Human nuclear membrane zinc metalloprotease ZMPSTE24 mutant (E336A) in complex with a synthetic CSIM tetrapeptide from the C-terminus of prelamin A

Target ID:

ZMPSTE24A

Aliases:

FACE-1FACE1FLJ14968STE24Ste24pZMPSTE24

Deposition Date:

Wednesday 31st October 2012

Families:

Membrane proteins

Structure type:

protein-ligand

Download Coordinates:

2YPT

Authors:

A.C.W.PIKE,Y.Y.DONG,A.QUIGLEY,L.DONG,P.SAVITSKY,C.D.O.COOPER,A.CHAIKUAD,S.GOUBIN,L.SHRESTHA,Q.LI,S.MUKHOPADHYAY,J.YANG,X.XIA,C.A.SHINTRE,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

2YPT

tabs

Structure Details

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).

We previously solved ZMPSTE24 at 3.4 Å resolution without bound substrate. 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.

Here we present the crystal structure of full-length inactivation mutant of ZMPSTE24 (E336A) complexed with a tetrapeptide Cys-Ser-Ile-Met at 3.8 Å resolution. The peptide comprises of the final 4 amino acids of prelamin A corresponding to the CAAX motif but lacking a farnesyl group on the cysteine residue. The CSIM tetrapeptide binds next to the catalytic zinc which faces into the intramembrane chamber. The binding mode is similar to that of an insect metalloprotease inhibitor in thermolysin (PDB: 3ssb) but differs with the peptide shifting one position in the binding register. This is supported by mass spectrometry based assays which show that the peptide is cleaved one position along (CS^IM) from the expected site (C^SIM).

Materials and Methods

ZMPSTE24A (2YPT) Materials & Methods

Entry Clone Source: MGC

Entry Clone Accession: IMAGE: 5269064

SGC Construct ID: ZMPSTE24A-c029

GI Number: 10269

Vector: pFB-CT10HF-LIC. Details [PDF ]; Sequence [ FASTA ] or [ GenBank ]

DNA sequence:
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
ACTAGGCCATGCACTGGGGCACTGGA
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):
MGMWASLDALWEMPAEKRIFGAVLLF
SWTVYLWETFLAQRQRRIYKTTTHVP
PELGQIMDSETFEKSRLYQLDKSTFS
FWSGLYSETEGTLILLFGGIPYLWRL
SGRFCGYAGFGPEYEITQSLVFLLLA
TLFSALAGLPWSLYNTFVIEEKHGFN
QQTLGFFMKDAIKKFVVTQCILLPVS
SLLLYIIKIGGDYFFIYAWLFTLVVS
LVLVTIYADYIAPLFDKFTPLPEGKL
KEEIEVMAKSIDFPLTKVYVVEGSKR
SSHSNAYFYGFFKNKRIVLFDTLLEE
YSVLNKDIQEDSGMEPRNEEEGNSEE
IKAKVKNKKQGCKNEEVLAVLGHALG
HWKLGHTVKNIIISQMNSFLCFFLFA
VLIGRKELFAAFGFYDSQPTLIGLLI
IFQFIFSPYNEVLSFCLTVLSRRFEF
QADAFAKKLGKAKDLYSALIKLNKDN
LGFPVSDWLFSMWHYSHPPLLERLQA
LKTMKQHAENLYFQ^shhhhhhhhhh
dykddddk

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% 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 55460Da, as expected from the primary sequence with a post translational modification of loss of 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. A three-fold excess of a farnesylated nonapeptide corresponding to the C-terminus of prelamin A (residues 656-664; QSPQNC(farn)SIM) was added prior to setting up crystallization plates

Crystallization: P1 crystals were obtained under similar conditions to the native enzyme at 20°C. Crystals grew from sitting drops (150nl) comprising 100nl of concentrated protein / peptide complex and 50nl of reservoir equilibrated against 20µl of reservoir solution (0.1M HEPES pH 7.0, 0.1M calcium chloride, 29% (v/v) PEG 400).

Data Collection:
Resolution: 3.8Å
Data were collected at 100K on the microfocus beamline I24 at Diamond Light Source using helical / straight line scans with a beamsize of 10µm x 10µm (wxh).

Structure Solution: Datasets collected from ZMPSTE24 / prelamin A peptide (Q656-M664) co-crystals showed that the peptide-binding site was empty. These co-crystals were then soaked with reservoir solution containing 5mM CSIM tetrapeptide and OGNG/CHS detergent for 24h at 6°C. The coordinates of the native P1 structure (4AW6) were used as a starting model for refinement with BUSTER. Clear difference Fo-Fc electron density in each of the four molecules in the asymmetric unit was interpreted based on the known binding mode of peptides and inhibitors to metalloproteases. The peptide 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. The entire tetrapeptide was modeled in two cases (molecules B&D) whereas only C661-I663 was modeled in molecules A & E. In all cases, the quality of the electron density was not sufficient to resolve the CD1 atom of Ile663 and this atom was not included in the model. Refinement was performed with BUSTER with TLS, NCS and LSSR restraints to the native structure.

References

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).