1 Experimentelle Kinderkardiologie, Deutsches Herzzentrum, Lazarettstrasse 36, D-80636 Munich, 3 GBF, 38124 Braunschweig, 4 MWG Biotech AG, 85560 Ebersberg, 5 EMBL, 69117 Heidelberg, Germany and 6 EBI, Hinxton, UK
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Abstract |
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Keywords: four-helix bundle/loop engineering/peptide presentation/topology
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Introduction |
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Here we present a protein engineering study in which we created a monomeric four-helix bundle starting from the Rop dimer. Our long-term goal is to create a small and stable molecule that can be used for in vitro and in vivo presentation studies of biologically active peptides.
Several successful topological reorganizations have been realized in different structural classes of proteins. They include circular permutations (Buchwalder et al., 1992; Zhang et al., 1993
; Ay et al., 1998
) and the transfer of loop modules (Hynes, 1989
). MacBeath et al. for example, redesigned the topology of chorismate mutase by directed evolution (MacBeath et al.,1998
). Finkelstein's group has ab initio designed a beta barrel protein with unusual topology, i.e. the strands are connected by loops in a way not seen before in proteins with known structure (Abdullaev et al., 1997
). These are all examples of more or less successful engineering and design projects. However, total ab initio protein design is still an interesting challenge (Bryson et al., 1995
; Walsh et al., 1999
).
Sander's group designed a monomeric four-helix bundle Rop (Sander, 1994). This bundle was designed to be left-handed and the success of the designed was confirmed by NMR (W.Eberle and C.Sander, personal communication). Regan's group (Predki and Regan, 1995
; Nagi and Regan, 1997
) have made an interesting right-handed monomer Rop design similar to the right-handed constructs that we present here.
Principally there exist four different ways in which a fully antiparallel monomeric Rop four-helix bundle topology can be organized when the three loops connect helices that are directly adjacent to each other. Figure 1 shows the loop engineering that is required to arrive at these four possibilities starting from the Rop dimer. It can be seen that three of the four possibilities require the design of only two new loops while one of the right-handed bundles can only be obtained by designing three new loops. Two of these bundles have a left-handed and two a right-handed super helical topology. The Rop wild-type four-helix bundle is left-handed.
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We constructed four left-handed and three right-handed Rop monomers. One of the right-handed constructs could not be isolated. The six successful constructs could be produced in milligram quantities and have been extensively characterized. As expected, our left-handed constructs are more stable than the right-handed ones. We therefore used the left-handed construct for the introduction of biologically interesting loops. In this stage it was our main goal to determine the boundaries within which we can move in this engineering project and we do not yet concentrate on specific applications. The results indicate that the Rop molecule is indeed a useful vehicle for the presentation of biologically interesting peptides. Preliminary studies show the applicability of our methodology to, for example, the presentation of inhibitor peptides to the HIV-1 proteinase.
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Materials and methods |
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The gene for pro-LM-Rop was kindly provided by S.C.E.Emery (Emery, 1990). We introduced the Pro59Asn mutation into LM-Rop in order to increase the stability and solubility. This mutation was introduced by a gapped-duplex single-strand mutagenesis method (Stanssens et al., 1989
) and the resultant pro-LM-Rop-Pro59Asn will be called LM-Rop throughout this study. For this mutagenesis we used the phagemid pMa/c 58 system (kindly provided by H.-J.Fritz) and PstI/EcoRI sites for insertion of the gene into this vector. The insertion variants were generated by ligation of synthetic NheI/BclI fragments into the NheI/BclI digested LM-ROP gene (all amino acid sequences are listed in Table I
). All oligonucleotide syntheses were performed on a Pharmacia gene assembler.
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Protein expression and purification
The monomer Rop genes were cloned into the pMAL-c fusion vector (New England Biolabs) via a PCR technique with a 21 bp non-mutagenic ATG(N)18 primer and the pMAL-c reverse sequencing primer for generation of a semi-blunt end/EcoRI fragment. The constructs were subcloned, sequenced and transformed. The proteins were expressed in the host strain TB 1 on a 2 l scale as C-terminal fusions of the maltose-binding protein (MBP-Rop) using a commercially available system (New England Biolabs) and following the manufacturer's protocols. After cell lysis in a 20 mM Tris buffer, pH 8.0 with a French Press and sonification (2x1 min, 50 mW, 4°C), the samples were centrifuged using a Sorvall SS-34 rotor (15 min at 4°C and 12 000 g). The crude cell extracts were subjected to anion-exchange chromatography (FPLC) at 4°C on a 40 ml Q-Sepharose FF column (Pharmacia) in 20 mM TrisHCl buffer, pH 8.0, and eluted with the same buffer with a 01 M NaCl gradient. The fusion protein was cleaved overnight at 20°C by 80 units of factor Xa protease (New England Biolabs) in elution buffer with 2 mM CaCl2. After cleavage, the NaCl concentration in the protein solution was reduced by ultrafiltration to 150 mM with a YM-10 membrane (Amicon) and addition of TrisHCl buffer at 4°C. For the cation-exchange chromatography of LM-Rop, the buffer was exchanged completely. LM-Rop eluate was loaded on a 20 ml Mono S column (Pharmacia) in 40 mM phosphate buffer, pH 6.4, containing 50 mM NaCl and eluted with this buffer with a 01 M NaCl gradient. The right-handed monomers and insertion variants were subjected, after the ultrafiltration, to anion-exchange chromatography on an 8 ml Mono Q column (Pharmacia) and eluted with a 01 M NaCl gradient in TrisHCl buffer. The monomer Rop fractions were combined and concentrated to 10 ml by ultrafiltration using a YM-5 membrane. In a final gel filtration step, the proteins were separated on a Superdex G 75 (26/60) column (Pharmacia) in 40 mM phosphate buffer containing 200 mM NaCl at a flow-rate of 0.5 ml/min. The protein fractions were analysed by 12% SDSPAGE according to the method of Schägger and von Jagow (1987) and stained with Coomassie Brilliant Blue. Eluate fractions were concentrated by ultrafiltration as described above. The identity of the Rop monomers was confirmed by Western blotting.
Analytical gel filtrations of partially enriched protein fractions were performed on a Superdex G 75 26/60 column under the buffer conditions described above at a flow-rate of 0.5 ml/min. A Pharmacia standard marker set and 0.5 mg each of BSA (67 kDa), ovalbumin (43 kDa), chymotrypsinogen (25 kDa) and RNase A (13.7 kDa) was used for molecular size determination.
Circular dichroism (CD) measurements in the 200260 nm range were routinely done with a JASCO J 600 spectropolarimeter, in a 0.5 or 1.0 mm quartz cuvette at a protein concentration of 0.1 mg/ml [except for HGHR-ROP (0.25 mg/ml) and ACHR-LM-Rop (0.71 mg/ml)] in 40 mM phosphate buffer, pH 6.4 (LM-Rop) or pH 7.6 (other LM-Rop and RM-Rop variants), containing 50 mM NaCl. The secondary structure contents were calculated using the CONTIN software (Provencher, 1982).
Loops were extracted from PDB files (Bernstein et al., 1977) using the WHAT IF loop search algorithm (Vriend, 1990
). This algorithm searches for loops that show a good overlap with the framework of the protein for three residues at either side of the insertion (i.e. the r.m.s. misfit for the six
-carbons is <1.0 Å). We introduced the additional constraint that the loop should be short and have the potential for tight packing between the residues in the loop and the helical framework of Rop. Obviously, the chance of finding the perfect loop by database searching only is very small and some adjustments were needed in all cases. The mutations required to convert the loops that were extracted from the database into the loops that were inserted in the Rop variants were predicted using a combination of WHAT IF's mutation prediction software and visual inspection. These adaptations were kept as conservative as possible.
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Results |
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The LM-Rop variant was created by connecting the loops in the order A1 A2 + B2 + B1. The two new loops (indicated by a +; bold face in Table I) were taken from the proLM-Rop variant designed by Sander's group (Emery, 1990
). Their design had a three-residue C-terminal extension AKG which they introduced to cap properly the fourth helix of the bundle. We kept this extension where appropriate.
The RM-Rop1 variant was created by connecting the loops in the order A1 + B1 B2 + A2. Visual inspection of the three-dimensional models indicated very little interaction between the A1 to B1 and B2 to A2 loops. Therefore, the A2 to B2 loop from the LM-Rop variant could also be used for the B2 to A2 connection in RM-Rop1 (remember that A2 and B2 have identical sequences so that the A2B2 and B2A2 connections are identical). The A1 to B1 loop was obtained using the WHAT IF loop search algorithm as described in the Materials and methods section. A well-fitting loop that would insert the five residues ESKRF was found in the PDB file 1RHD (Ploegman et al., 1978).
Previous studies indicated that the A2 to B2 loop in LM-Rop can easily be modified without great loss of stability. We exploited this fact by making three insertions in this six-residue loop leading to the variants HGHR-LM-Rop (16 residues; from the human growth hormone receptor), ACHR-LM-Rop (20 residues; from the nicotinic acetylcholine receptor) and GLOOP-LM-Rop (20 residues; the immunogenic G loop from lysozyme) (Table I). These loops were chosen solely because they were biologically `interesting'; and their N- and C-terminal residues must be close to each other because of cysteine bridges that connect residues at or near these loop termini.
We decided not to vary the B2 to B1 loop because this loop connects two helix ends that are terminal in the wild-type variant. We did not mutate the A1 to A2 and B1 to B2 loops because they are native loops and we wanted to minimize the differences between the monomeric constructs and the native dimer. The B2 to A2 loop in RM-Rop1 was not varied because this loop is identical with the A2 to B2 loop in LM-Rop and variation of this loop would not provide new information.
The RM-Rop2 and RM-Rop3 variants were constructed using the loop VESNGT from 1OVO (Papamokos et al., 1982) and the loop AGGDATE from 2B5C (Mathews et al., 1972
), respectively. Both loops were adapted manually by residue exchanges to their new environment (Table I
).
The maltose binding protein fusion protein expression yielded stable Rop monomers for all constructs except GLOOP-LM-Rop, which is stable but nicked, and RM-Rop3, which could not be isolated. The cleavage in the A2 to B2 loop of GLOOP-LM-Rop could be monitored during the purification. The typical yields from a 2 l fermentation were 25 mg. SDSPAGE analysis confirmed the homogeneity of the final products. All stable monomers were soluble at concentrations of 5 mg/ml.
Figure 2 shows the results of the Superdex gel filtrations of the Rop monomers. Table II
shows the apparent molecular masses calculated from the elution volumes. Each Rop protein eluted as a single, well-defined peak, and LM-Rop was determined immunologically in the fractions. The GLOOP-LM-Rop also eluted as a single peak, but a subsequent SDSPAGE indicated that cleavage had occurred in the A2 to B2 loop. The wild-type Rop variant (nominal molecular mass of the dimer: 14.3 kDa) displayed an apparent molecular mass of 1718 kDa in the gel filtrations of earlier workers (Lacatena et al., 1984
; Kokkinidis et al., 1993). Obviously, the rod-shaped molecular structure of Rop leads to unusual chromatographic behaviour. The LM-Rop molecule shows a slightly (
2 kDa) higher apparent molecular mass compared with wtRop. The apparent molecular masss of our right-handed monomers and insertion variants are increased by several kDa (Figure 2
, Table II
).
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Discussion |
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Our studies on the LM-Rop variants suggest that if the residues directly adjacent to the helix are carefully selected, the loop can tolerate a wide variety of loop insertions.
Predki and Regan designed a right-handed Rop monomer variant like we did, but they had to introduce a stabilizing Asp30Gly mutation, leading to an increase in the Tm of 13°C (Predki and Regan, 1995
). Despite this stabilization, their initially designed protein showed strong aggregation behaviour. Consequently, Regan's group varied the lengths of the two newly inserted loops. For the A1B1 loop they found that the six-residue GGGGTK loop (using GGGGTK where we use ESKAG; see Table I
) yielded a monomer Rop of higher stability (they measured the melting temperatures, helix content and molecular volumes) than A1B1 loops with five or seven residues. However, we found that the five-residue ESKAG A1B1 loop of RM-Rop1 yielded more compact structures with higher helix content than the six-residue SQSNGS A1B1 loop of RM-Rop2 or the seven-residue AGGDATK construct in the presumably highly unstable RM-Rop3. For the B2A2 loop, Predki and Regan found that the five-residue loop GGGGA (using GGGGA where we use KKNGQI with six residues; see Table I
) yielded a structure of higher stability than loops with more or fewer glycines. Our B2KKNGQIA2 helixloophelix structure module in RM-Rop has the same peptide sequence as the A2KKNGQIB2 structure in LM-Rop where we used this peptide and longer structures for loop insertion.
These differences once again confirm the importance of the actually used amino acids. Loop length surely plays an important role, but the loop length effects are supplemented by the interactions made by the loop residues.
It can be concluded that the influence of the loops on the Rop monomer secondary structure and the apparent molecular volume is small provided that the new loops are designed carefully. Further studies have to reveal if the observations made in this study and the studies by Regan's group regarding loop lengths are general rules or whether those loops for which these observations were made will also tolerate many more modifications once we have found the ideal residues. In summary, the Rop monomer can serve well as a vehicle for the presentation of bioactive peptides in an in vivo system. Preliminary studies, in which HIV-1 proteinase-inhibiting peptides derived from proteinase recognition sequences were inserted into the A2B2 loop context of an HGHR-LMR variant, seem to confirm this conclusion. We constructed seven new monomers by introduction of heptamer peptides into the loop with flexible three-residue linkers on each side. The linker sequences and the overall loop lengths were homologous with the HGHR-LMR. Upon expression in E.coli, five of the seven monomers were biologically stable.
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Notes |
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Acknowledgments |
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References |
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Received September 7, 2000; revised May 31, 2001; accepted June 18, 2001.