Structural Biology Laboratory, Salk Institute and Division of Biology, University of California at San Diego, San Diego, CA 92037, USA
1 To whom correspondence should be addressed. E-mail: choe{at}salk.edu
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Abstract |
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Keywords: integral membrane protein/maltose binding proteinmembrane protein crystallization/potassium channel
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Introduction |
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Few alternatives to the detergent dilemma for crystallization have been suggested (Landau and Rosenbusch, 1996; Ostermeier and Michel, 1997
; Faham and Bowie, 2002
). It has been argued that semi-polar organic solvents, able to accommodate both the hydrophobic and hydrophilic portions of a membrane protein, might provide one solution, but to date such procedures have rarely been successfully applied (Garavito et al., 1996
). More intriguing is the suggestion by Michel that increasing the ratio of hydrophilic to hydrophobic surface area by binding solubilizing antibodies to the target protein may improve the chances of successful crystallization (Hunte and Michel, 2002
). This technique has been successfully applied to cytochrome c oxidase (Ostermeier et al., 1995
), cytochrome bc1 complex (Hunte et al., 2000
) and more recently the potassium channels KcsA (Zhou et al., 2001
) and KvAP (Jiang et al., 2003
). However, this method does not abolish the need for detergent solubilization with its inherent limitations. Additionally, the expense and difficulty of raising the necessary monoclonal antibodies have severely restricted the utility of this protocol.
A novel possibility, given today's advances in recombinant techniques, knowledge concerning protein folding and the explosion of genomic data, is that one might be able to re-engineer a membrane protein into a soluble protein by strategic site-directed mutagenesis of its transmembrane domain, while retaining its native structure (Rees et al., 1989). The primary difficulty in such an approach is that usually an intimate knowledge of the protein's structure is a prerequisite for intelligently modifying its composition (Slovic et al., 2004
). Although this paradox would seem to present an insurmountable challenge towards using this approach to solve novel structures, thanks to the genomic era sufficient information may now be available to overcome this obstacle through systematic analysis of diverse homologous members within a protein family. Most critical is the successful identification of lipid-exposed residues on transmembrane helices. Theoretically such residues, while invariably hydrophobic, would display little conservation beyond this chemical property in comparison with neighboring residues that contribute to proteinprotein folding interactions and those inward-facing amino acids that may display a high degree of conservation of a specific chemical character, contributing directly to the function of the transmembrane domain (Senes et al., 2004
). This hypothesis suggests that the external, lipid-exposed amino acids can be elucidated by a careful analysis of the amino acid conservation pattern within transmembrane helices. Consistent with this approach, it has been demonstrated that manipulation of the lipid-facing residues of an integral membrane protein to polar and charged amino acids can be accomplished without significantly altering the overall structure of the targeted molecule (Chen and Gouaux, 1997
; Zhou and Bowie, 2000
) and that the lipid-facing residues of phospholamban can be modified to more polar alternatives without disrupting the pentameric helical assembly of this protein (Li et al., 2001
; Slovic et al., 2003
). More recently, computational design of a water-soluble analogue of a K+ channel of known structure has been accomplished (Slovic et al., 2004
). Satisfactory re-engineering not only abolishes the need for detergent solubilization, but also enhances the opportunity for success at both the expression and crystallization stages of a structural study.
A target for exploring such possibilities was selected in a short (104 amino acid) open reading frame from Archeaoglobus fulgidus (Klenk et al., 1997). This sequence, conserving without an exception the 16 residues of the potassium pore signature motif (Figure 1), putatively encodes a potassium-selective ion channel, KchAfu104. It is comparable to the viral channel Kcv, a 94 amino acid protein that has been shown to form a conducting, K+-selective pore in oocytes (Plugge et al., 2000
). KchAfu104 may be unique, however, as it C-terminus is surprisingly polar in contrast to all other known simplistic K+ channel homologues. Either this channel architecturally lacks a second transmembrane helix or, alternatively, has an unusually hydrophilic composition for this second transmembrane domain. As all potassium channel family members are thought to assemble into tetramers (Jan and Jan, 1997
), KchAfu104 provided an unparalleled opportunity for re-engineering. First, the high degree of selectivity of these channels for potassium over other monovalent cations (particularly Na+) of atomically similar radii demands that the selectivity filter be rigidly maintained at a nearly fixed radius (Doyle et al., 1998
). Further, as the source of this channel is a hyperthermophilic organism, such rigidity must be maintained even at extremely high temperatures. Together these criteria suggest that the constituent monomers are held together very tightly through proteinprotein interactions, allowing for some leeway in re-engineering, such that weakly destabilizing mutations may not readily denature the native complex. Further, the relatively small size of each monomer means that the necessary peptide chain can be assembled and manipulated entirely through a synthetically constructed template, allowing ultimate flexibility in redesigning the protein's genetic coding. Finally, the homotetrameric nature of the target implies that an appropriate modification at a specific site is in fact placed through four-fold symmetry around the molecule, thus significantly reducing the number of alterations that are necessary to affect solubilization. Here we present data that led to the successful solubilization of this small putative K+ channel of unknown structure.
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Materials and methods |
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To determine the appropriate residues for modification within this channel, a sequence alignment of all identifiable prokaryotic potassium channels available at the time was conducted with the aid of transmembrane prediction tools SOSUI (Hirokawa et al., 1998) and DAS (Cserzo et al., 1997
). Such analysis concludes that the protein-facing surface of the first, outer helix is composed of small or hydrophilic residues along the edges of the interface (Senes et al., 2004
), with large aromatic residues positioned down the center of the pore (Figure 2A and B). This result was reached independently of structural knowledge, but has subsequently been supported by several experimentally determined structures of potassium channels (Doyle et al., 1998
; Jiang et al., 2002
), in addition to scanning mutagenesis experiments conducted in yeast on the eukaryotic potassium channel, IRK (Minor et al., 1999
). Modeling of KchAfu104's outer transmembrane helix emphasizes the unique conservation pattern within this family of channels and highlights weakly conserved, hydrophobic residues that are appropriate targets for mutagenesis (Figure 2C).
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Synthetic genes for the expression of KchAfu104 were assembled using eight contiguously overlapping 60 base pair oligonucleotides, combined with PCR extension and amplification (Figure 1A). The oligonucleotides were designed such that the critical transmembrane helix could be altered by substitution of the third and fourth oligonucleotides within the synthetic template (Figure 1B). Additional changes were made utilizing site-directed mutagenesis protocols or by subcloning fusion domains, as appropriate. Codons were modified from the native sequence for optimal Escherichia coli expression when appropriate (Nakamura et al., 2000). All constructs were cloned into an octahistidine-tagged variant of pET-28 (Novagen) for N-terminally His-tagged expression. Escherichia coli BL-21 (DE3) cells were transformed with each plasmid, grown at 37°C to an optical density of 1.0 at 600 nm, induced with 0.1 mM IPTG and harvested 3 h after induction. Iterative rounds of mutagenesis or modification of the construct were employed followed by biochemical analysis of resulting recombinant protein by SDSPAGE and gel filtration on either a FPLC Superdex S-200 column (Pharmacia) or on a GFC-1300 analytical HPLC column (Supelco). For certain samples, analytical centrifugation was carried out on a Beckman (Palo Alto, CA) Optima-XLA instrument, run with three protein concentrations (1, 0.5 and 0.25 mg/ml) spun at two different speeds (20 000 and 14 000 r.p.m.) overnight in 8 M ultra-pure guanidinium hydrochloride (Sigma). These data were analyzed with the program Origin (adapted for the Optima-XLA by Beckman) and fitted as a single species. A summary of all constructs analyzed, and their nomenclature as discussed in this report, is provided in Figure 1B.
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Results |
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Wild-type KchAfu104, as with many membrane proteins, is not suited to study by traditional recombinant methods, as it does not accumulate to detectable levels when expressed in bacteria. In order to modify this protein's solubility while maintaining the integrity of its structure, a stepwise process of mutagenesis and analysis was utilized. Six sites towards the extracellular leaflet of the cell membrane were first altered, as the orientation of the transmembrane helix could be more definitively assigned in this region (Figures 1B and 2C). Both rational and combinatorial redesigns were employed to find a combination of changes that would not disrupt the protein's ability to fold. Hydrophilic residues (K, E/D, Q and S) with -helical propensity, and also the capacity to form stabilizing salt bridges through i to i + 3 interactions, were chosen to substitute for the hydrophobic lipid-facing residues of the helix. All of the constructs produced in the first round of re-engineering, as exemplified by construct KchAfu104-I, yielded large quantities of recombinant protein in insoluble inclusion bodies.
KchAfu104-I retains tetrameric stoichiometry in 8 M guanidinium hydrochloride (Gdm-HCl)
The rationally redesigned construct, KchAfu104-I, was selected for further study and inclusion body protein from this construct was solubilized in 8 M Gdm-HCl, purified and analyzed. Surprisingly, this protein retains substantial tetrameric structure even in this high concentration of denaturing chaotrope, as measured by both gel filtration chromatography (Figure 3A) and analytical ultracentrifugation (Figure 3B). Further experiments showed that the monomeric species assembles entirely into tetramers as Gdm-HCl concentration is lowered to 6.5 M, followed by aggregation and precipitation when the chaotrope concentration is further reduced (data not shown). These results imply that the channel assembly is extraordinarily stable and has been maintained through the first round of mutagenesis. This preliminary result supported further probing to improve the solubility of the construct.
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Mutagenesis of residues interfacing the cytoplasmic lipid monolayer was initially avoided owing to the less definitive assignment of pore-facing versus lipid-facing residues along this portion of the helix, especially along the stretch encoding AILIYA (Figure 1B). However, with the initial success of KchAfu104-I, assignment was extrapolated to this area with reasonable confidence and further changes were introduced (Figure 1B). These new constructs, KchAfu104-II, -III and -IV, resulted in proteins that, while still expressed in inclusion bodies, remained soluble upon complete removal of chaotrope by slow dialysis. Nevertheless, despite the first transmembrane helix being at this point highly hydrophilic, the protein remains aggregated.
KchAfu104-III and -
IV express as soluble proteins extractable from supernatant
Further modification of the channel was undertaken through deletion of the first 19 residues (Figure 1B). This stretch of 17 amino acids is predicted to form a highly amphipathic helix (Figure 2C), similar to the membrane peripheral slide helix also observed for KcsA (Cortes et al., 2001) and Kcv (Gazzarrini et al., 2004
) and may be a nucleus for aggregation in the re-engineered channel. These constructs, KchAfu104-
III and -
IV, produced recombinant proteins that could now be extracted from the lysed bacterial supernatant at significant quantities,
10 mg of protein per liter of culture (Figure 4A). However, gel filtration analysis still revealed these proteins to be mildly aggregated, unsuitable for further structural study. In an effort to try to stabilize the cytoplasmic face of the channel, a fusion construct linking the eukaryotic K+ channel tetramerization domain (T1) (Roosild et al., 2004
) in place of the native 19 N-terminal residues was made next. This chimera, T1-KchAfu104-
III, expresses only in inclusion bodies. Although it appears tetrameric by analytical gel filtration when refolded by gradual removal of chaotrope in very dilute solutions (<0.1 mg/ml), the T1 domain only marginally helps the re-engineered channel maintain a native, soluble assembly at higher protein concentrations. We hypothesized that a small hydrophobic patch remains capable of nucleating undesirable aggregation and precipitation.
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To convert this integral membrane channel into a well-behaved soluble protein, a fusion construct with maltose binding protein (MBP) in place of T1 was made, MBP-KchAfu104-IV (Figure 1B). MBP has been shown to improve the solubility of various proteins, putatively in part by enveloping insoluble or misfolded portions of fused proteins within its carbohydrate-binding pocket (Kapust and Waugh, 1999
). We hypothesized that MBP could seek out and cover the elusive sticky patch remaining on the re-engineered channel. This construct, with MBP linked to the N-terminus of the channel via a long flexible chain, produces soluble protein with no evidence of inclusion body formation. Further, the purified protein is no longer aggregated, appearing to exist in equilibrium between monomeric and tetrameric forms by gel filtration (Figure 4B). The tetrameric assembly of the protein is entirely dependent on the channel portion of the construct as co-purified free MBP, lacking the fusion partner owing to proteolysis within the flexible linker, is monomeric. Although we have not directly demonstrated its functional integrity after modification owing to the absence of appropriate assays, this result is consistent with maintenance of the oligomeric state predicted for KchAfu104 in its native tertiary structure as it exists in the cell membrane.
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Discussion |
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It has been demonstrated previously that lipid-facing residues are more tolerant to amino acid substitutions (Zhou and Bowie, 2000; Bowie, 2001
) and can be mutated to more hydrophilic amino acids without affecting the native structure of a protein (Chen and Gouaux, 1997
; Slovic et al., 2004
). Our results strongly support this conclusion. Certainly, site-directed mutagenesis of putative lipid-facing residues rapidly makes a targeted membrane protein incapable of membrane insertion and can substantially improve its water solubility. However, it is clear from our experience that solubility and monodispersity, prerequisites for the application of most structural characterization techniques, are goals of disparate difficulty. This is likely due to an inability to predict accurately enough all of the residues in need of alteration, as well as the precise, optimal nature of each change necessary, so as to create a completely uniform hydrophilic surface with no residual hydrophobic patches that can nucleate protein aggregation. Although a solution to this problem may eventually emerge from various innovative techniques currently under development that harness the power of natural selection and allow screening and selection of large molecular populations, here we show that MBP fusion is potentially capable of surmounting this obstacle. Being uniquely effective at promoting solubilization of fusion partners, it is likely that MBP is able to clamp over residual hydrophobic patches remaining after redesign and hence deter undesirable associations.
Our re-engineered channel protein was developed progressively based on biochemical analysis and characterization of multiple intermediates, each displaying gradual improvement in solubility. When flexibly linked to MBP, the resulting fusion is soluble in aqueous buffer as a stable tetrameric complex, suggesting that a substantial degree of structural similarity to its native precursor might also be present. This methodology may lead to new avenues for obtaining high-resolution information regarding this family of potassium-selective channels. Eventually it might also prove valuable for studies of other classes of integral membrane proteins, such as transporters and signal transducers that also have well-defined transmembrane core domains, suitable to similar manipulation.
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Acknowledgments |
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Received November 19, 2004; revised February 14, 2005; accepted February 15, 2005.
Edited by William DeGrado
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