Designing proteins to crystallize through ß-strand pairing

Christer Wingren1,2, Allen B. Edmundson3 and Carl A.K. Borrebaeck1

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


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Inherent difficulties in growing protein crystals are major concerns within structural biology and particularly in structural proteomics. Here, we describe a novel approach of engineering target proteins by surface mutagenesis to increase the odds of crystallizing the molecules. To this end, we have exploited our recent triad-hypothesis using proteins with crystallographically defined ß-structures as the principal models. Crystal packing analyses of 182 protein structures belonging to 21 different superfamilies implied that the propensities to crystallize could be engineered into target proteins by replacing short segments, 5–6 residues, of their ß-strands with ‘cassettes’ of suitable packing motifs. These packing motifs will generate specific crystal packing interactions that promote crystallization. Key features of the primary and tertiary structures of such packing motifs have been identified for immunoglobulins. Further, packing motifs have been engineered successfully into six model antibodies without disturbing their capabilities to be produced, their immunoreactivity and their overall structure. Preliminary crystallization analyses have also been performed. Taken together, the procedures outline a rational protocol for crystallizing proteins by surface mutagenesis. The importance of these findings is discussed in relation to the crystallization of proteins in general.

Keywords: crystal engineering/directed crystallization/protein crystallization/structural proteomics/triad-hypothesis


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Structural proteomics have taken structural biology into a new and exciting era and aim to produce a three-dimensional structure for every protein encoded in all completed genomes (Burley et al., 1999Go; Skolnick et al., 2000Go). Ultimately, this enormous effort will provide the scientific community with atomic details of the molecular machines that drive the cellular processes, as well as key insights into biological function (Domingues et al., 2000Go; Skolnick et al., 2000Go). Important advances to meet this ambitious effort have been made within X-ray crystallography (Stevens, 2000Go), currently the prime method for generating structural data for proteins. For example, new procedures have emerged for collecting X-ray diffraction data with micron-sized crystals (Cusack et al., 1998Go; Nava, 1999Go). Introduction of robotic technology has allowed automation, miniaturization and parallelization of crystallization trials (Stevens, 2000Go). In spite of the rapid progress, a major concern remains the unpredictable and sometimes insurmountable crystallization step (Gaasterland, 1998Go; Burley et al., 1999Go). In fact, we still only have a fragmentary understanding of the principles governing protein crystallization (Durbin and Feher, 1996Go), but key factors have proved to be the purity and homogeneity of the proteins under study (McPherson, 1999Go). To date, the general procedure for crystallizing a protein includes many trial-and-error steps, often involving crystallization screens dominated by empirical conditions known to enhance crystallization (Stura et al., 1992Go; Ducruix and Giege, 1999Go; McPherson, 1999Go). New theories and technologies to increase the probabilities for crystallizing target molecules and enabling directed crystallization would thus be of lasting importance.

Here, we address the task of generating protein crystals by designing the propensity to crystallize into the target molecules (D’Arcy, 1994Go; Carugo and Argos, 1997Go). To this end, rational surface mutagenesis will be used to target intermolecular protein–protein crystal packing contacts that can improve macromolecular crystallization (Lawson et al., 1991Go; D’Arcy, 1994Go; Longenecker et al., 2001Go). 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, 1998Go; Edmundson et al., 1998Go, 1999Go). 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 5–6 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., 1978Go; Fan et al., 1992Go; Faber et al., 1998Go).

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., 1989Go; Shirai et al., 1999Go; Kumar et al., 2000Go). 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.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Antibodies

Samples of DNA encoding {lambda}-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., 2000Go). DNA samples for {kappa}-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 {kappa}-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 {lambda}-packing motif

To generate a free N-terminus in VL, the constructs were first modified from VH–linker–VL to VL–linker–VH 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

{kappa}-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.

{lambda}-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% SDS–PAGE, western blot analysis and dot-blot analysis. Briefly, samples were applied in serial dilutions. The SDS–PAGE 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 {kappa}-ITC88-D3, {kappa}-ITC88-D3-triad, {lambda}-CT17, {lambda}-SMUC159 and {lambda}-FITC8 were produced as described above. The {kappa}-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 {lambda}-scFvs were purified from the periplasmic fraction by His-tag affinity chromatography on Ni-NTA. {lambda}-FITC8 was further purified by affinity chromatography on FITC–BSA–Sepharose. The purity and homogeneity of the samples were assessed by the means of SDS–PAGE, 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 10–50 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., 1992Go) 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, 1997Go) and O (Jones et al., 1991Go).


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Exploring the triad-hypothesis

To explore the triad-hypothesis, we performed crystal packing analyses of 62 ß-structures available in the Protein Data Bank (PDB) (Tables IGo–IIIGo). The selected models, collectively referred to as variable-domain proteins (VDPs), included scFv and Fv antibody fragments, as well as variable domains from Bence–Jones proteins, each carrying either {kappa}- or {lambda}-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 IGo–IIIGo).


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Table I. Frequency of {kappa}-VDPs listed in the Protein Data Bank (PDB) that pack according to the triad-hypothesis, using residues 8–13 in VL ß-strand 4–1 as packing motif. Although not an integral part of the packing motif, residue 17 is identified because it may interfere with the triad-interactions

 

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Table III. Frequency of {lambda}-VDPs listed in the PDB that pack according to the triad-hypothesis, using VL residues 1–5 in the N-terminal segment (motif 1) and residues 17–21 in ß-strand 4–2 in VL (motif 2) as packing motifs

 
Fifteen {kappa}-VDPs conformed to the pattern predicted by the triad-hypothesis and formed extended, antiparallel ß-pleated sheets between neighboring molecules (Table IGo). In all 15 examples, adjacent molecules interacted exclusively via hydrogen bond formation between pairs of packing motifs consisting of residues 8–13 in the outermost ß-strands (designated 4–1) in the variable domain of the light chain (VL). This special VL segment with a sequence of PSSLSA was also proposed as a potential packing motif when the triad-hypothesis was formulated (Edmundson et al., 1998Go). Indeed, 10 of 14 (71%) VDPs carrying this particular sequence motif followed the triad-hypothesis (Table IGo).

In Figure 1aGo, this type of crystal packing is shown for a {kappa}-Fv. As for all {kappa}-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 1bGo).



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Fig. 1. Crystal packing of {kappa}-VDPs that conformed to the triad-hypothesis. (a) The {kappa}-Fv (PDB code 1FVC) packed as a dimer. The VL domain is colored cyan, the VH domain pink, the packing motif red (residues P8SSLSA13) and the cross-molecule hydrogen bonds yellow. (b) The {kappa}-Fv (PDB code 1BVK) bound to the protein antigen lysozyme packed as a dimer. The VL domain is colored cyan, the VH domain pink, the antigen white, the packing motif red (residues P8SSLSA13) and the cross-molecule hydrogen bonds yellow. (c) Close-up view of the packing motif interaction in (a). The interchain hydrogen bonds are colored yellow. Figures 1Go–3Go were prepared using SwissPdbViewer (Guex and Peitsch, 1997Go) and WebLab Viewer (MSI, San Diego, CA).

 
A close-up view of the antiparallel triad-interactions is presented in Figure 1cGo for two adjacent {kappa}-Fv carrying the P8SSLSA13 motif. Hydrogen bonds were formed between the main-chain atoms of residues S9, S10 and S12, i.e. the packing triad. In general, the number of interchain hydrogen bonds ranged from two to four, depending on the exact orientation of the motifs. In the most favorable orientation, the backbone carbonyl group and amide group of S12 (motif 1) formed hydrogen bonds with the backbone amide group of S9 and carbonyl group of S10 (motif 2) and vice versa. The side-chain residues of the interacting motifs were partially exposed and accessible and those of S10 and S12 strengthened the triad-interactions by forming two additional intermolecular hydrogen bonds. Thus, this packing motif, composed of small non-polar and polar residues, provided the main driving force in the crystal packing.

Exceptions to the triad-hypothesis were identified by analyzing the four {kappa}-VDPs with the P8SSLSA13 motif that deviated from the proposed crystal packing pattern (Table IGo). 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 VL–VL 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 {kappa}-VDPs (Table IGo). 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{kappa}-exons were examined (Tomlinson et al., 1998Go; Lefranc et al., 1999Go; Ruiz et al., 2000Go). 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 {kappa}-VDPs, as well as by modeling of the motif interactions in groups 5–15 (Table IGo). 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 8–12 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 IGo). Further, structural analysis implied that residue 17 was also important, although it was not an integral part of the assigned packing motif (Table IGo). 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 {kappa}-VDPs.

Alternative triad-interactions of {kappa}-VDPs

The crystal packing features of the 35 {kappa}-VDPs lacking the four sequence motifs discussed above are summarized in Table IIGo. We found that 60% (21 of 35) of these {kappa}-VDPs displayed alternative triad-interactions (Table IIGo), again demonstrating the importance of cross-molecular ß-strand pairing for crystallization. Three {kappa}-VDPs interacted in triad-like fashion, but utilized different packing motifs (Table IIGo). In one of the cases, the crystal packing motifs were provided by the variable domains of the antigen, a T-cell receptor. Further, 18 {kappa}-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 {kappa}-VDPs, the crystal packing was primarily dependent on shape complementarity alone.


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Table II. Crystal packing features of the 35 {kappa}-VDPs (groups 5–15 in Table IGo) that carried other sequences at VL positions 8–13 in ß-strand 4–1
 
Packing motifs in {lambda}-VDPs

Of the eight {lambda}-VDPs analyzed, two displayed triad-interactions, four participated in protein-specific triad-like interactions and two relied upon shape complementarity for crystallization (Table IIIGo). Compared with the {kappa}-VDPs, the {lambda}-VDPs utilized different packing motifs in VL to generate triad-interactions (cf. Tables IGo and IIIGo). The Bence–Jones {lambda}-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 4–2) (motif 2) (Figure 2aGo). 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 2aGo).



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Fig. 2. Crystal packing of {lambda}-VDPs that conformed to the triad-hypothesis. (a) The {lambda}-light chain dimer (PDB code 1JVK) packed as ribbon of indefinite length. The VL domain 1 is colored white, the VL domain 2 pink, the packing motif 1 red (residues PCA1TALT5), the packing motif 2 green (residues S17ITVS21), the cross-molecule hydrogen bonds yellow. (b) Close-up view of the packing motif interaction in (a). The interchain hydrogen bonds are colored yellow. (c) The {lambda}-VL domain (PDB code 2RHE) packed as a dimer. The VL domain 1 is colored white, the VL domain 2 cyan, the packing motif red (residues A44PKLL48) and the cross-molecule hydrogen bonds yellow.

 
A close-up view of the triad-interactions is shown in Figure 2bGo. It should be noted that the N-terminal Gln residue had spontaneously cyclized to form pyrrolidonecarboxylic acid (PCA). Two cross-molecular hydrogen bonds were formed between PCA1 and T19 and between A3 and S17. Only one VL domain of each 1JVK dimer was capable of participating in the triad-interactions (Figure 2aGo), because the orientation of the second VL unit shielded its N-terminal segment from adjacent molecules. Interestingly, significant structural differences were observed between the N-terminal segments of the two VL units of each 1JVK dimer, thus indicating that a conformational change of this segment must have occurred to allow triad-interactions.

The remaining seven {lambda}-VDPs carried other sequences at positions 1–5 and 17–21 and none of them participated in such triad-interactions (Table IIIGo). Several lacked the N-terminal Gln or its PCA derivative and all exhibited major variations in the structures of their N-terminal segments (Table IIIGo). Other steric complications may be attributed to residues with long side chains at position 17, e.g. R17 in the 2RHE {lambda}-VL structure (Table IIIGo).

The latter used packing motifs of yet a different design in VL to establish triad-interactions (Figure 2cGo). 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 {lambda}-VDPs differed from 2RHE in the sequence of positions 44 to 48 and did not participate in such triad-interactions (Table IIIGo). 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 VL–VH association will reduce the accessibility of the motif.

To examine the natural occurrence of the three {lambda}-packing motifs, we examined the sequences of all potentially functional human germline V{lambda}-exons (Tomlinson et al., 1998Go). 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 IGo–IIIGo). 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 IVGo). In general, five randomly selected structures within each family/superfamily were analyzed.


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Table IV. Prevalence of ß-structures belonging to different families and superfamilies listed in the PDB to pack according to the triad-hypothesis. The proteins are classified (numbered) according to SCOP (Murzin et al., 1995Go). The crystal packing features of, on average, five randomly selected structures per family/superfamily were analyzed
 
The results showed that numerous ß-structures belonging to different classes, folds, superfamilies and families were capable of displaying triad-interactions (Table IVGo), thus illustrating the general phenomena of the triad-hypothesis. In the first class, denoted all ß-proteins, two of three folds analyzed were found to display triad-interactions (Table IVGo). While the {gamma}-crystallin-like fold proteins analyzed failed to exhibit triad-patterns, 11 of 16 (69%) superfamilies within the immunoglobulin-like ß-sandwich fold and two of two within the lipocalin fold conformed well. Noteworthy, five of five families within the immunoglobulin superfamily displayed triad-patterns. In the second class, denoted small proteins, structures within the small Kunitz-type inhibitors and BPTI-like toxins family also were found to exhibit triad-packing behavior.

In Figure 3Go, representative examples of crystal packing patterns for proteins belonging to the retinol-binding protein-like family (lipocalin superfamily) (Figure 3aGo), fatty acid-binding protein-like family (lipocalin superfamily) (Figure 3bGo) and the small Kunitz-type inhibitors and BPTI-like toxin family (BPTI-like superfamily) (Figure 3cGo) 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 3aGo). 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 3bGo). 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 3cGo illustrates the crystal packing of the ß-protein precursor (BPTI-like superfamily) forming deleterious amylofibrils in Alzheimer’s 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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Fig. 3. Crystal packing of ß-structures belonging to superfamilies other than the immunoglobulins that conformed to the triad-hypothesis. (a) ß-Lactoglobulin (PDB code 1B0O), belonging to the retinol binding protein-like family within the lipocalin superfamily, packed as a dimer. Molecule 1 is colored pink, molecule 2 cyan, the packing motif red (residues H146IRLSF151) and the cross-molecule hydrogen bonds yellow. (b) The cellular retinol-binding protein II (PDB code 1OPA), belonging to the fatty acid binding protein-like family within the lipocalin superfamily, packed as a dimer. Molecule 1 is colored pink, molecule 2 white, the packing motif 1 red (residues G67VEF70), the packing motif 2 green (residues T87WEG90) and the cross-molecule hydrogen bonds yellow. (c) The Alzheimer’s amyloid ß-protein precursor (PDB code 1AAP), belonging to the small Kunitz-type inhibitors and BPTI-like toxin family within the BPTI-like superfamily, packed as ribbon of indefinite length. Molecules 1 and 3 are colored pink, molecule 2 white, the packing motif 1 green (residues S6EQ8), the packing motif 2 red (residues A16MISR20) and the cross-molecule hydrogen bonds yellow.

 
In summary, these observations expanded the generality of the triad-hypothesis and indicated the general applicability of the triad crystallization methodology.

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 {kappa}- and three {lambda}-scFvs by site-directed mutagenesis without affecting their functional and structural integrity (Table VGo). Two mutations, P8ATLSA13 to P8SSLSA13, were introduced into the {kappa}-molecules, while five substitutions were made in the {lambda}-scFvs: Q1SVLT5 and R17VTIS21 to Q1TALT5 and S17ITVS21. As judged by SDS–PAGE, 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 VGo). Furthermore, antigen-specific ELISA showed that the triad-mutants retained the immunoreactivity of the native proteins (Table VGo).


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Table V. Comparison of triad-mutated vs native scFv antibodies with respect to their ability to be expressed, immunreactivity, and overall structure. Of the six model scFvs, three carried {kappa}-light chain isotype and three carried {lambda}-light chain isotype. Suitable packing motifs ({kappa}-P8ATLS13 -> P8SSLS13; {lambda}-Q1SVLT5 -> Q1TALT5, R17VTIS21 -> S17ITVS21) were introduced into VL by directed mutagenesis
 
Homology models of the six native scFvs and their triad-mutated counterparts were generated and compared (Table VGo). As expected, the folds were verified to be structurally most similar to antibody molecules according to the Dali program (Holm and Sanders, 1993Go); the best matches giving a Z-score of ~21.0 and an r.m.s. deviation for C{alpha} rigid-body superimposition of ~0.7 Å. In all six models, only minor structural differences were detected between the native and triad-mutated scFvs (Table VGo). These differences were concentrated in three CDRs, L1, L3 and/or H3. However, these changes were more likely a consequence of the modeling than the introduced triad-mutations.

Crystallization trials were initiated for four native scFvs ({lambda}-FITC8, {lambda}-CT17, {lambda}-SMUC159 and {kappa}-ITC88-D3) and one triad-mutated variant ({kappa}-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 {lambda}-FITC8, {lambda}-CT17 and {lambda}-SMUC159. These three scFvs are all devoid of known packing motifs. Moreover, their VL sequences contain R17, which may be incompatible with the proposed {lambda}-triad interactions (Table IIIGo). 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.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
This paper describes a new way of engineering target proteins to increase the odds of crystallizing the molecules: in effect to permit directed crystallization. The challenge to generate protein crystals has in general been addressed by the development of new crystallization techniques, supplemented by ‘footprint screens’ covering established crystallization conditions (Stura et al., 1992Go; Ducruix and Giege, 1999Go; McPherson, 1999Go; Alvarado et al., 2001Go). However, few initiatives have been devoted to the crystallization process itself, particularly in those aspects concerned with designing propensities to crystallize into target proteins (D’Arcy, 1994Go; Carugo and Argos, 1997Go). Some examples have been reported in which protein–protein contacts have been engineered to generate crystals or to improve crystal quality (Lawson et al., 1991Go; D’Arcy, 1994Go; Longenecker et al., 2001Go). In these cases, protein-specific mutations were introduced into mainly those regions responsible for the intermolecular contacts in the crystal lattice. Instead of engineering the propensity to crystallize into a protein, it has been more common to target the properties of the proteins empirically found often to interfere with the crystallization process, e.g. removal of carbohydrate moieties (Stura et al., 1992Go; Zhan et al., 1999Go) or removal of flexible regions of the proteins (Dale et al., 1999Go).

The concept of crystal engineering has been hampered by the lack of rational approaches (Longenecker et al., 2001Go) and incomplete understanding of protein–protein crystal packing interactions (D’Arcy, 1994Go; Carugo and Argos, 1997Go). 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., 2001Go). The packing motifs identified in this study, which were composed of mainly small uncharged non-polar and polar residues (Tables IGo–IIIGo), 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 IGo–IIIGo).

However, it has previously been argued that most crystal packing contacts are non-specific, opportunistic and possibly randomized (Janin and Rodier, 1995Go; Carugo and Argos, 1997Go; Janin, 1997Go). 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., 1992Go). In contrast, the highly ordered triad-interactions were displayed by a large set of proteins (Tables IGo–IVGo) 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 protein–protein crystal packing interactions.

Crystal engineering projects have traditionally emphasized packing motifs tailored for individual proteins (Lawson et al., 1991Go; D’Arcy, 1994Go; Longenecker et al., 2001Go). Our triad-hypothesis could provide a platform for designing specific crystal packing contacts in entire groups of molecules with ß-structures. For example, all {kappa}-VDPs analyzed could be assigned to one of only four sequence variants of a packing motif, while the {lambda}-VDPs were confined to one of three motifs (Tables IGo–IIIGo). With the VL domains, however, the structures and exact locations of the motifs (Tables IGo–IIIGo) varied with the framework 1 substitutions (residues 1–20) in different subgroups of {kappa}- and {lambda}-chains. Participation of VH in triad-type packing (Harris et al., 1998Go) was too infrequent to be divided into specific categories (Tables IIGo and IIIGo).

Packing patterns incompatible with the triad-hypothesis fell into three categories (Tables IGo and IIIGo): (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 IGo–IIIGo) 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 IVGo) 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., 2000Go), 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., 1997Go; Byrne et al., 2000Go). In this study, we have identified single VL domains as the smallest units suitable for use as potential triad-fusion partners (Tables IGo and IIIGo). However, the recent design of a stable monomeric, three-stranded, antiparallel ß-sheet (Kortemme et al., 1998Go) 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., 1992Go; Hunte et al., 2000Go). 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.


    Acknowledgments
 
This study was supported by grants from the Crafoord Research Foundation (to C.W.), the Foundation Lars Hiertas Minne (to C.W.), the Magnus Bergwall Foundation (to C.W.), the Royal Swedish Academy of Science (to C.W.), the Royal Physiographic Society in Lund (to C.W.), the Swedish Council for Engineering Sciences (TFR) (to C.A.K.B.) and the National Cancer Institute, Department of Health and Human Services, Bethesda, MD (to A.B.E.). C.W. was in part supported by a grant from the Swedish Natural Science Research Council (NFR).


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Received January 9, 2002; revised February 28, 2003; accepted March 7, 2003.





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