Crystallization of Proteins that bind phosphate moieties

The Reverse Screening Technique:

Rule 2 in reverse screening states that "It is desired that the precipitant play no role other than that of inducing supersaturation." In order to better control the parameters that affect crystallization, we attempt to separate out the actual "precipitating" effect from any "specific binding" effect of each precipitant."
Phosphate is a commonly used precipitant, ammonium sulfate is another commonly used precipitant. Phosphate and in particular PBS (phosphate buffered saline) is a commonly used buffer. Sulfate is a phosphate mimic. Cacodylate (methyl arsonate) is another phosphate mimic, and a commonly used buffer; commonly used in the crystallization of DNA and RNA, molecules which are rich in phosphates.
Clearly, the use of phosphate for the crystallization of a protein that binds phosphate moieties, does not follow rule 2. The application of reverse screening requires that "precipitants and buffers are checked by replacing them with others to determine whether they have, or have not, a specific action on the crystallization. Precipitants or buffers found to have a specific effects necessary in the crystallization are then added to the crystallization in precisely determined quantities as additives, while using a similar (non-specific) precipitant to provide most of the precipitating action. In this way the specific role of the precipitants in crystallization can be maintained independently from their role of controlling the pH (for buffers) or in inducing supersaturation (in the case of precipitants)."
However, reverse screening does not prohibit the use of phosphate or phosphate mimics in the crystallization of proteins that bind phosphate moieties. It suggests that we should be aware that phosphate is affecting the state of the protein at the same time that it may be inducing supersaturation. We may be loosing control of the crystallization. Using high phosphate concentrations will ensure that the phosphate sites are fully saturated. This might provide a high degree of homogeneity (and heterogeneity is bad for crystallization). We will look at this point in further detail below.


Phosphate and Phosphate Mimics:
Are Proteins that bind phosphate moieties commonly crystallized with phosphate as a precipitant?:

Let's look at some examples:

1. Glycinamide Ribotide Transformylase : YES: In the "Native" form.
Crystals of glycinamide ribonucleotide transformylase were grown from 0.4 to 1 M ammonium sulfate, 0.6 to 1 M sodium-potassium phosphate. (click on the word Native above to see the result) Changing the precipitant to 0.65 to 1 M citrate in the pH range 4.5-7.0 did not help because the preparation involved a step in which an ammonium sulfate cut had been made.
In the crystallization of the complex with the multisubstrate adduct 1476U89 polyethylene glycol, and no phosphate was used.

2. Deoxyribose-5-phosphate Aldolase
If we survey the literature on the crystallization of aldolases we find that ammonium sulfate and phosphate are commonly used.
In our work we chose to use polyethylene glycol and 200mM imidazole malate buffer (pH 6.0). In fact this has enabled us to use Tungstate as a heavy atom derivative. Tungstate (WO4) is a phosphate mimic.

3. FIV dUTPase
Two crystal forms of FIV dUTPase were obtained using ammonium sulfate. The form that diffracts to the highest resolution (1.8A) is obtained with 13% MPEG, 50 mM sodium cacodylate, pH 6.5. Cacodylate is a phosphate mimic. Crystallization is inhibited by adding phosphate to the mother liquor. Crystals dissolve when vanadate, molybdate and tungstate are added. (This is also true for di- and tri- nucleotides). Another form, grown from 1.0 M sodium citrate, pH 6.5 is consistent with di- and tri- phosphates of nucleotides.

4. ADP-Ribosyl Cyclase
We have observed different crystal forms with Lithium sulfate additions to the crystallization in polyethylene glycol.

5. Glycogen Phosphorylase b
Crystals of Glycogen Phosphorylase b grown from A.S. are in the tetrameric active form.

6. Dynamin
From studies that Amy Muhlberg has carried out it appears that phosphate and its mimics will play a role in the crystallization of this GTPase.


Magnesium and other Divalent metals:
Magnesium and other divalent metals may be utilized in the binding or in the catalysis of phosphate moieties. Magnesium dependence is typical in enzymes that bind tri-phosphates. Zinc is often an inhibitor of this reaction. On the other hand zinc is required for activity in alkaline phosphatase.
Hence, in many cases it is important to investigate various divalent metals as
additives .