. G. Bricogne et al., BUSTER, version 2.8 (Global Phasing Ltd, Cambridge, 2011).
44. V. B. Chen et al., MolProbity: All-atom structure validation for macromolecular crystallography. Acta Crystallogr. D Biol. Crystallogr. 66, 12 (2010).
45. E. F. Pettersen et al., UCSF Chimera—a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605 (2004).
46. B. Hess, C. Kutzner, D. van der Spoel, E. Lindahl, GROMACS 4: Algorithms for highly efficient, load-balanced, and scalable molecular simulation. J. Chem. Theory Comput. 4, 435 (2008).
47. L. Monticelli et al., The MARTINI coarse-grained force field: Extension to proteins. J. Chem. Theory Comput. 4, 819 (2008).
48. K. A. Scott et al., Coarse-grained MD simulations of membrane protein-bilayer self-assembly. Structure 16, 621 (2008).
49. P. J. Stansfeld, M. S. Sansom, Molecular simulation approaches to membrane proteins. Structure 19, 1562 (2011).
50. L. Holm, P. Rosenström, Dali server: Conservation mapping in 3D. Nucleic Acids Res. 38, (Web Server issue), W545 (2010).
51. V. Pelmenschikov, M. R. Blomberg, P. E. Siegbahn, A theoretical study of the mechanism for peptide hydrolysis by thermolysin. J. Biol. Inorg. Chem. 7, 284 (2002).
52. C. S. Bond, A. W. Schüttelkopf, ALINE: A WYSIWYG protein-sequence alignment editor for publication-quality alignments. Acta Crystallogr. D Biol. Crystallogr. 65, 510 (2009).