Human Nuclear Membrane Zinc Metalloprotease ZMPSTE24 (FACE1)

Target ID:

ZMPSTE24A

Aliases:

FACE-1FACE1FLJ14968STE24Ste24pZMPSTE24

Deposition Date:

Thursday 31st May 2012

Families:

Membrane proteins

Structure type:

apo

Download Coordinates:

4AW6

Authors:

A.C.W.PIKE,Y.Y.DONG,A.QUIGLEY,L.DONG,C.D.O.COOPER,A.CHAIKUAD,S.GOUBIN,L.SHRESTHA,Q.LI,S.MUKHOPADHYAY,J.YANG,A.J.BARR,G.BERRIDGE,R.CHALK,J.E.BRAY,F.VONDELFT,A.BULLOCK,C.BOUNTRA,C.H.ARROWSMITH,A.EDWARDS,N.BURGESS-BROWN,E.P.CARPENTER

Site:

Oxford

4AW6

tabs

Structure Details

Read the article in Science (Open Access): The Structural Basis of ZMPSTE24-Dependent Laminopathies

ZMPSTE24 is a nuclear zinc metalloprotease required for the processing of prelamin A to form mature lamin A (1). Lamin A is one of the three lamins which make up the intermediate filaments of the nuclear lamina of higher eukaryotic cells. The lamina maintain the shape of the nuclei and are also essential for the correct segregation of chromosomes (2). Prelamin A is processed in four steps. Initially a cysteine residue in the C-terminal CAAX sequence (where C is a cysteine residue, A is any aliphatic residue and X is any residue) is farnesylated by farnesyl transferase. Then the C-terminal AAX residues are remove by either ZMPSTE24 (3) or RCE1. The C-terminus is then carboxymethylated by ICMT. This highly hydrophobic farnesylated and carboxymethylated cysteine residue anchors the C-terminus of prelamin A in the membrane. However a further cleavage by ZMPSTE24, which removes the C-terminal 15 residues, including the farnesyl cysteine, removes this hydrophobic anchor, thus liberating ZMPSTE24 from the membrane, creating mature soluble lamin A within the nucleoplasm of cells (1).

Failure of prelamin A processing leads to a series of diseases called laminopathies. The severity of these diseases depends on the extent of residual prelamin A processing. Mutations which cause a complete loss of ZMPSTE24 activity result in restrictive dermopathy, a condition in which the skin does not stretch, causing death before or shortly after birth (4, 5). Mutations in prelamin A, which disrupt the secondary ZMPSTE24A-specific cleavage site, result in accumulation of a permanently farnesylated, membrane-bound form of prelamin A called progerin which leads to Hutchinson Gilford progeria syndrome (HGPS) (6, 7). HGPS patients have many of the symptoms of an 80 year old at the age of 10, and they often die of heart disease or strokes before they reach their 20s. Point mutations in ZMPSTE24 lead to an atypical form of HGPS (8). Mutations in ZMPSTE24 which lead to a reduction, rather than complete loss of protease activity (9), lead to relatively mild laminopathies such as mandibuloacral dysplasia type B, a condition in which the skeleton, skin and lipid metabolism is abnormal (10). A mutation in ZMPSTE24 was also identified in one patient with metabolic syndrome in a study of 100 cases (11). Metabolic syndrome affects 20-30% of the human population and so ZMPSTE24 may have a significant role in this relatively common disease. During normal ageing the levels of ZMPSTE24 decrease, particularly in vascular smooth muscle cells, leading to changes in the nuclear membrane and a decrease in effective cell division (12) These is also evidence that HIV protease inhibitors, used in the treatment of AIDS, may also inhibit ZMPSTE24, potentially causing some of the adverse side effects of certain HIV protease inhibitors (13)

Here we present the crystal structure of full-length ZMPSTE24 at 3.4 Å resolution. The structure reveals a novel seven transmembrane helical barrel fold surrounding an extensive chamber, which spans the nuclear membrane. The barrel is capped by a zinc metalloprotease domain with a similar fold to thermolysin on the nucleoplasmic side of the membrane. On the opposite side of the membrane, facing the endoplasmic reticulum lumen, the chamber is closed by the presence of two helices which connect transmembrane helices.

The catalytic site of the zinc metalloprotease faces into the intramembrane chamber. The C-terminus of prelamin A will enter the chamber from the interface between the nucleoplasm where the protein resides and the inner leaflet of the inner nuclear membrane, where the farnesyl tail lies. The cavity is not completely closed and there is a possible entry route for the C-terminus of prelamin A, which would direct the substrate from the membrane to the zinc atom at the centre of the zinc metalloprotease active site. The C-terminal three residues are then cleaved and leave the chamber by one of several openings in the chamber walls. The partially processed prelamin A would then leave the chamber, with the farnesyl group still attached to the C-terminal cysteine and embedded in the membrane. The intramembrane enzyme ICMT will then carboxymethylate the C-terminus of prelamin A. This hydrophobic tail would then enter the chamber of ZMPSTE24 again and cleavage at the second site will remove the C-terminal 15 residues from the partially processed prelamin A, resulting in loss of the farnesyl tail from the C-terminus, thus giving mature soluble lamin A which is released from the chamber of ZMPSTE24 into the nucleoplasm.

Materials and Methods

ZMPSTE24A (2YPT) Materials & Methods
Entry Clone Source: MGC
Entry Clone Accession: IMAGE: 5269064
SGC Construct ID: ZMPSTE24A-c000 / ZMPSTE24A-c011
GI Number: 10269
Vector: pFB-CT10HF-LIC. Details [PDF ]; Sequence [ FASTA ] or [ GenBank ]
DNA sequence: c000: CTTAAGAAGGAGATATACTATGGGGA TGTGGGCATCGCTGGACGCTTTGTGG GAGATGCCGGCCGAGAAGCGTATCTT CGGGGCCGTGCTGCTCTTTTCCTGGA CAGTGTATCTTTGGGAGACCTTCCTA GCACAGCGGCAGAGAAGGATATATAA AACAACAACTCATGTACCACCGGAGT TAGGACAGATCATGGATTCTGAAACA TTTGAGAAATCTCGACTCTATCAACT GGATAAAAGCACTTTCAGCTTCTGGT CAGGACTCTATTCAGAGACTGAAGGC ACTCTTATTCTTCTCTTTGGAGGAAT ACCTTATCTCTGGAGACTTTCTGGAC GGTTCTGTGGTTATGCTGGCTTTGGA CCAGAATATGAGATCACTCAGTCCCT GGTGTTTCTGCTGTTGGCTACACTTT TCAGTGCATTGGCTGGTTTGCCATGG AGTCTTTATAATACTTTTGTGATAGA AGAAAAACATGGCTTCAATCAACAGA CTTTGGGGTTCTTCATGAAAGATGCA ATCAAGAAATTTGTTGTGACTCAGTG CATTTTGTTGCCTGTGTCTTCACTTC TACTTTACATTATTAAAATTGGGGGT GACTATTTTTTTATTTATGCCTGGCT GTTCACATTAGTTGTGTCTCTGGTTC TTGTCACAATCTATGCTGATTATATT GCCCCTTTATTTGACAAATTCACACC TCTGCCTGAGGGAAAGCTTAAAGAAG AAATTGAAGTAATGGCAAAGAGTATT GACTTTCCTTTGACGAAGGTGTATGT TGTGGAAGGATCTAAACGCTCTTCCC ACAGCAATGCTTATTTTTATGGCTTC TTCAAGAACAAGCGAATAGTTTTGTT TGACACTCTACTAGAAGAGTACTCTG TACTAAACAAAGACATCCAGGAGGAT TCTGGCATGGAACCCCGCAATGAGGA AGAAGGGAACAGTGAAGAAATAAAAG CTAAAGTTAAAAATAAGAAACAAGGA TGTAAAAATGAGGAGGTACTCGCTGT ACTAGGCCATGAACTGGGGCACTGGA AGTTGGGACATACAGTCAAAAATATC ATTATTAGCCAGATGAATTCTTTCCT GTGTTTTTTTTTATTTGCTGTATTAA TTGGTCGAAAGGAGCTTTTTGCTGCA TTTGGTTTTTATGATAGCCAACCCAC TCTTATTGGACTATTGATCATCTTCC AGTTTATTTTTTCACCTTACAATGAG GTTCTTTCTTTTTGCCTAACAGTCCT AAGCCGCAGATTTGAGTTTCAAGCTG ATGCATTTGCCAAGAAACTTGGGAAG GCTAAAGACTTATATTCTGCTTTAAT CAAACTTAACAAAGATAACTTGGGAT TCCCTGTTTCTGACTGGTTGTTCTCA ATGTGGCATTATTCTCATCCTCCACT GCTAGAGAGACTTCAAGCTTTGAAAA CTATGAAGCAACACGCAGAGAACCTC TACTTCCAATCGCACCATCATCACCA TCACCATCACCACCATGATTACAAGG ATGACGACGATAAGTGAGGATCC ww	DNA sequence: c011: CTTAAGAAGGAGATATACTATGGGGA TGTGGGCATCGCTGGACGCTTTGTGG GAGATGCCGGCCGAGAAGCGTATCTT CGGGGCCGTGCTGCTCTTTTCCTGGA CAGTGTATCTTTGGGAGACCTTCCTA GCACAGCGGCAGAGAAGGATATATAA AACAACAACTCATGTACCACCGGAGT TAGGACAGATCATGGATTCTGAAACA TTTGAGAAATCTCGACTCTATCAACT GGATAAAAGCACTTTCAGCTTCTGGT CAGGACTCTATTCAGAGACTGAAGGC ACTCTTATTCTTCTCTTTGGAGGAAT ACCTTATCTCTGGAGACTTTCTGGAC GGTTCTGTGGTTATGCTGGCTTTGGA CCAGAATATGAGATCACTCAGTCCCT GGTGTTTCTGCTGTTGGCTACACTTT TCAGTGCATTGGCTGGTTTGCCATGG AGTCTTTATAATACTTTTGTGATAGA AGAAAAACATGGCTTCAATCAACAGA CTTTGGGGTTCTTCATGAAAGATGCA ATCAAGAAATTTGTTGTGACTCAGTG CATTTTGTTGCCTGTGTCTTCACTTC TACTTTACATTATTAAAATTGGGGGT GACTATTTTTTTATTTATGCCTGGCT GTTCACATTAGTTGTGTCTCTGGTTC TTGTCACAATCTATGCTGATTATATT GCCCCTTTATTTGACAAATTCACACC TCTGCCTGAGGGAAAGCTTAAAGAAG AAATTGAAGTAATGGCAAAGAGTATT GACTTTCCTTTGACGAAGGTGTATGT TGTGGAAGGATCTAAACGCTCTTCCC ACAGCAATGCTTATTTTTATGGCTTC TTCAAGAACAAGCGAATAGTTTTGTT TGACACTCTACTAGAAGAGTACTCTG TACTAAACAAAGACATCCAGGAGGAT TCTGGCATGGAACCCCGCAATGCTGC TGCTGGGAACAGTGAAGAAATAAAAG CTAAAGTTAAAAATAAGAAACAAGGA TGTAAAAATGAGGAGGTACTCGCTGT ACTAGGCCATGAACTGGGGCACTGGA AGTTGGGACATACAGTCAAAAATATC ATTATTAGCCAGATGAATTCTTTCCT GTGTTTTTTTTTATTTGCTGTATTAA TTGGTCGAAAGGAGCTTTTTGCTGCA TTTGGTTTTTATGATAGCCAACCCAC TCTTATTGGACTATTGATCATCTTCC AGTTTATTTTTTCACCTTACAATGAG GTTCTTTCTTTTTGCCTAACAGTCCT AAGCCGCAGATTTGAGTTTCAAGCTG ATGCATTTGCCAAGAAACTTGGGAAG GCTAAAGACTTATATTCTGCTTTAAT CAAACTTAACAAAGATAACTTGGGAT TCCCTGTTTCTGACTGGTTGTTCTCA ATGTGGCATTATTCTCATCCTCCACT GCTAGAGAGACTTCAAGCTTTGAAAA CTATGAAGCAACACGCAGAGAACCTC TACTTCCAATCGCACCATCATCACCA TCACCATCACCACCATGATTACAAGG ATGACGACGATAAGTGAGGATCC
Expressed sequence (small letters refer to tag sequence): c000 MGMWASLDALWEMPAEKRIFGAVLLF SWTVYLWETFLAQRQRRIYKTTTHVP PELGQIMDSETFEKSRLYQLDKSTFS FWSGLYSETEGTLILLFGGIPYLWRL SGRFCGYAGFGPEYEITQSLVFLLLA TLFSALAGLPWSLYNTFVIEEKHGFN QQTLGFFMKDAIKKFVVTQCILLPVS SLLLYIIKIGGDYFFIYAWLFTLVVS LVLVTIYADYIAPLFDKFTPLPEGKL KEEIEVMAKSIDFPLTKVYVVEGSKR SSHSNAYFYGFFKNKRIVLFDTLLEE YSVLNKDIQEDSGMEPRNEEEGNSEE IKAKVKNKKQGCKNEEVLAVLGHELG HWKLGHTVKNIIISQMNSFLCFFLFA VLIGRKELFAAFGFYDSQPTLIGLLI IFQFIFSPYNEVLSFCLTVLSRRFEF QADAFAKKLGKAKDLYSALIKLNKDN LGFPVSDWLFSMWHYSHPPLLERLQA LKTMKQaenlyfq^shhhhhhhhhhd ykddddk	Expressed sequence (small letters refer to tag sequence): c011 MGMWASLDALWEMPAEKRIFGAVLLF SWTVYLWETFLAQRQRRIYKTTTHVP PELGQIMDSETFEKSRLYQLDKSTFS FWSGLYSETEGTLILLFGGIPYLWRL SGRFCGYAGFGPEYEITQSLVFLLLA TLFSALAGLPWSLYNTFVIEEKHGFN QQTLGFFMKDAIKKFVVTQCILLPVS SLLLYIIKIGGDYFFIYAWLFTLVVS LVLVTIYADYIAPLFDKFTPLPEGKL KEEIEVMAKSIDFPLTKVYVVEGSKR SSHSNAYFYGFFKNKRIVLFDTLLEE YSVLNKDIQEDSGMEPRNAAAGNSEE IKAKVKNKKQGCKNEEVLAVLGHELG HWKLGHTVKNIIISQMNSFLCFFLFA VLIGRKELFAAFGFYDSQPTLIGLLI IFQFIFSPYNEVLSFCLTVLSRRFEF QADAFAKKLGKAKDLYSALIKLNKDN LGFPVSDWLFSMWHYSHPPLLERLQA LKTMKQHAENLYFQaenlyfq^shhh hhhhhhhdykddddk
Tags and additions: C-terminal, TEV cleavable decahistidine / FLAG tag.
Host: Spodoptera frugiperda (SF9) insect cells
Growth Medium & Induction Protocol: Insect cells with a density of 2x10⁶ per litre of cell culture in SF900 medium (Invitrogen) were infected with recombinant baculovirus (5ml P2 virus per litre cell culture) and incubated for 72 hours. Cells were harvested by centrifugation and the pellet was flash frozen in liquid N₂.
Extraction buffer, extraction method: Frozen pellets were thawed and re-suspended in extraction buffer supplemented with 1% DDM or 1% OGNG/ 0.1% CHS and incubated at 4°C on a tube rotator. The extracted protein was separated from insoluble membranes by centrifugation and collected for purification.
Extraction/wash buffer: 50 mM HEPES, pH 7.5; 200 mM NaCl
Column 1: Co-affinity. Cobalt Talon (Clontech), 0.75 ml of 50 % slurry/ 1L of cells in 1.5 x 10 cm column, washed with extraction/ wash buffer.
Column 1 Buffers: Extraction/wash buffer: 50 mM HEPES, pH 7.5; 200 mM NaCl; 20 mM imidazole, detergent at 3x CMC. Elution buffer: 50 mM HEPES, pH 7.5; 200 mM NaCl; 250 mM imidazole, detergent at 3xCMC
Column 1 Procedure: The solubilised membrane protein was batch bound to Cobalt Talon resin equilibrated with extraction buffer at 4°C for one hour and subsequently poured into a gravity flow glass column. The column was then washed with 30CV of wash buffer followed by elution in 1CV fractions until all the protein was eluted.
Column 2: Size Exclusion Chromatography. Superdex200 (GE healthcare)
Column 2 Buffer: Gel Filtration buffer: 20 mM HEPES pH 7.5, 200 mM NaCl,detergent at 1.2xCMC
Column 2 Procedure: The protein was concentrated and applied to a Superdex 200 gel filtration column equilibrated with gel filtration buffer using an AKTA purifier system.
Enzymatic Treatment: TEV protease (1:4, TEV:protein) was added overnight at 4°C to purified protein. The protease was removed and tag cleaved protein was collected by binding to Cobalt talon and collecting the flow-through.
Mass spec characterization: The purified protein was homogeneous and had an experimental mass of 55517Da (c000) or 55345Da (c011), as expected from the primary sequence including loss of the N-terminal methionine. Masses were determined by LC-MS, using an Agilent LC/MSD TOF system with reversed-phase HPLC coupled to electrospray ionisation and an orthogonal time-of-flight mass analyser. Proteins were desalted prior to mass spectrometry by rapid elution off a C3 column with a gradient of 5-95% methanol in water with 0.1% formic acid.
Protein Concentration: Protein was concentrated to ~20mg/ml using a 100kDa cut-off concentrator and back diluted to 9-11mg/ml.
Crystallization: Crystals were grown at 20°C from sitting drops (150-300nl). Two crystal forms were observed depending on the purification detergent used. Monoclinic (P21) crystals were obtained from protein (c011) purified in 0.015% 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) crystals were obtained from protein (c000) purified in 0.12% OGNG/0.012% CHS, and crystallised with a reservoir solution containing 30-42%(v/v) PEG 400, 0.1M HEPES pH 7.5, 0.1M calcium chloride. Crystals were cryocooled by transfer into liquid nitrogen without the addition of a cryoprotectant. Diffraction of the P1 crystal form was improved by transferring crystallization plates to 6°C and equilibration for at least 24 hours prior to direct flash cooling of crystals.
Data Collection: Resolution: 3.4Å All 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 10µm x 10-30µm (wxh).
Structure Solution: Phases were obtained using SIRAS with data from P1 crystals derivatised with 1mM EMTS. A total of 12 mercury sites were located with SHELXD (4 ZMPSTE24 molecules per asymmetric unit) and phase refinement was carried out by SOLVE/RESOLVE to 3.8Å resolution. Initial electron density maps, calculated after four-fold averaging / phase extension to 3.4Å, allowed an initial backbone trace to be built. Subsequent cross-crystal averaging between the P1 (4 mols/AU) and P2₁ (4 mols/AU) forms gave a substantial improvement in the quality of the experimental phases and allowed tracing and sequence assignment for the transmembrane and protease domains. The structure was refined with BUSTER using all data to 3.4Å with appropriate NCS and TLS restraints to final R/Rfree values of 24.6 and 26.4% respectively. The final P1 model comprises 4 copies of ZMPSTE24 with each copy encompassing residues 10-107, 116-285, 322-472, a single zinc ion and two phospholipid alkyl chains.

References

1. J. Barrowman, S. Michaelis, ZMPSTE24, an integral membrane zinc metalloprotease with a connection to progeroid disorders. Biol Chem 390, 761 (Aug, 2009).
T. A. Dittmer, T. Misteli, The lamin protein family. Genome biology 12, 222 (2011).
D. P. Corrigan et al., Prelamin A endoproteolytic processing in vitro by recombinant Zmpste24. Biochem J 387, 129 (Apr 1, 2005).
C. L. Moulson et al., Homozygous and compound heterozygous mutations in ZMPSTE24 cause the laminopathy restrictive dermopathy. J Invest Dermatol 125, 913 (Nov, 2005).
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 (Oct 15, 2004).
M. Eriksson et al., Recurrent de novo point mutations in lamin A cause Hutchinson-Gilford progeria syndrome. Nature 423, 293 (May 15, 2003).
R. D. Goldman et al., Accumulation of mutant lamin A causes progressive changes in nuclear architecture in Hutchinson-Gilford progeria syndrome. Proc Natl Acad Sci U S A 101, 8963 (Jun 15, 2004).
J. Denecke et al., A homozygous ZMPSTE24 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 (Jun, 2006).
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 (Sep 15, 2012).
A. K. Agarwal, J. P. Fryns, R. J. Auchus, A. Garg, Zinc metalloproteinase, ZMPSTE24, is mutated in mandibuloacral dysplasia. Hum Mol Genet 12, 1995 (Aug 15, 2003).
A. Dutour et al., High prevalence of laminopathies among patients with metabolic syndrome. Hum Mol Genet 20, 3779 (Oct 1, 2011).
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 (May 25, 2010).
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 (Aug 14, 2007).

Human Nuclear Membrane Zinc Metalloprotease ZMPSTE24 (FACE1)

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ZMPSTE24A (2YPT) Materials & Methods