Department of Molecular Physiology and Biological Physics, University of Virginia, Health Sciences Center, Charlottesville, VA 229060011, USA
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Abstract |
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Keywords: bi-cistronic/crystal structure/expression vector/heterodimer/hydrolase
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Introduction |
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Defects in neuronal migration result in various syndromes, which are primarily genetic in nature. Some of the causal genes for neuronal migration syndromes have been identified and their respective products are subjects of ongoing studies. Arguably, of the nearly 30 syndromes known to date, the most dramatic in appearance is lissencephaly, a developmental disorder that prevents the formation of normal cortex, leading to smooth cerebral surface and severe mental retardation (Dobyns et al., 1993; Dobyns and Truwit, 1995
). The causal gene for MillerDieker lissencephaly (a form of lissencephaly associated with dysmorphic facial appearance), known as LIS1, has been identified and characterized (Reiner et al., 1993
, 1995
; Mizuguchi et al., 1995
). It encodes a 45 kDa polypeptide chain with homology to members of the WD-repeat family, a group of predominantly regulatory proteins which include, among others, the ß-subunits of trimeric G-proteins (Reiner et al., 1993
). It is uncertain what the exact biological function of the LIS1 protein is, but it is thought to function via interactions with other proteins involved in the regulation of the cytoskeleton (Sapir et al., 1997
, 1999
; Morris et al., 1998
). Most notably it associates in tissues with a unique intracellular isoform of phospholipase A2, the brain platelet-activating factor acetylhydrolase (PAF-AH) (Hattori et al., 1994b
). This brain PAF-AH is a dimer made up of one or both types of two homologous (63% amino acid identity) polypeptide chains of ~26 kDa, denoted the
1 and
2 chains (Hattori et al., 1994a
, 1995
). The enzyme was originally identified for its ability to hydrolyze the potent intra- and extracellular phospholipid messenger, platelet-activating factor (PAF, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine), but is now known to hydrolyze such substrates as 1-O-alkyl-2-acetyl-sn-glycero-3-phosphorylethanolamine (AAGPE) or 1-O-alkyl-2-acetyl-sn-glycero-3-phosphoric acid (AAGPA) (Manya et al., 1999
). The relative catalytic activities depend on the composition of the dimer, so that the
2/
2 homodimer is significantly more active against PAF and AAGPE than the other two dimeric species,
1/
1 and
1/
2. Also, while both homodimers have comparable catalytic efficiency against PAF, in the heterodimer, only the
1-subunit appears to be active. The association with LIS1 (also referred to as the ß-subunit) further modulates substrate specificity of the brain PAF-AH (Manya et al., 1999
).
The composition of the catalytic PAF-AH dimer appears to be tightly regulated during development: the 1 subunit and consequently the
1/
2 heterodimer are expressed in rat specifically in neurons of fetal and neonatal brains and are not found in adult tissues, although the exact expression pattern may be different in other species (Manya et al., 1998
). There is mounting evidence that this regulatory mechanism is indeed physiologically relevant and plays a role during cortical development (Albrecht et al., 1996
; Sweeney et al., 2000
). Mutations in LIS1 that are known to impair neuronal migration have recently been shown to abolish interaction with the PAF-AH catalytic dimers (Sweeney et al., 2000
).
In order to understand the regulatory mechanisms involving LIS1 and PAF-AH it is necessary to gain insight into the molecular structure of the physiologically relevant complexes. Previously, we have determined the crystal structure of the recombinant bovine 1 homodimer at 1.7 Å resolution (Ho et al., 1997
). In general terms, the tertiary fold of the protein resembles that of the small G-proteins (Ras and Rho). The catalytic site contains a chymotrypsin-like triad of SerHisAsp and the mechanism of hydrolytic cleavage of the sn-2 ester bond is identical with the serine proteinases and neutral lipases (Derewenda, 1994
). The dimer interface contains a significant number of charged and polar residues and we also showed that the specificity for an acetyl moiety as the leaving acyl group is determined by a unique binding site (Ho et al., 1999
). Finally, we were able to show that the dissociation of the dimeric protein into monomers leads to the inactivation of the protein and its destabilization (McMullen et al., 2000
).
In this paper we describe the preparation and structure determination (at 2.1 Å resolution) of the 1/
2 heterodimer, which appears to be functionally important during fetal brain development. The preparation of the heterodimer was accomplished by co-expression of the two chains using a specially designed bi-cistronic construct containing two ribosome binding sites (RBS) and two affinity tags, maltose binding protein (MBP) and His6, fused with the two
-chains via linkers containing Factor Xa and rTEV proteolytic cleavage sites, respectively. This relatively novel construct may prove useful as a general tool for preparation of heterodimeric complexes by co-expression of individually unstable components, particularly those proteins which are found in equilibrium between homo- and heterodimeric association.
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Materials and methods |
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To obtain a bi-cistronic plasmid, the His6-1 cDNA from pHIS:
1 was subcloned immediately downstream of the
2 cDNA in pMBP:
2 as an XbaI fragment. Excision of the
1 gene from pHIS:
1 as an XbaI fragment allowed the
1 cDNA to be isolated along with the ribosome binding site (RBS) from the pHIS vector. The resulting construct is shown in Figure 1a
. This construct presented problems during purification (see Results). Therefore, the His6-
1 cDNA from this construct was transferred as a SalI fragment to pMal-C2::
2 to generate the construct shown in Figure 1b
, in which MBP is fused to the
2 gene via a linker containing the Factor Xa site.
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The bi-cistronic plasmids were transformed into Escherichia coli strain XL1Blue (Stratagene). Expression was carried out in 12 l of Luria broth. Growth was initiated at 37°C until mid-log phase (OD600 nm 0.40.6), expression was induced by the addition of IPTG (final concentration 0.5 mM) and growth continued for a further 16 h at 37 or 30°C (see Results). Cells were harvested by centrifugation and resuspended in buffer I (50 mM TrisHCl, pH 8.0, 500 mM NaCl) and lysed by sonication. Soluble protein was recovered by centrifugation (30 min at 15 000 g). Purification of the heterodimer was achieved by sequential use of Ni-NTA affinity chromatography and size exclusion chromatography techniques. His6-1 homodimer and His6-
1/
2-MBP heterodimer were isolated from the MBP-
2 homodimer by Ni-NTA chromatography. The heterodimer was then purified from the
1 homodimer by size exclusion, taking advantage of the size of the MBP tag. The tags from the second construct were removed sequentially to aid final purification: the MBP tag was removed first by treatment with factor Xa (NEB) and the His6-tagged heterodimer was re-isolated from the MBP protein by Ni-NTA affinity chromatography. The His6 tag was then removed by treatment with rTEV protease and pure
1/
2 heterodimer was isolated after the final size exclusion chromatography step.
Size exclusion chromatography
Size exclusion chromatography was performed on a BioCad Sprint system (PerSeptive Biosystems) using a Superdex 75 column (Pharmacia). A standard Tris buffer (50 mM TrisHCl, pH 8.0, 200 mM NaCl) was used throughout the purification.
Ni-NTA metal affinity chromatography
His6-tagged fusion proteins were isolated by binding to 5 ml Ni-NTA-agarose columns (Qiagen) pre-equilibrated with 100 ml of His buffer I (50 mM TrisHCl, pH 8.0, 500 mM NaCl). The soluble protein fraction was allowed to bind to the column for 2 h at 4°C before being drained and washed with 2 l of His buffer I at 4°C. Fusion protein was eluted in 20 ml of His buffer II (His buffer I + 250 mM imidazole, pH 8.5).
Crystallization
Crystallization experiments were performed by the hanging-drop vapor-diffusion method at 21°C using Linbro 24-well tissue culture plates. Initial screening was done using the Hampton Research crystal screen I. Crystallization experiments were done using protein concentration of 6 mg/ml, as determined by the Bradford assay (Bio-Rad).
Data collection
X-ray data were collected using synchrotron radiation on beamline 19 ID (Structural Biology Center, Argonne National Laboratory) to 2.1 Å resolution. Data were processed using HKL2000 (Otwinowski and Minor, 1997).
Structure determination
The structure of 1/
2 heterodimer was solved by Molecular Replacement with AMoRe (Navaza, 1994
) using data in the 15.04.0 Å resolution range. The
1 homodimer (PDB accession number 1WAB) including all side chains was used as a search model. Rotation and translation searches yielded a clear solution with a correlation coefficient of 55.1% and an R-factor of 39.3%. The resulting model was subjected to rigid body refinement and residues in the model that are different from
2 were replaced. The final refinement using CNS (Brunger et al., 1998
) converged with Rwork 21.8% and Rfree 27.7%. The quality of the model was checked using PROCHECK (Laskowski et al., 1993
). The CCP4 program package was used for all other purposes (CCP4, 1994
).
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Results |
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Analysis of post-induction protein expression in E.coli by SDSPAGE (Figure 2a) under denaturing conditions revealed two expected proteins bands: one corresponding to His6-
1 (~32 kDa) and the other to MBP-
2 (~68 kDa). The existence of the heterodimer in solution was confirmed by an initial Ni+-NTA agarose extraction, which isolated both His6-
1 and MBP-
2, implying that MBP-
2, which could not have been purified by Ni+-NTA extraction on its own, was in complex with His6-
1 (Figure 2b
). Isolation of pure heterodimer should have been completed by passing the Ni+-NTA eluate through an amylose resin column to separate His6-
1 homodimer from MBP-
2/
1-His6 heterodimer. Unfortunately, the MBP tagged heterodimer did not bind well to the amylose resin and the majority remained in the flow through. Purification of tagged heterodimer was consequently achieved by size exclusion chromatography, which allowed for separation of the 60 kDa
1 homodimer from the 105 kDa heterodimer, although resolution was less than perfect owing to the limited capability of the Superdex 75 column.
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Crystallization
Crystals were obtained in 1618% PEG 8000, 100 mM TrisHCl pH 6.8, 10 mM CaCl2 and in 2530% (NH4)2SO4, 100mM sodium acetate pH 6.4. Further trials were performed around these conditions and diffracting crystals were obtained in 17% PEGMME 2000, 100 mM sodium acetate, 10 mM CaCl2 with an 1/
2 concentration of 12 mg/ml.
Data collection
Details of data processing are shown in Table I.
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The stereochemical details of the final model and the crystallographic R-factors are given in Table I.
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Discussion |
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The issue of efficient preparation of recombinant complexes (dimeric and of higher order) is of vital importance in the post-genomic era of `combinatorial' crystallography of signal transduction pathways. The simplest possible solution is the reconstitution of the complex from individual components but this does not always work, particularly if the dissociated components are unstable. Co-expression, either using different but compatible vectors or the use of bi-cistronic constructs, may increase the yields by several orders of magnitude (Jonckheere et al., 1996; Halling et al., 1999
). It should be remembered, however, that co-expression using independent plasmids carries potential dangers. For example, if the copy numbers of the two plasmids are not balanced, the yield of the complex may be low (Tsao and Waugh, 1997
). Placing the genes under the control of one promoter ensures translation with similar yields. Bi-cistronic vectors of this type and containing two target genes each preceded by ribosome binding sites (RBS) and fused to affinity tags have been used in the past, e.g. for the preparation of nuclear receptor partners RAR and RXR (Li et al., 1997
) and in the analysis of the NF
B p50/p65 heterodimer (Chen et al., 1999
). However, these vectors either lacked tags or cleavage sites, making purification of the wild-type complex either difficult or impossible. In contrast, the vector construction described in this work provides more options with respect to efficient purification. First, we used two tags, markedly different with respect to molecular weight, i.e. MBP and His6. This allows one to use gel filtration as a fast and efficient means of separating different species of dimers, whilst still leaving the option of using affinity chromatography. We deliberately avoided the use of GST, which forms it own dimers and is likely to interfere with the interactions of the target proteins. Second, the use of highly specific and different proteolytic cleavage sites in the linker regions, i.e. Xa and rTEV, allowed for sequential removal of the tags to facilitate purification. These additional features could prove useful in other investigations.
Fortuitously, the pairing of the -subunits was highly temperature sensitive and we were able to use this phenomenon to increase the efficiency of heterodimer recovery. In other cases, such as the T-cell receptors and MHC-DR2, it was necessary to resort to enhancement of affinity by adding heterodimeric leucine zippers (Chang et al., 1994
; Kalandadze et al., 1996
). While this was not required for our purposes, it is conceivable to engineer such zippers immediately upstream of the target genes in our construct to ensure proper pairing. Finally, as pointed out by others (Chen et al., 1999
), the strategy may be extended to ternary and higher order complexes.
Regrettably, the crystal structure of the PAF-AH(Ib) catalytic heterodimer does not shed much light on the biological differences between the two -chains, nor does it reveal any potential mechanism for regulation by the ß-subunit. The two
-subunits are very similar, as expected from the relatively high amino acid similarity levels (Figure 4
). The only significant difference relates to the region between residues 53 and 61, which is found to be disordered in the
1-subunit in both the
1-homodimer and the heterodimer. Nevertheless, this region is clearly distinguishable in the electron density map of the
2-subunit (Figure 5
). It is not clear whether this difference has any biological relevance. A comparison of the C
- of the
-subunits coordinates (excluding the disordered region mentioned above) overlapped by least-squares procedure shows that in both crystal forms the
1 and
2 subunits are very similar. The only exceptions are residues 164 to 168 in
2, which are displaced when compared to their counterparts in
1. We attribute this to crystal packing effects. The structures of the active sites in the two subunits are remarkably similar and it is difficult to see why only
1 can be fully competent in the heterodimer. Perhaps the structure of the heterodimer complexed with a substrate analogue would help, but to date we were unable to succeed in the preparation of suitable crystals.
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The functional link between cytoskeletal regulation and PAF hydrolysis has never been defined. PAF is found in abundance in the central nervous system (Bito et al., 1992) and when generated in response to neurotransmitters or electrical stimuli (Sogos et al., 1990
), PAF acts as a synapse messenger, transcriptional inducer of the early-response genes c-fos, c-jun and zif-268 and a mediator in long-term potentiation (Squinto et al., 1990
; Morgan and Curran, 1991
; Bazan et al., 1993
; Kato et al., 1994
). PAF has also been implicated in brain damage caused by ischemia and seizures (Panetta et al., 1987
). It is not known, however, if PAF plays any direct role in neuronal migration during development, although there is some circumstantial evidence that it might. It has been reported that certain PAF-acetylhydrolase inhibitors, which do not bind to PAF receptors, inhibit migration of neurons in cultures (Adachi et al., 1997
). Expression patterns of the catalytic subunits are also consistent with a role played in neuronal migration (Albrecht et al., 1996
). Finally, mutations in the LIS1 protein, that are associated with the onset of lissencephaly, critically affect the protein's ability to interact with the catalytic dimer (Sweeney et al., 2000
).
Although the evidence in favor of physiological relevance of the association of LIS1 and the -subunits with PAF-AH activity appears very strong, it is not clear what role PAF plays in this phenomenon. Recent studies have shown that the
-subunits will hydrolyze substrates similar to PAF, but with different head groups (Manya et al., 1999
). Furthermore, it is now recognized that the
-subunit is a member of an old family of hydrolytic proteins, identified some years ago as a family of lipolytic enzymes (Brick et al., 1995
; Upton and Buckley, 1995
). One of its members, rhamnogalacturonan acetylhydrolase, had its crystal structure recently determined (Molgaard et al., 2000
) and shown to be very similar to that of the
-subunit. It is therefore conceivable that the natural substrate of the presumed brain PAF-AH could be an as yet unidentified molecule with an acetyl moiety attached via an ester bond.
The story became even more complicated with the discovery that the Drosophila genome contains an -subunit homologue which lacks catalytic activity (Sheffield et al., 2000
). So far this is the only known non-mammalian eukaryotic gene with homology to the
-subunit. The protein is co-expressed with the Drosophila LIS1 homologue during oogenesis and embryogenesis, consistent with a functional role during development. Is the catalytic function of the
-subunit separate from a role in developmental signal transduction? The question will have to be addressed as more data on the biology of the system are accumulated. We hope that our structural data including the study presented in this paper will ultimately help in conclusive elucidation of structurefunction relationships in this interesting protein complex.
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Notes |
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2 To whom correspondence should be addressed. E-mail: zsd4n{at}virginia.edu
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Acknowledgments |
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References |
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Received October 1, 2000; revised March 9, 2001; accepted March 19, 2001.