Crystallization of Protein DNA Complexes

Adrian H Batchelor, Walter and Eliza Hall Institute, Royal Melbourne Hospital, Victoria, Australia Derek E Piper, Howard Hughes Medical Institute and the Johns Hopkins University School of
Medicine, Baltimore, Maryland, USA

Secondary article
Article Contents
. Introduction . Protein Sample Preparation . The DNA: Sequence Choice and Sample Preparation . Complex Preparation . Crystallization Conditions . Summary of What Has Worked for Others

Cynthia Wolberger, Howard Hughes Medical Institute and the Johns Hopkins University School
of Medicine, Baltimore, Maryland, USA

A well-ordered crystal of the complex in question is a prerequisite for the determination of the three-dimensional structure of a proteinnucleic acid complex by X-ray crystallography. Techniques of crystallizing macromolecules remain essentially empirical and a trial-and-error approach is typically used. ProteinDNA complexes pose even more problems than pure proteins, but accumulated experience from many investigations makes it possible to offer basic guidelines for procedure.

Introduction
X-ray crystallography has provided a wealth of information on the three-dimensional structures of proteins and nucleic acids, and on the atomic details of how these two types of biological macromolecules interact. With the rapid improvements in data collection and computational methods that occurred in the 1990s, the principal obstacle that lies in the path to determining an atomic structure is the crystallization of the molecule or complex of interest. Typically, a trial-and-error approach is used to identify conditions under which single crystals will grow from a concentrated solution of macromolecules. The approach to crystallizing macromolecules remains rather empirical. In the case of proteinDNA complexes, there are many more considerations than for the case of the crystallization of proteins alone, making the task seem daunting. In addition to considerations of protein choice and purity, one must consider the composition and length of the DNA and the way in which the complex is formed. Fortunately, the accumulated experience of the many investigators who have studied proteinDNA complexes makes it possible to come up with basic guidelines for anyone embarking upon the crystallization of a new proteinDNA complex. While this article focuses on complexes between proteins and DNA, many of the considerations outlined here will be the same for crystallizing complexes between protein and RNA.

Protein Sample Preparation

The rst consideration in crystallizing a proteinDNA complex is the protein. While it is always most desirable to

crystallize the intact protein or proteins bound to DNA, it is frequently the case that disordered or exible regions of the protein will interfere with the formation of a wellordered crystal lattice. A common procedure for identifying the minimal folded protein fragment to use is to carry out limited proteolysis experiments. In these experiments, serial dilutions of proteases such as subtilisin or trypsin are incubated with the protein in the presence and absence of DNA. Reactions are stopped by the appropriate protease inhibitor and analysed by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE). The major products of the protease digestion can be identied by excising bands from the gel and subjecting them to analysis by N-terminal sequencing and mass spectroscopy. Specic cleavage products must also be analysed for biological activity such as DNA binding or cooperative interactions with partner proteins. If these experiments identify a smaller fragment that retains the activities of interest, polymerase chain reaction (PCR) cloning methods are used to construct an expression vector to express this fragment. For the purpose of crystallization, it is desirable to have a highly pure, concentrated preparation of protein. Ideally, the protein should be  99% homogeneous and at a concentration of at least 5 mg mL 2 1, but preferably 10 20 mg mL 2 1. The protein solution should be buered with 2550 mmol L 2 1 of a nonphosphate buer in the pH range 6.08.0 and should contain the minimum of salt required to maintain protein solubility. In addition, the solution should contain 1 mmol L 2 1 EDTA and 0.001% sodium azide to inhibit bacterial growth. Reducing agents such as dithiothreitol (DTT) should be included if the protein contains any cysteines. If signicantly higher levels of salt or buer are needed to maintain protein solubility,
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Crystallization of ProteinDNA Complexes

attempts to reduce the ionic strength can be made after the proteinDNA complex has been formed (see below).

The DNA: Sequence Choice and Sample Preparation

Like the choice of which protein fragment to use, the choice of the DNA fragment plays a signicant role in the success of a crystallization experiment. The experience of numerous investigators has shown that the precise length and composition of the oligonucleotide is the most critical variable and must be experimentally determined for each new protein of interest. Since many proteinDNA complexes crystallize under a rather narrow range of solution conditions, an ecient approach is to focus ones eorts on trying an exhaustive variety of DNA fragments under a relatively limited set of crystallization conditions. While, in theory, one should also try to be exhaustive in exploring potential crystallization conditions, experience has shown the eort is better spent on trying more DNA sequences. In the choice of the DNA fragment to use in complex formation, a critical variable is the length of the DNA. DNA has a strong preference to stack end-to-end in a crystal, so the precise length of the DNA fragment and the nature of the stacking interactions between fragments are critical determinants of the unit cell size and crystalline order. The lower limit on the length of the DNA fragments will be determined by the size of the minimal binding site necessary for tight complex formation between the protein and DNA. Since protein contacts with the DNA backbone generally extend beyond the minimal DNA sequence recognized by the protein, it is important to use data on DNA binding anity and not rely solely on studies that identify the base pairs most critical for DNA sequence recognition. Beginning with the minimal double-stranded sequence needed for binding, one can then experiment with sequences of increasing length. Lengths of DNA that have been particularly successful in crystallization trials (11, 15, 16, 20, 21 and 26 base pairs) correspond to multiples of approximately integral or half integral turns of the DNA. Initial crystallization trials should be carried out with a series of DNA fragments of these lengths containing the protein binding site near the centre of a DNA fragment. If these oligonucleotides are unsuccessful, additional bases should be added or removed until the whole spectrum of lengths is examined. One should also consider modifying the binding-site sequence, altering the position of the binding site within the oligonucleotide, or adding binding sites to the oligonucleotide. Another important sequence variable is the composition of the DNA ends. Blunt-ended oligonucleotides have yielded high-quality crystals in a number of cases, although single or double complementary overhanging bases at either the 3 or 5 end are frequently included to promote
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end-to-end stacking of oligonucleotides in the crystal. While a number of dierent types of overhangs have been used successfully, the most common one is a single 5 overhanging A on one strand and a 5 overhanging T on the other. Another approach is to engineer DNA ends that promote triplex interactions through Hoogsteen basepairing of an overhanging C with a G.C base pair in the opposing fragment. Double-stranded oligonucleotides containing the sequence CpC at the 5 end of each strand and a G at each 3 end can potentially form these interactions. DNA for crystallization trials can be produced by 1 mmole synthesis of each strand on commercially available DNA synthesizers. Purity of greater than 99% can be achieved by two rounds of purication by reversed-phase high-performance liquid chromatography. In the rst step, the synthetic single-stranded oligonucleotide with the 5 trityl group is loaded onto a reversed-phase Varian PureDNA column in the presence of 0.1 mol L 2 1 triethylamine acetate (TEAA), pH 7, and eluted with a gradient of increasing acetonitrile (2030%) in 0.1 mol L 2 1 TEAA. The pooled peak fractions are dialysed into 0.01 mol L 2 1 triethylamine bicarbonate (TEAB), pH 7, and lyophilized. The DNA is then resuspended in 0.1 mol L 2 1 TEAA and reloaded onto the column. The trityl group is removed by owing 0.5% triuoroacetic acid (TFA) over the column for 10 minutes, after which the TFA is washed away with 0.1 mol L 2 1 TEAA and the column is developed with an acetonitrole gradient in 0.1 mol L 2 1 TEAA. The peak fractions are pooled, dialysed into 0.01 mol L 2 1 TEAB, then lyophilized until needed. Complementary strands are annealed by resuspending each strand in 0.5 ml of 0.01 mol L 2 1 TEAB and combining stoichiometric amounts, heating the solution for 5 minutes in a 708C water bath, and then allowing the DNA to come slowly to room temperature. The concentration of the annealed DNA is determined spectroscopically and the DNA is aliquoted in xed amounts, lyophilized, and stored at 2 808C until needed.

Complex Preparation
The proteinDNA complex is prepared by adding the concentrated protein solution directly to the lyophilized DNA. The goal is to have a slight excess of DNA (typically 1020%) over the amount needed for a 1:1 (or 2:1 or 3:1, as the case may be) ratio of protein to DNA. For more complex systems involving two dierent proteins, it is advisable to look closely at the ratio of the proteins, examining the complex at a series of dierent protein protein ratios by gel shift to maximize complex formation. If there is more than one DNA binding site and binding of the proteins is not cooperative, it may not be advisable to have much excess DNA. In this case, the presence of extra

Crystallization of ProteinDNA Complexes

DNA may lead to a solution containing a mixture of 1:1 and 2:1 proteinDNA complexes, which may not be conducive to crystal growth. Once a complex of the appropriate stoichiometry is formed, it is dialysed into the most minimal buer conditions under which the complex remains soluble. Since the conditions for the complex might be quite dierent from those for either the protein or the DNA alone, these conditions must be determined for each complex, although not necessarily for each new DNA sequence. The minimal buer would ideally be distilled water, although that is usually not possible. A more achievable minimal buer might be 10 mmol L 2 1 buer, 1 mmol L 2 1 EDTA (if you are concerned about nucleases), 0.001% sodium azide, and 1 mmol L 2 1 DTT if necessary. The underlying idea is that certain components might interfere with crystallization, so it is best to eliminate as much as possible from the solution. Moreover, it is easier to manipulate the pH in crystallization trials if there is little or no buer present in the complex solution. The precise buer conditions that will allow the complex to remain soluble will depend somewhat on the concentration of the protein and DNA. Sometimes, the solubility of the complex is signicantly higher than that of the protein alone, making it possible to further concentrate the complex at this stage. This is particularly useful if it has not been possible to concentrate the protein to more than a few mg ml 2 1. In that case, a second concentration step following formation of the proteinDNA complex would be advisable. The entire complex can then be further puried chromatographically, but this has been determined to be unnecessary in the majority of cases. After the nal preparation of each complex, a small sample should be analysed on a nondenaturing polyacrylamide gel to ascertain whether a complex with the desired proportion of excess DNA is present.

Crystallization Conditions
A large proportion of proteinDNA complexes whose structures have been determined were crystallized under a relatively limited set of conditions. These include poly(ethylene glycol) (PEG) as the primary precipitant, 20 100 mmol L 2 1 buer between pH 5 and 8, and frequently some amount of salt (either NaCl of KCl) and/or divalent cation (usually from CaCl2 or MgCl2). In addition, small amounts of various additives are sometimes required to produce high-quality crystals. These additives include polyamines such as spermine or cobaltic hexamine chloride, nonvolatile organics such as glycerol or 2methyl-2,4-pentanediol (MPD), and ions such as zinc. Crystallization trials should employ the hanging-drop vapour diusion method, which allows rapid screening of many conditions and oligonucleotides. In this technique, a

small amount of the proteinDNA complex is pipetted onto a siliconized coverslip. An equal amount of crystallization solution is added to the drop and the coverslip is inverted over a well containing the same crystallization solution. Equilibration of conditions between the drop and the well solution occurs by diusion of vapour within the sealed well. Since the precipitation points for complexes composed of various oligonucleotides are frequently similar, a simple screen can be used to quickly test the conditions required for crystallization. Owing to the exibility of the number of drops that can be placed on a single coverslip, up to four complexes with dierent oligonucleotides can be tested simultaneously with the same crystallization solution. It is frequently the case that once the correct oligonucleotide is found, crystallization occurs under numerous conditions. Therefore, a limited crystallization screen can be used to rapidly test the various oligonucleotides used in complex formation. First, a 24-well tray should be set up to determine the precipitation point of the various complexes. Various concentrations of a specic PEG can be screened as a function of four dierent pH values. For example, the concentration of PEG 4000 can be varied from 5% to 30% (w/v) in intervals of 5%, in the presence of 100 mmol L 2 1 buer: citrate pH 5, Mes pH 6, Hepes pH 7, and Tris-HCl pH 8 (Figure 1). Once the precipitation point is found for the specic precipitant and pH, a second tray can be set up to address the eect of dierent additives. In this tray, the range of precipitant concentrations should be chosen so that the maximum concentration of precipitant is just below that required for complex precipitation. A good starting point is to test 2 mmol L 2 1 spermine, 5 mmol L 2 1 cobaltic hexamine chloride, 100 mmol L 2 1 NaCl, 10 mmol L 2 1 MgCl2 and 100 mmol L 2 1 NaCl 1 10 mmol L 2 1 MgCl2. Another tray, in which CaCl2 is used in the presence and absence of other salts, should also be set up. Precipitation and crystal formation from these solutions should then be used to direct the choice of conditions for further crystallization trials. While conditions to be explored are limited only by ones imagination, it is advisable to start by exploring several dierent molecular masses of PEG, varying the type and concentration of salt, and experimenting with the presence of dierent divalent cations. Once some sign of crystal formation has been observed, crystal quality can sometimes be dramatically improved by adding small amounts of some type of salt, organic solvent or other compound. A little creativity and adventurousness in exploring the chemical shelf can pay o in the form of large, single crystals. Once crystals have been obtained, it is important to verify that they indeed contain the proteinDNA complex of interest. Crystal content can be analysed by washing a crystal in well solution, then dissolving the crystal in dilute buer and subjecting the solution to electrophoresis in a nondenaturing polyacrylamide gel as described above. Standard SDS-PAGE of dissolved crystals can also be used
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Crystallization of ProteinDNA Complexes

PEG 4000 (w/v): 5% Buffer: Citrate, pH 5

5% PEG pH 5 10% PEG pH 5 15% PEG pH 5 20% PEG pH 5 25% PEG pH 5 30% PEG pH 5

10%

15%

20%

25%

30%

Mes, pH 6

5% PEG pH 6

10% PEG pH 6

15% PEG pH 6

20% PEG pH 6

25% PEG pH 6

30% PEG pH 6

Hepes, pH 7

5% PEG pH 7

10% PEG pH 7

15% PEG pH 7

20% PEG pH 7

25% PEG pH 7

30% PEG pH 7

Tris, pH 8

5% PEG pH 8

10% PEG pH 8

15% PEG pH 8

20% PEG pH 8

25% PEG pH 8

30% PEG pH 8

Figure 1 Sample crystallization tray showing conditions in each well for an initial screen for precipitation points of the proteinDNA complexes in question. A skilled practitioner can easily fit four drops in a single cover slip, thereby facilitating the rapid screening of many different oligonucleotides.

to verify that the protein in the crystal has not become degraded in the crystal drop. If degradation appears to have occurred, further analysis of the sample by mass spectrometry may be necessary.

Summary of What Has Worked for Others

It is not possible in the scope of this article to review the crystallization conditions for all proteinDNA complexes whose stuctures have been determined. We summarize here some of the ndings from a survey of 72 crystal structures determined between 1988 and 1999. Of these DNA protein complexes, three-quarters were crystallized with PEG as the primary precipitant, with about 10% utilizing MPD and the remainder a variety of salts and organic solvents. Complexes were crystallized over a broad pH range, extending from pH 4 to 9, with 6.4 being the average

pH. Over half of the complexes were crystallized in the presence of on average 0.2 mol L 2 1 salt, with the concentration of NaCl or KCl ranging from 0.02 to 0.6 mol L 2 1. About 65% of the co-crystals were formed in the presence of divalent cations. They are, in decreasing order of popularity: Mg2 1 , Ca2 1 , Zn2 1 , Ba2 1 and Cd2 1 . The average divalent cation concentration was 0.05 mol L 2 1, with a range of 2 mmol L 2 1 to 2 mol L 2 1. Another commonly occurring additive was a polyamine: either 0.13 mmol L 2 1 spermine or 250 mmol L 2 1 cobaltic hexamine chloride. Organic solvents such as glycerol, MPD or ethylene glycol, which are most commonly used as primary precipitating agents, have also been used in small amounts (less than 15% w/v) as additives that improve crystal quality.

Further Reading
McPherson A (1999) Crystallization of Biological Macromolecules. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.