1 Department of Immunotechnology, Lund University, P.O. Box 7031, SE-220 07 Lund, Sweden and 3 Crystallography Program, Oklahoma Medical Research Foundation, 825 NE 13th Street, Oklahoma City, OK 73104, USA
2 To whom correspondence should be addressed. E-mail: christer.wingren{at}immun.lth.se
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Abstract |
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Keywords: crystal engineering/directed crystallization/protein crystallization/structural proteomics/triad-hypothesis
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Introduction |
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Here, we address the task of generating protein crystals by designing the propensity to crystallize into the target molecules (DArcy, 1994; Carugo and Argos, 1997
). To this end, rational surface mutagenesis will be used to target intermolecular proteinprotein crystal packing contacts that can improve macromolecular crystallization (Lawson et al., 1991
; DArcy, 1994
; Longenecker et al., 2001
). Our approach is based on our recent triad-hypothesis, which proposes that proteins with ß-structures can be directed toward specific crystal packing interactions that promote crystallization (Edmundson and Borrebaeck, 1998
; Edmundson et al., 1998
, 1999
). In these studies, the analyses of a limited number of antibody structures indicated that the main driving force in their crystal packing was the formation of extended ß-pleated sheets across symmetry-related molecules. These sheets were stabilized by hydrogen bonding between specific sequences, packing motifs, located on the outermost ß-strands of adjacent molecules. Within the motifs of 56 residues, cross-molecule hydrogen bonds were formed between main-chain atoms of on average three pairs of residues (hence the name triads). When secured to a pleated sheet network, the ß-strands containing the motifs pulled the rest of the molecules with them to produce pre-crystalline, three-dimensional aggregates. These aggregates may be considered directed forms of crystal nucleation. Such highly ordered nuclei can then work independently of or in concert with shaped-based complementarity to produce stable crystal lattices (Ely et al., 1978
; Fan et al., 1992
; Faber et al., 1998
).
The aim of this study was to explore and exploit the triad-hypothesis. First, we performed crystal packing analyses of 182 model proteins from 21 different superfamilies with crystallographically defined ß-structures. Notably, database investigations are frequently used to support hypotheses emerging from three-dimensional studies of macromolecules (e.g. Chothia et al., 1989; Shirai et al., 1999
; Kumar et al., 2000
). The model proteins were selected to provide (i) a large set (62) of small, well-defined proteins with similar structures allowing the molecular features of the triad-hypothesis to be explored and evaluated in detail, and (ii) a broad set (120) of structures allowing the general phenomena of the triad-hypothesis to be evaluated. Next, the feasibility of the triad-hypothesis was tested by applying protein engineering and crystallization trials to six model scFv antibody proteins. Our results provided strong support for our triad-hypothesis. By combining the triad-hypothesis with protein engineering and crystallization trials, we have taken the first steps towards a rational protocol for crystallizing proteins by surface mutagenesis.
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Materials and methods |
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Samples of DNA encoding -scFvs against fluorescein isothiocyanate (FITC: clone FITC8), mucine-1 (clone SMUC159) and cholera toxin (clone CT17) were kindly provided by BioInvent Therapeutic (Lund, Sweden). All were derived from the n-CoDeR library (Söderlind et al., 2000
). DNA samples for
-scFvs against cytomegalovirus (clones ITC88-AE11, ITC88-F3C and ITC88-D3) were generously supplied by Dr M.Ohlin (Department of Immunotechnology, Lund University, Sweden).
Engineering of -packing motif
The packing motif was introduced by overlap-extension PCR using the sequences P1for 5'-TACTACTATTGCCAGCATTGC-3' and P2rew 5'-TTCAGATCCTCTTCTGA-GATG-3' as external primers and P3for 5'-CCATCCTCCCTGTCTTTGTCT-3' and P4rew 5'-CAAAGACAGGGAGGATGGAGACTG-3' as internal primers. Assembled gene fragments were purified by agarose gel electrophoresis and using a QIAquick Gel Extraction Kit (Qiagen, Hilden, Germany), digested with NotI (New England Biolabs, Beverly, MA) and XhoI (Boehringer Mannheim, Mannheim, Germany). They were then ligated into the NotI/XhoI sites in pPICZaA (Invitrogen, Groningen, The Netherlands) and transformed into RbCl-competent Top10F' Escherichia coli cells. Positive clones were linearized by PmeI (New England Biolabs) and transformed into electrocompetent P.pastoris cells.
Engineering of -packing motif
To generate a free N-terminus in VL, the constructs were first modified from VHlinkerVL to VLlinkerVH by overlap-extension PCR using the sequences P5for 5'-TCAGG-CGGAGGTGGATCCGGCGGTGGCGGATCGGAGGTGCAGCTGTTGGAGTCT-3' and P6rew 5'-GCCTTTAGCGTCAG-ACTGTAGGCGGCCGCTTTATCATCATCATCTTTATAAT-CACCGCTGCTCACGGTGACCAGTGT-3' as VH primers and P7for 5'-GGCCGATTCATTAATGCAGCGGCCCAGCCGGCCATGGCGCAGTCTGTGCTGACTCAGCCA-3' and P8rew 5'-ACCGCCGGATCCACCTCCGCCTGAACCGCCTCCACCTAGGACCGTCAGCTTGGTTCC-3' as VL primers. The packing motifs were introduced into these new constructs by another round of overlap-extension PCR using the sequences P9for 5'-GGCCGATTCATTAATGCAGC-3' and P10rew 5'-GTAAAACGACGGCCAGT-3' as external primers and P11for 5'-ACCCTCAGCGTCTGGGACCCCCGGGCAGAGCATC-ACCGTCTCTTGTTCT-3' (SMUC159, CT17), P12for 5'-AC-CCTCAGCGTCTGGGACCCCCGGGCAGAGCATCACCG-TCTCTTGCACT-3' (FITC8) and P13rew 5'-CCGGGGGT-CCCAGACGCTGAGGGTGGCTGAGTCAGCGCAGTCTGCGCCT-3' as internal primers. Assembled gene fragments were purified by agarose gel electrophoresis and using a QIAquick Gel Extraction Kit and digested with NotI and SfiI. They were then ligated into the SfiI/EagI-digested pFAB5c. His vector and transformed into RbCl-competent Top10F' E.coli cells.
Genetic analysis
DNA sequencing was performed using a Big Dye Terminator Kit (PE Biosystems, Warrington, UK). The reactions were analyzed with the ABI377 automated sequencing equipment (Applied Biosystems, Foster City, CA, USA) and the results were evaluated with Mac vector 6.5.3 (Oxford Molecular Group, Oxford, UK).
Protein expression
-scFvs were expressed in 10 ml of BMMY medium with 0.5% methanol. The cells were incubated at 30°C for 2 days with vigorous shaking. After centrifugation, the supernates were collected and stored at 4°C.
-scFvs were expressed in 10 ml of 2xYT with 0.5 mM isopropyl-ß-D-thiogalactopyranoside. After the cells had been grown overnight at 30°C, soluble scFv were retrieved by purification of the periplasmic fraction and subsequently stored at 4°C.
The levels of expressed proteins were assessed by 15% SDSPAGE, western blot analysis and dot-blot analysis. Briefly, samples were applied in serial dilutions. The SDSPAGE gels were developed by Coomassie Brilliant Blue and/or silver staining. The western blots were processed by standard procedures using anti-FLAG M2 monoclonal antibody (Sigma, St. Louis, MO) and horseradish peroxidase-conjugated rabbit anti-mouse Ig antibodies (Dako, Glostrup, Denmark). The color reactions were developed with an ECL substrate kit (Amersham-Pharmacia Biotech, Solna, Sweden). The dot-blots were processed and developed in a similar manner using either anti-FLAG M2 monoclonal antibody or anti-his monoclonal antibody (Qiagen, Valencia, CA) as primary reagents. Spot density was quantified in a GS-710 densitometer (Bio-Rad, Hercules, CA).
Antigen-specific ELISA
Immunoreactivities of native and triad-mutated scFvs were assessed and compared by antigen-specific ELISA. Briefly, the antigen was coated on to 96-well ELISA microtiter plates (Costar, Corning, NY). Samples were added to the wells as serial dilutions and bound antibodies were detected colorimetrically by the sequential addition of anti-FLAG M2 monoclonal antibody and horseradish peroxidase-conjugated rabbit anti-mouse Ig antibodies. o-Phenylenediamine was used as the enzyme chromogen and the absorbance was read at 490 nm.
Crystallization of antibodies
Samples of -ITC88-D3,
-ITC88-D3-triad,
-CT17,
-SMUC159 and
-FITC8 were produced as described above. The
-scFvs were isolated from crude supernates by a two-step ion-exchange chromatographic procedure on SP Sepharose, followed by size-exclusion chromatography on Superdex 75 (Amersham-Pharmacia Biotech). The
-scFvs were purified from the periplasmic fraction by His-tag affinity chromatography on Ni-NTA.
-FITC8 was further purified by affinity chromatography on FITCBSASepharose. The purity and homogeneity of the samples were assessed by the means of SDSPAGE, analytical size-exclusion chromatography (Rainin Hydroporec-5-SEC; Varian, Walnut Creek, CA) and dynamic light scattering (DynaPro-99; kindly provided by Dr U.Alkner, Astra-Zeneca, Lund, Sweden). The scFvs were concentrated to 1050 mg/ml (Microcon, 10K; Millipore, Bedford, MA). Crystallization trials were performed at 24°C by vapor diffusion, using mainly sitting drops. Crystal screen 1, crystal screen 2 and crystal screen cryo (50 conditions each) (Hampton Research, Laguna Niguel, CA) and/or a 24-well crystallization footprint screen designed for antibodies (Stura et al., 1992
) were used.
Structure modeling
The Web Antibody Modelling (WAM) procedure (http://antibody.bath.ac.uk) was used to generate homology models for the six native scFvs and their triad-mutated counterparts.
Crystal packing analyses
Crystal packing analyses were performed using the programs SwissPDBviewer (Guex and Peitsch, 1997) and O (Jones et al., 1991
).
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Results |
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To explore the triad-hypothesis, we performed crystal packing analyses of 62 ß-structures available in the Protein Data Bank (PDB) (Tables IIII
). The selected models, collectively referred to as variable-domain proteins (VDPs), included scFv and Fv antibody fragments, as well as variable domains from BenceJones proteins, each carrying either
- or
-light chain isotype. In 42 of 62 (68%) VDP structures, cross-molecular ß-strand pairing was found to be a prominent feature for crystallization (Tables I
III
).
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In Figure 1a, this type of crystal packing is shown for a
-Fv. As for all
-VDPs, the triad-interactions resulted in the formation of a dimer, which constituted the smallest crystal packing unit. Notably, the Fv 1BVK, that was bound to its protein antigen lysozyme, also proved capable of adopting these favorable crystal packing interactions between two VL domains (Figure 1b
).
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Exceptions to the triad-hypothesis were identified by analyzing the four -VDPs with the P8SSLSA13 motif that deviated from the proposed crystal packing pattern (Table I
). First, the N-terminal strand of VL of the 1BWW Fv contained two residues not present in the other structures. These additional residues made contact with neighboring molecules that created a 7 Å gap between the designated packing motifs, thereby disabling any ordered close packing interactions. Second, positioning of the C-terminal segment of the 1BVL Fv prevented the triad-interactions, either directly through steric interference or indirectly by crystal contacts made with VL in an adjacent molecule. Third, the protruding CDR-H3 loop in the 1DQL Fv blocked any VLVL triad-interactions. Fourth, potential triad-interactions were disabled by a subtle conformational change in the packing motif around residue P8 in the 1DVF Fv.
Further, we found three variants of the P8SSLSA13 motif, PSSLSV, PSSLAM and PATLSL, that also could act as packing motifs for -VDPs (Table I
). In a group of five examples, four unliganded variants and one complexed with p-nitrophenol, all followed the triad-hypothesis. To assess the natural occurrence of these packing motifs, the sequences of all potentially functional murine and human germline V
-exons were examined (Tomlinson et al., 1998
; Lefranc et al., 1999
; Ruiz et al., 2000
). While PSSLSA was expressed in both murine and human (at a frequency of 28.3% in human) proteins, PSSLSV and PSSLAM were present only in mice. PATLSL was restricted to humans at a frequency of 8.4%.
Designs of packing motifs
The structural requirements for functional packing motifs were defined by comparing the primary and tertiary structures of all 15 groups of -VDPs, as well as by modeling of the motif interactions in groups 515 (Table I
). In functional motifs, P8 and L11 were conserved, while S9, S10 and S12 were semi-conserved. A13 was subject to a wider range of substitutions, but only small uncharged residues were tolerated. Sequence variations in positions 812 resulted in subtle, but significant, conformational changes that affected the triad-interactions. For example, replacements of S9 and/or S12 with proline disabled any triad-interactions through disallowed clashes of juxtaposed prolines (Table I
). Further, structural analysis implied that residue 17 was also important, although it was not an integral part of the assigned packing motif (Table I
). Residues with long side-chains in position 17 (e.g. Glu, Gln and Asn) could sterically interfere with the triad-interactions. By identifying key features of the primary and tertiary structures of functional packing motifs, we have defined a structural framework for rational design of packing motifs involving
-VDPs.
Alternative triad-interactions of -VDPs
The crystal packing features of the 35 -VDPs lacking the four sequence motifs discussed above are summarized in Table II
. We found that 60% (21 of 35) of these
-VDPs displayed alternative triad-interactions (Table II
), again demonstrating the importance of cross-molecular ß-strand pairing for crystallization. Three
-VDPs interacted in triad-like fashion, but utilized different packing motifs (Table II
). In one of the cases, the crystal packing motifs were provided by the variable domains of the antigen, a T-cell receptor. Further, 18
-VDPs displayed protein-specific triad-like interactions. In these examples, pairing of ß-strands or ß-strands and loops were prominent features, but the interactions typically involved only one interchain hydrogen bond and no general motifs or patterns were discernible. Among the remaining 14
-VDPs, the crystal packing was primarily dependent on shape complementarity alone.
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Of the eight -VDPs analyzed, two displayed triad-interactions, four participated in protein-specific triad-like interactions and two relied upon shape complementarity for crystallization (Table III
). Compared with the
-VDPs, the
-VDPs utilized different packing motifs in VL to generate triad-interactions (cf. Tables I
and III
). The BenceJones
-light chain dimer 1JVK used two packing motifs in VL consisting of residues PCA1TALT5 in the N-terminal segment (motif 1) and S17ITVS21 in the second ß-strand (designated 42) (motif 2) (Figure 2a
). Cross-molecular, antiparallel hydrogen bonds were formed between motifs 1 and 2 located on symmetry-related molecules. This process continued ad infinitum to produce a packing ribbon of indefinite length in the crystal lattice (Figure 2a
).
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The remaining seven -VDPs carried other sequences at positions 15 and 1721 and none of them participated in such triad-interactions (Table III
). Several lacked the N-terminal Gln or its PCA derivative and all exhibited major variations in the structures of their N-terminal segments (Table III
). Other steric complications may be attributed to residues with long side chains at position 17, e.g. R17 in the 2RHE
-VL structure (Table III
).
The latter used packing motifs of yet a different design in VL to establish triad-interactions (Figure 2c). A packing dimer was generated through antiparallel pairing of a packing motif consisting of residues A44PKLL48 in ß-strand C'. Reciprocal cross-molecular main-chain hydrogen bonds were formed between P45 and L47. All other
-VDPs differed from 2RHE in the sequence of positions 44 to 48 and did not participate in such triad-interactions (Table III
). Moreover, large variations were observed in the conformations of these segments, mainly because of the flexible loop preceding the C' ß-strand. The 2RHE packing motif is likely to be restricted to free VL units, since VLVH association will reduce the accessibility of the motif.
To examine the natural occurrence of the three -packing motifs, we examined the sequences of all potentially functional human germline V
-exons (Tomlinson et al., 1998
). The PCA1TALT5 and S17ITVS21 sequences were not found in any germline sequence. In contrast, the A44PKLL48 sequence was expressed with a high frequency (29.7%).
Expansion of the triad-hypothesis to other superfamilies
Taken together, we found 21 VDPs that carried putative packing motifs, of which 17 (81%) conformed to patterns predicted by the triad-hypothesis (Tables IIII
). In order to examine the prevalence (not the frequency) of ß-structures in general to pack according to the triad-hypothesis, additional crystal packing analysis of 120 ß-structures, widely distributed between two classes, four folds and 21 superfamilies were performed (Table IV
). In general, five randomly selected structures within each family/superfamily were analyzed.
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In Figure 3, representative examples of crystal packing patterns for proteins belonging to the retinol-binding protein-like family (lipocalin superfamily) (Figure 3a
), fatty acid-binding protein-like family (lipocalin superfamily) (Figure 3b
) and the small Kunitz-type inhibitors and BPTI-like toxin family (BPTI-like superfamily) (Figure 3c
) are shown. The retinol-binding protein ß-lactoglobulin formed a packing dimer through antiparallel pairing of packing motifs consisting of residues H146IRLSF151 in ß-strand 10 (Figure 3a
). Cross-molecular hydrogen bonds were formed between the main-chain atoms of H146, R148 and S150. The cellular retinol-binding protein II (fatty acid-binding protein-like family) used two packing motifs consisting of residues G67VEF70 in ß-strand 5 (motif 1) and T87WEG90 in ß-strand 6 (motif 2) to produce a packing dimer (Figure 3b
). Antiparallel hydrogen bonds were formed between motifs 1 and 2 located on symmetry-related molecules involving the main-chain atoms of G67, E69 and W88. Figure 3c
illustrates the crystal packing of the ß-protein precursor (BPTI-like superfamily) forming deleterious amylofibrils in Alzheimers disease. A packing ribbon that continued ad infinitum was initiated via interactions between two antiparallel tripeptide segments, S6EQ8, with ß-strand characteristics. Hydrogen bonds were formed between main-chain atoms of S6 and Q8. Next, this dimer interacted with a second dimer through parallel pairing of packing motifs consisting of residues A16MISR20 on ß-strand 1. Interchain cross-molecular hydrogen bonds were formed between main-chain atoms of residues M17 and S19.
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Feasibility of the triad-hypothesis
In order to evaluate experimentally the feasibility of the triad-hypothesis, protein engineering and crystallization trials were applied to six model scFv antibody proteins. The results showed that suitable packing motifs could be introduced into three - and three
-scFvs by site-directed mutagenesis without affecting their functional and structural integrity (Table V
). Two mutations, P8ATLSA13 to P8SSLSA13, were introduced into the
-molecules, while five substitutions were made in the
-scFvs: Q1SVLT5 and R17VTIS21 to Q1TALT5 and S17ITVS21. As judged by SDSPAGE, western blot and dot-blot analyses, the mutated scFvs were expressed as molecular constructs of the correct size (~25 kDa) and at levels similar to those of the native molecules (Table V
). Furthermore, antigen-specific ELISA showed that the triad-mutants retained the immunoreactivity of the native proteins (Table V
).
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Crystallization trials were initiated for four native scFvs (-FITC8,
-CT17,
-SMUC159 and
-ITC88-D3) and one triad-mutated variant (
-ITC88-D3-triad). To date, 174 crystallization conditions, set up at two different protein concentrations (10 or 30 mg/ml), have failed to produce crystals of
-FITC8,
-CT17 and
-SMUC159. These three scFvs are all devoid of known packing motifs. Moreover, their VL sequences contain R17, which may be incompatible with the proposed
-triad interactions (Table III
). More importantly, the crystallization behavior of ITC88-D3-triad was found to be significantly different from that of its native counterpart in a 24-well crystallization footprint screen. Most strikingly, large crystals (0.3x0.3x0.2 mm) were obtained for ITC88-D3-triad in 22.5% polyethylene glycol 10 000 at pH 8.5, that failed to produce crystals of the native scFv.
Taken together, the results provided strong support for our triad-hypothesis and we have taken the first step towards a rational protocol for crystallizing proteins by surface mutagenesis.
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Discussion |
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The concept of crystal engineering has been hampered by the lack of rational approaches (Longenecker et al., 2001) and incomplete understanding of proteinprotein crystal packing interactions (DArcy, 1994
; Carugo and Argos, 1997
). Protein crystallization occurs at the expense of entropy, including the conformational entropy of initially exposed surface residues which become ordered during the formation of crystal contacts. Therefore, it has been proposed that protein crystallization could be designed by creating epitopes (more) favorable for the formation of crystal contacts with little or neglible loss of conformational entropy (Longenecker et al., 2001
). The packing motifs identified in this study, which were composed of mainly small uncharged non-polar and polar residues (Tables I
III
), may thus be thermodynamically well suited for interactions within the crystal lattices. Other factors (e.g. removal/alterations of steric hindrance, hydration shell, etc.) may also contribute to the crystallization process, but irrespective of rationale, the frequency of potential motifs participating in crystal packing interactions is striking (Tables I
III
).
However, it has previously been argued that most crystal packing contacts are non-specific, opportunistic and possibly randomized (Janin and Rodier, 1995; Carugo and Argos, 1997
; Janin, 1997
). In one earlier study, in which a protein was crystallized in a variety of space groups, it was concluded that nearly the entire protein surface could be involved in crystal packing contacts (Crosio et al., 1992
). In contrast, the highly ordered triad-interactions were displayed by a large set of proteins (Tables I
IV
) crystallized in a wide variety of space groups and over an extensive range of crystallization conditions (data not shown). Thus, the triad-hypothesis could provide us with the means to design specific proteinprotein crystal packing interactions.
Crystal engineering projects have traditionally emphasized packing motifs tailored for individual proteins (Lawson et al., 1991; DArcy, 1994
; Longenecker et al., 2001
). Our triad-hypothesis could provide a platform for designing specific crystal packing contacts in entire groups of molecules with ß-structures. For example, all
-VDPs analyzed could be assigned to one of only four sequence variants of a packing motif, while the
-VDPs were confined to one of three motifs (Tables I
III
). With the VL domains, however, the structures and exact locations of the motifs (Tables I
III
) varied with the framework 1 substitutions (residues 120) in different subgroups of
- and
-chains. Participation of VH in triad-type packing (Harris et al., 1998
) was too infrequent to be divided into specific categories (Tables II
and III
).
Packing patterns incompatible with the triad-hypothesis fell into three categories (Tables I and III
): (i) non-specific and/or steric interference by side chains in the N- and C-terminal regions of VL; (ii) protruding loop structures; and (iii) conformational changes in the motifs. Additional data are required before these pitfalls can be predicted and circumvented. However, the observation that 81% of the VDPs that carried the putative packing motifs actually displayed triad-interactions (Tables I
III
) demonstrated the potential of the triad-hypothesis.
The observations that packing motifs could be introduced into six model scFvs without affecting their structural or functional integrities (Table IV) are in line with the facts that the motifs are (i) located spatially remote from the active site, and (ii) composed of sequences typical of naturally occurring ß-pleated sheets. Therefore, the antigen-binding site and the packing motif can be manipulated independently without forfeiture of performance levels. Nevertheless, it is important to choose the most optimal time for introducing the packing motifs. When working with antibody or other protein libraries (Söderlind et al., 2000
), the motif should be included into the original design of the library before any selection has been made for individual functional proteins. Such a strategy would allow the protein library to be based on a master framework that promotes high expression of fully folded, functional proteins with increased propensities to crystallize.
Our crystal engineering approach can be used independently of or in concert with traditional methods, to crystallize proteins in general or to target, for example, troublesome proteins that have resisted crystallization. The methodology can be implemented in different ways. First, it can be applied in a direct manner by introducing the prescribed packing motif(s) into the target protein. Second, the method can be applied in an indirect manner by adding a triad-protein (i) as a fusion partner or (ii) as a specific ligand. Previously, non-triad proteins, such as protein Z and gluthathione-S-transferase, have been successfully used as fusion partners to produce crystals of higher quality (Kuge et al., 1997; Byrne et al., 2000
). In this study, we have identified single VL domains as the smallest units suitable for use as potential triad-fusion partners (Tables I
and III
). However, the recent design of a stable monomeric, three-stranded, antiparallel ß-sheet (Kortemme et al., 1998
) indicates that triad-units smaller than VL domains could be the building blocks of the future. Further, the general approach to add a specific ligand that binds tightly to the target protein to generate (better) crystals has also previously been successfully used (Stura et al., 1992
; Hunte et al., 2000
). Interestingly, antibodies have frequently been used as the ligands in such experiments. Thus, adopting the triad-hypothesis and using antibodies containing packing motifs as specific ligands for the target proteins may thus promote the crystallization of targeted proteins even further.
In conclusion, our results have provided strong support for our triad-hypothesis, implying that the propensity to crystallize can be engineered into target proteins by simply replacing a short segment of their ß-strands with cassettes of suitable packing motifs. We have outlined a rational protocol for crystallizing proteins by surface mutagenesis. To establish the triad-hypothesis fully, we obviously need to determine the crystal packing features for a group of proteins before and after engineering of packing motifs. This work is under way. Ultimately, these insights could pave the way for the development of novel technologies in structural biology, particularly in the crystallization and X-ray crystallographic analysis of proteins with ß-structures.
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Acknowledgments |
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Received January 9, 2002; revised February 28, 2003; accepted March 7, 2003.