Preparation and crystal structure of the recombinant {alpha}1/{alpha}2 catalytic heterodimer of bovine brain platelet-activating factor acetylhydrolase Ib

Peter J. Sheffield,1, Todd W.P. McMullen, Jia Li, Yew-Seng Ho, Sarah M. Garrard, Urszula Derewenda and Zygmunt S. Derewenda,2

Department of Molecular Physiology and Biological Physics, University of Virginia, Health Sciences Center, Charlottesville, VA 22906–0011, USA


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The intracellular form of mammalian platelet activating factor acetylhydrolase found in brain (PAF-AH Ib) is thought to play a critical role in control in neuronal migration during cortex development. This oligomeric complex consists of a homodimer of the 45 kDa (ß) LIS1 protein, the product of the causative gene for type I lissencephaly, and, depending on the developmental stage and species, one of three possible pairs of two homologous ~26 kDa {alpha}-subunits, which harbor all of the catalytic activity. The exact composition of this complex depends on the expression patterns of the {alpha}1 and {alpha}2 genes, exhibiting tissue specificity and developmental control. All three possible dimers ({alpha}1/{alpha}1, {alpha}1/{alpha}2 and {alpha}2/{alpha}2) were identified in tissues. The {alpha}1/{alpha}2 heterodimer is thought to play an important role in fetal brain. The structure of the {alpha}1/{alpha}1 homodimer was solved earlier in our laboratory at 1.7 Å. We report here the preparation of recombinant {alpha}1/{alpha}2 heterodimers using a specially constructed bi-cistronic expression vector. The approach may be useful in studies of other systems where pure heterodimers of recombinant proteins are required. The {alpha}1/{alpha}2 dimer has been crystallized and its structure was solved at 2.1 Å resolution by molecular replacement. These results set the stage for a detailed characterization of the PAF-AH Ib complex.

Keywords: bi-cistronic/crystal structure/expression vector/heterodimer/hydrolase


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The cerebral cortex, the seat of cognitive functions in mammals, forms from progenitor neurons, which migrate over large distances, up to 1000 times the length of the cell, in successive waves to create a six-layered structure. This process proceeds in such a manner that layers of neurons are laid out from the deepest to the outermost, with the younger generations of neurons migrating through the layers of older cells (McConnell, 1995Go). The underlying molecular mechanisms, the elucidation of which is vital for the understanding of many aspects of neurophysiology, are only beginning to be unraveled (Pearlman, et al., 1998Go).

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., 1993Go; Dobyns and Truwit, 1995Go). The causal gene for Miller–Dieker lissencephaly (a form of lissencephaly associated with dysmorphic facial appearance), known as LIS1, has been identified and characterized (Reiner et al., 1993Go, 1995Go; Mizuguchi et al., 1995Go). 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., 1993Go). 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., 1997Go, 1999Go; Morris et al., 1998Go). 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., 1994bGo). 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 {alpha}1 and {alpha}2 chains (Hattori et al., 1994aGo, 1995Go). 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., 1999Go). The relative catalytic activities depend on the composition of the dimer, so that the {alpha}2/{alpha}2 homodimer is significantly more active against PAF and AAGPE than the other two dimeric species, {alpha}1/{alpha}1 and {alpha}1/{alpha}2. Also, while both homodimers have comparable catalytic efficiency against PAF, in the heterodimer, only the {alpha}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., 1999Go).

The composition of the catalytic PAF-AH dimer appears to be tightly regulated during development: the {alpha}1 subunit and consequently the {alpha}1/{alpha}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., 1998Go). There is mounting evidence that this regulatory mechanism is indeed physiologically relevant and plays a role during cortical development (Albrecht et al., 1996Go; Sweeney et al., 2000Go). 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., 2000Go).

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 {alpha}1 homodimer at 1.7 Å resolution (Ho et al., 1997Go). 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 Ser–His–Asp and the mechanism of hydrolytic cleavage of the sn-2 ester bond is identical with the serine proteinases and neutral lipases (Derewenda, 1994Go). 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., 1999Go). 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., 2000Go).

In this paper we describe the preparation and structure determination (at 2.1 Å resolution) of the {alpha}1/{alpha}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 {alpha}-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.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Construction of the bi-cistronic plasmids for coexpression of {alpha} 1 and {alpha}2 chains

To obtain a bi-cistronic plasmid, the His6-{alpha}1 cDNA from pHIS:{alpha}1 was subcloned immediately downstream of the {alpha}2 cDNA in pMBP:{alpha}2 as an XbaI fragment. Excision of the {alpha}1 gene from pHIS:{alpha}1 as an XbaI fragment allowed the {alpha}1 cDNA to be isolated along with the ribosome binding site (RBS) from the pHIS vector. The resulting construct is shown in Figure 1aGo. This construct presented problems during purification (see Results). Therefore, the His6-{alpha}1 cDNA from this construct was transferred as a SalI fragment to pMal-C2::{alpha}2 to generate the construct shown in Figure 1bGo, in which MBP is fused to the {alpha}2 gene via a linker containing the Factor Xa site.



View larger version (38K):
[in this window]
[in a new window]
 
Fig. 1 (a) DNA arrangement of first bi-cistronic construct, containing two rTEV protease cleavage sites, and location of ribosome binding sites (rbs). (b) Schematic of second bi-cistronic construct containing a Factor Xa cleavage site between MBP-{alpha}2 and an rTEV cleavage site between the His6 tag and {alpha}1.

 
Expression of the bi-cistronic constructs in E.coli

The bi-cistronic plasmids were transformed into Escherichia coli strain XL1Blue (Stratagene). Expression was carried out in 1–2 l of Luria broth. Growth was initiated at 37°C until mid-log phase (OD600 nm 0.4–0.6), 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 Tris–HCl, 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-{alpha}1 homodimer and His6-{alpha}1/{alpha}2-MBP heterodimer were isolated from the MBP-{alpha}2 homodimer by Ni-NTA chromatography. The heterodimer was then purified from the {alpha}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 {alpha}1/{alpha}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 Tris–HCl, 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 Tris–HCl, 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, 1997Go).

Structure determination

The structure of {alpha}1/{alpha}2 heterodimer was solved by Molecular Replacement with AMoRe (Navaza, 1994Go) using data in the 15.0–4.0 Å resolution range. The {alpha}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 {alpha}2 were replaced. The final refinement using CNS (Brunger et al., 1998Go) converged with Rwork 21.8% and Rfree 27.7%. The quality of the model was checked using PROCHECK (Laskowski et al., 1993Go). The CCP4 program package was used for all other purposes (CCP4, 1994Go).


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Expression and purification of the heterodimer

Analysis of post-induction protein expression in E.coli by SDS–PAGE (Figure 2aGo) under denaturing conditions revealed two expected proteins bands: one corresponding to His6-{alpha}1 (~32 kDa) and the other to MBP-{alpha}2 (~68 kDa). The existence of the heterodimer in solution was confirmed by an initial Ni+-NTA agarose extraction, which isolated both His6-{alpha}1 and MBP-{alpha}2, implying that MBP-{alpha}2, which could not have been purified by Ni+-NTA extraction on its own, was in complex with His6-{alpha}1 (Figure 2bGo). Isolation of pure heterodimer should have been completed by passing the Ni+-NTA eluate through an amylose resin column to separate His6-{alpha}1 homodimer from MBP-{alpha}2/{alpha}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 {alpha}1 homodimer from the 105 kDa heterodimer, although resolution was less than perfect owing to the limited capability of the Superdex 75 column.



View larger version (77K):
[in this window]
[in a new window]
 
Fig. 2 (a) Coomassie Brilliant Blue-stained SDS–PAGE gels of {alpha}1/{alpha}2 heterodimer purification. M = markers; 1 = total cell protein uninduced; 2 = total cell protein induced; 3 = soluble protein fraction; 4 = flow-though from Ni+-NTA column; 5 = Ni+-NTA column elution with 250 mM imidazole. (b) Coomassie Brilliant Blue-stained SDS–PAGE gels of {alpha}1/{alpha}2 heterodimer purification. M = markers; 1 = Ni+-NTA column elution with 250 mM imidazole; 2 = heterodimer after treatment with Factor Xa; 3 = flow-though from Ni+-NTA column; 4 = second Ni+-NTA column elution with 250 mM imidazole.

 
The ratios of His6-{alpha}1 homodimer to His6-{alpha}1/{alpha}2-MBP heterodimer, as determined by size exclusion chromatography for expression runs performed at different temperatures, revealed that the post-induction expression temperature had a significant effect on heterodimer formation. At 37°C (initial experiments) the relative ratio of heterodimer to {alpha}1-homodimer, after Ni-NTA extraction, was 4:6. However, when the post-induction temperature was dropped to 30°C the ratio was 9:1 in favor of heterodimer (Figure 3Go). Purification was significantly improved by exploiting the temperature-dependent formation of heterodimer and the ability to isolate predominantly heterodimer after one round of Ni-NTA extraction.



View larger version (19K):
[in this window]
[in a new window]
 
Fig. 3. (A) Size exclusion elution profile showing the effect of temperature on the relative amounts of MBP-{alpha}2/{alpha}1-His6 heterodimer and His6-{alpha}1/{alpha}1-His6 homodimer after first Ni+-NTA extraction. (B) Elution profile showing homogeneity of MBP-{alpha}2/{alpha}1-His6 heterodimer after size exclusion chromatography to remove contaminating His6-{alpha}1/{alpha}1-His homodimer.

 
Removal of both tags by rTEV treatment produced another purification problem in that the 60 kDa heterodimer could not easily be isolated from the 45 kDa MBP tag owing to the insufficient binding affinity of the latter. This problem was eliminated by replacing the rTEV site between the MBP and {alpha}2 with a factor Xa site (Figure 1bGo). This allowed for the two tags to be removed sequentially, allowing isolation of the heterodimer from the MBP tag by a second round of Ni+-NTA agarose extraction before removal of the His6 tag by rTEV treatment. Pure heterodimer was isolated by size exclusion chromatography, which removed any remaining contaminating proteins (i.e. rTEV protease ~36 kDa, His6 tag ~2 kDa).

Crystallization

Crystals were obtained in 16–18% PEG 8000, 100 mM Tris–HCl pH 6.8, 10 mM CaCl2 and in 25–30% (NH4)2SO4, 100mM sodium acetate pH 6.4. Further trials were performed around these conditions and diffracting crystals were obtained in 17% PEG–MME 2000, 100 mM sodium acetate, 10 mM CaCl2 with an {alpha}1/{alpha}2 concentration of 12 mg/ml.

Data collection

Details of data processing are shown in Table IGo.


View this table:
[in this window]
[in a new window]
 
Table I. Data collection and refinement statistics
 
Structure determination and refinement

The stereochemical details of the final model and the crystallographic R-factors are given in Table IGo.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The preparation of the {alpha}1/{alpha}2 catalytic heterodimer of the brain PAF-AH (Ib) was essential for the crystallization of the intact recombinant PAF-AH (Ib) complex. However, this proved to be a challenge, since the individual {alpha}-subunits are expressed as tightly associated homodimers, which confer stability and catalytic competence on the protein (McMullen et al., 2000Go). However, our unpublished data suggested that the recombinant {alpha}1 and {alpha}2 homodimers could be dissociated to a degree at high salt concentration (>500 mM) and high pH (>=9). The high ionic strength appeared to have a stabilizing effect on the monomers, since attempts to isolate the monomers under normal conditions (50 mM salt and pH 7.0) resulted in either dimer reformation or precipitated protein. In an attempt to generate the heterodimer by monomer exchange within a mixture of homodimers, differently tagged {alpha} homodimers (His6-{alpha}1 and a MBP-{alpha}2) were mixed in equimolar amounts and dissociated into monomers at high pH and high salt concentration. Re-association of the monomers was achieved by removal of the salt and re-adjusting the pH to 7.0 by sequential dialysis. This process produced, as expected, three distinct soluble species and by successive rounds of affinity chromatography it was possible to isolate the His6-{alpha}1/{alpha}2-MBP heterodimer from the homodimers. However, the efficiency of heterodimer formation was very low and it was clear that it was not an efficient enough method to produce the heterodimer in amounts required for crystallization.

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., 1996Go; Halling et al., 1999Go). 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, 1997Go). 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., 1997Go) and in the analysis of the NF{Phi}B p50/p65 heterodimer (Chen et al., 1999Go). 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 {alpha}-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., 1994Go; Kalandadze et al., 1996Go). 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., 1999Go), 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 {alpha}-chains, nor does it reveal any potential mechanism for regulation by the ß-subunit. The two {alpha}-subunits are very similar, as expected from the relatively high amino acid similarity levels (Figure 4Go). The only significant difference relates to the region between residues 53 and 61, which is found to be disordered in the {alpha}1-subunit in both the {alpha}1-homodimer and the heterodimer. Nevertheless, this region is clearly distinguishable in the electron density map of the {alpha}2-subunit (Figure 5Go). It is not clear whether this difference has any biological relevance. A comparison of the C{alpha}- of the {alpha}-subunits coordinates (excluding the disordered region mentioned above) overlapped by least-squares procedure shows that in both crystal forms the {alpha}1 and {alpha}2 subunits are very similar. The only exceptions are residues 164 to 168 in {alpha}2, which are displaced when compared to their counterparts in {alpha}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 {alpha}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.



View larger version (30K):
[in this window]
[in a new window]
 
Fig. 4. Stereo view of the C{alpha} trace of {alpha}1/{alpha}2 heterodimer (colored black) superimposed with {alpha}1/{alpha}1 homodimer (colored gray).

 


View larger version (36K):
[in this window]
[in a new window]
 
Fig. 5. Electron density map of region from residues 54–61 in {alpha}2 subunit. This region is disordered in the {alpha}1 subunit.

 
The natural history and physiology of the catalytic subunits of the PAF-AH(Ib) complex are far from clear. The oligomeric complex was originally identified for its ability to hydrolyze PAF (Hattori et al., 1993Go). It was soon realized that the catalytic activity resides in the two smaller subunits (Hattori et al., 1994aGo, 1995Go) and that, surprisingly, the larger subunit is the product of the previously identified causal gene for Miller–Dieker lissencephaly, LIS1 (Hattori et al., 1994bGo). LIS1 is a protein apparently involved in protein–protein interaction cascades, possibly regulating the cytoskeleton, although its physiologically relevant partners have not been identified with certainty (Morris et al., 1998Go; Reiner and Sapir, 1998Go; Sapir et al., 1999Go).

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., 1992Go) and when generated in response to neurotransmitters or electrical stimuli (Sogos et al., 1990Go), 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., 1990Go; Morgan and Curran, 1991Go; Bazan et al., 1993Go; Kato et al., 1994Go). PAF has also been implicated in brain damage caused by ischemia and seizures (Panetta et al., 1987Go). 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., 1997Go). Expression patterns of the catalytic subunits are also consistent with a role played in neuronal migration (Albrecht et al., 1996Go). 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., 2000Go).

Although the evidence in favor of physiological relevance of the association of LIS1 and the {alpha}-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 {alpha}-subunits will hydrolyze substrates similar to PAF, but with different head groups (Manya et al., 1999Go). Furthermore, it is now recognized that the {alpha}-subunit is a member of an old family of hydrolytic proteins, identified some years ago as a family of lipolytic enzymes (Brick et al., 1995Go; Upton and Buckley, 1995Go). One of its members, rhamnogalacturonan acetylhydrolase, had its crystal structure recently determined (Molgaard et al., 2000Go) and shown to be very similar to that of the {alpha}-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 {alpha}-subunit homologue which lacks catalytic activity (Sheffield et al., 2000Go). So far this is the only known non-mammalian eukaryotic gene with homology to the {alpha}-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 {alpha}-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 structure–function relationships in this interesting protein complex.



View larger version (21K):
[in this window]
[in a new window]
 
Fig. 6. Root mean square deviation of the main-chain atoms for each of the subunit in {alpha}1/{alpha}2 heterodimer and {alpha}1/{alpha}1 homodimer.

 

    Notes
 
1 Present address: Department of Pharmacology, Markey Center for Cell Signaling, University of Virginia, Charlottesville, VA 22908, USA Back

2 To whom correspondence should be addressed. E-mail: zsd4n{at}virginia.edu Back


    Acknowledgments
 
Use of the Argonne National Laboratory Structural Biology Center beamlines at the Advanced Photon Source was supported by the US Department of Energy, Office of Biological and Environmental Research, under Contract No. W-31-109-ENG-38. The study was funded by NIH, grant NS36267. We thank Professor Keizo Inoue (University of Tokyo) for many useful discussions. Coordinates have been deposited with the Protein Data Bank, accession code 1FXW.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Adachi,T., Aoki,J., Manya,H., Asou,H., Arai,H. and Inoue,K. (1997) Neurosci. Lett., 235, 133–136.[ISI][Medline]

Albrecht,U., Abu-Issa,R., Ratz,B., Hattori,M., Aoki,J., Arai,H., Inoue,K. and Eichele,G. (1996) Dev. Biol., 180, 579–593.[ISI][Medline]

Bazan,H.E., Tao,Y. and Bazan,N.G. (1993) Proc. Natl Acad. Sci. USA, 90, 8678–8682.[Abstract/Free Full Text]

Bito,H., Nakamura,M., Honda,Z., Izumi,T., Iwatsubo,T., Seyama,Y., Ogura,A., Kudo,Y. and Shimizu,T. (1992) Neuron, 9, 285–294.[ISI][Medline]

Brick,D.J., Brumlik,M.J., Buckley,J.T., Cao,J.X., Davies,P.C., Misra,S., Tranbarger,T.J. and Upton,C. (1995) FEBS Lett., 377, 475–480.[ISI][Medline]

Brunger,A.T. et al. (1998) Acta Crystallogr., D54, 905–921.[ISI]

CCP4 (1994) Acta Crystallogr., D50, 760–763.[ISI]

Chang,H.C. et al. (1994) Proc. Natl Acad. Sci. USA, 91, 11408–11412.[Abstract/Free Full Text]

Chen,F.E., Kempiak,S., Huang,D.B., Phelps,C. and Ghosh,G. (1999) Protein Eng., 12, 423–428.[Abstract/Free Full Text]

Derewenda,Z.S. (1994) Adv. Protein Chem., 45, 1–52.[ISI][Medline]

Dobyns,W.B. and Truwit,C.L. (1995) Neuropediatrics, 26, 132–147.[ISI][Medline]

Dobyns,W.B., Reiner,O., Carrozzo,R. and Ledbetter,D.H. (1993) J. Am. Med. Assoc., 270, 2838–2842.[Abstract]

Halling,B.P., Yuhas,D.A., Eldridge,R.R., Gilbey,S.N., Deutsch,V.A. and Herron,J.D. (1999) Protein Express. Purif., 17, 373–386.[ISI][Medline]

Hattori,M., Arai,H. and Inoue,K. (1993) J. Biol. Chem., 268, 18748–18753.[Abstract/Free Full Text]

Hattori,M., Adachi,H., Tsujimoto,M., Arai,H. and Inoue,K. (1994a) J. Biol. Chem., 269, 23150–23155.[Abstract/Free Full Text]

Hattori,M., Adachi,H., Tsujimoto,M., Arai,H. and Inoue,K. (1994b) Nature, 370, 216–218.[ISI][Medline]

Hattori,M., Adachi,H., Aoki,J., Tsujimoto,M., Arai,K. and Inoue,K. (1995) J. Biol. Chem., 270, 31345–31352.[Abstract/Free Full Text]

Ho,Y.S. et al. (1997) Nature, 384, 89–93.

Ho,Y.S., Sheffield,P.J., Masuyama,J., Arai,H., Li,J., Aoki,J., Inoue,K., Derewenda,U. and Derewenda,Z.S. (1999) Protein Eng., 12, 693–700.[Abstract/Free Full Text]

Jonckheere,H., De Vreese,K., Debyser,Z., Vandekerckhove,J., Balzarini,J., Desmyter,J., De Clercq,E. and Anne,J. (1996) J. Virol. Methods, 61, 113–125.[ISI][Medline]

Kalandadze,A., Galleno,M., Foncerrada,L., Strominger,J.L. and Wucherpfennig,K.W. (1996) J. Biol. Chem., 271, 20156–20162.[Abstract/Free Full Text]

Kato,K., Clark,G.D., Bazan,N.G. and Zorumski,C.F. (1994) Nature, 367, 175–179.[ISI][Medline]

Laskowski,R.A., McArthur,M.W., Moss,D.S. and Thornton,J.M. (1993) J. Appl. Crystallogr., 26, 282–291.

Li,C., Schwabe,J.W., Banayo,E. and Evans,R.M. (1997) Proc. Natl Acad. Sci. USA, 94, 2278–2283.[Abstract/Free Full Text]

Manya,H., Aoki,J., Watanabe,M., Adachi,T., Asou,H., Inoue,Y., Arai,H. and Inoue,K. (1998) J. Biol. Chem., 273, 18567–18572.[Abstract/Free Full Text]

Manya,H., Aoki,J., Kato,H., Ishii,J., Hino,S., Arai,H. and Inoue,K. (1999) J. Biol. Chem., 274, 31827–31832.[Abstract/Free Full Text]

McConnell,S.K. (1995) Neuron, 15, 761–768.[ISI][Medline]

McMullen,T.W., Li,J., Sheffield,P.J., Aoki,J., Martin,T.W., Arai,H., Inoue,K. and Derewenda,Z.S. (2000) Protein Eng., 13, 865–871.[Abstract/Free Full Text]

Mizuguchi,M., Takashima,S., Kakita,A., Yamada,M. and Ikeda,K. (1995) Am. J. Pathol., 147, 1142–1151.[Abstract]

Molgaard,A., Kauppinen,S. and Larsen,S. (2000) Struct. Fold. Des., 8, 373–383.[ISI][Medline]

Morgan,J.L. and Curran,T. (1991) Annu. Rev. Neurosci., 14, 421–451.[ISI][Medline]

Morris,S.M., Albrecht,U., Reiner,O., Eichele,G. and Yu-Lee,L.Y. (1998) Curr. Biol., 8, 603–606.[ISI][Medline]

Navaza,J. (1994) Acta Crystallogr., A50, 157–163.[ISI]

Otwinowski,Z. and Minor,W. (1997) Methods Enzymol., A276, 307–326.[ISI]

Panetta,T., Marcheselli,V.L., Braquet,P., Spinnewyn,B. and Bazan,N.G. (1987) Biochem. Biophys. Res. Commun., 149, 580–587.[ISI][Medline]

Pearlman,A.L., Faust,P.L., Hatten,M.E. and Brunstrom,J.E. (1998) Curr. Opin. Neurobiol., 8, 45–54.[ISI][Medline]

Reiner,O. and Sapir,T. (1998) Int. J. Mol. Med., 1, 849–853.[ISI][Medline]

Reiner,O., Carrozzon,R., Shen,Y., Wehnert,M., Faustinella,F., Dobyns,W.B., Caskey,C.T. and Ledbetter,D.H. (1993) Nature, 364, 717–721.[ISI][Medline]

Reiner,O. et al. (1995) J. Neurosci., 15, 3730–3738.[Abstract]

Sapir,T., Elbaum,M. and Reiner,O. (1997) EMBO J., 16, 6977–6984.[Abstract/Free Full Text]

Sapir,T., Cahana,A., Seger,R., Nekhai,S. and Reiner,O. (1999) Eur. J. Biochem., 265, 181–188.[Abstract/Free Full Text]

Sheffield,P.J., Garrard,S., Caspi,M., Aoki,J., Arai,H., Derewenda,U., Inoue,K., Suter,B., Reiner,O. and Derewenda,Z.S. (2000) Proteins: Struct. Funct. Genet., 39, 1–8.[ISI][Medline]

Sogos,V., Bussolino,F., Pilia,E., Torelli,S. and Gremo,F. (1990) J. Neurosci. Res., 27, 706–711.[ISI][Medline]

Squinto,S.P., Braquet,P., Block,A.L. and Bazan,N.G. (1990) J. Mol. Neurosci., 2, 79–84.[ISI][Medline]

Sweeney,K.J., Clark,G.D., Prokscha,A., Dobyns,W.B. and Eichele,G. (2000) Mech. Dev., 92, 263–271.[ISI][Medline]

Tsao,K.L. and Waugh,D.S. (1997) Protein Express. Purif., 11, 233–240.[ISI][Medline]

Upton,C. and Buckley,J.T. (1995) Trends Biochem. Sci., 20, 178–179.[ISI][Medline]

Received October 1, 2000; revised March 9, 2001; accepted March 19, 2001.





This Article
Abstract
FREE Full Text (PDF)
Alert me when this article is cited
Alert me if a correction is posted
Services
Email this article to a friend
Similar articles in this journal
Similar articles in ISI Web of Science
Similar articles in PubMed
Alert me to new issues of the journal
Add to My Personal Archive
Download to citation manager
Search for citing articles in:
ISI Web of Science (4)
Request Permissions
Google Scholar
Articles by Sheffield, P. J.
Articles by Derewenda, Z. S.
PubMed
PubMed Citation
Articles by Sheffield, P. J.
Articles by Derewenda, Z. S.