[ccp4bb]: summary of sulphate binding at ATP binding sites

["N.Manoj" <nm77@cornell.edu>]

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Hi all,
        Thanks a lot for all the replies. Here is a compilation of all
the replies that I got. Sorry, its not in a condensed form.

The question was 

>            I am trying to get cocrystals with ATP bound in the active
> site.  But the electron density maps show no evidence of ATP binding. I
> have ~200mM ammonium sulphate, PEG and 4mM ATP in the crystallization
> conditions and most probably ammonium sulphate competes with the ATP,
> preventing its binding.
> Does anyone know of a case where the ammonium sulphate has been
> exchanged or replaced from the crystals by soaking etc and found
> competing substrates being bound after that?


I did see electron density for sulphate ions in the expected ATP binding
site. I am trying now to replace AMS with NaCl/Na Citrate etc as
suggested.

manoj


Replies:

>
This isn't an answer to your question directly, but I know of a
case where AmSO4 was also found in the active site of an ATPase.
Ivan Rayment's work on chicken myosin (Science, 1993) used
crystals grown from AmSO4 and had a sulfate in the active site.
He was only able to get the nucleotide in by finding crystals
that grew in the absence of AmSO4, in his case it required using
myosin from a different source (Dictyostelium in Biochemistry, 1996).
I'd try seeing if you could exchange the crystals from the PEG/AMS
to PEG + another salt, keeping ionic strength constant or
a little higher, to wash out the AMS, and replace in the active site
with ATP.

Andrew M. Gulick, Ph.D.

>
In some cases it helps if you substitute ammonium sulphate with sodium
potassium tartrate. Tartrate is much bigger than sulphate and usually
does not bind at the active site. I am not sure about the concentration
but I usually try the same concentration as the ammonium sulphate first.
However, KNa Tartrate and PEGs do not mix well and the salt precipitates
in high concentrations (e.g. greater that 1.0 M KNaTartrate and 30 %
PEG4000). In your case since you only use 0.2 M NH4(SO4)2 there should
not be any problem. Have a look at
 Leonidas et al (2001). J. Biol. Chem. 276, 15009-15017.

Demetres D. Leonidas, Ph.D.

>

I observed pcp-AMP and pnp-AMP binding under soaking in 1.8M AS. Are you
sure ATP is not cleaved by your enzyme?

Dr. Dmitriy Alexeev

>
the last year I have had a problem similar to you; the solution has been
to find a new crystallization's condition. My crystals grew in 1.5M
ammonium sulfate, glycerol 12%, TRIS 0.1M pH=8.5: I have calculated the
ionic strength of this solution then I have prepared a new solution with
a
new precipitant (citrate or phosphate). I have prepared a little screen
about concentration and pH ....... I have achieved a new crystals and I
have soaked AMP-PNP (similar to ATP) without competition/problem!!!!
Good luck

Simone (Italy, Genoa)

>
what is the kd for ADP/ATP binding ? did you try ADP ?
I find it highly unlikely for AS to compete with ATP since that would
mean
that the binding constant of AS is ONLY =B150 times less than ATP.
I think we had much more AS and binding ADP/ATP was never a problem.
AS does compete out heavy atoms for sure and maybe lipid head groups
that have PO4 ... but the binding of ATP is different ....
Do you have also =B120mM Mg in your xtallisation buffer ?????
I would guess that is needed, unless you really have a strange
ATP binding protein that needs no Mg and has very low affinity for ATP
binding.

tassos

>
I have indeed one ATPase that crystallizes from 2 M ammonium sulfate and
upon solving the structure I found sulfate at the position equivalent to
the
beta-phosphate of ATP.  I believe this is a general affinity of many
ATPases
as there are numerous examples in the literature where this has
happened.
Now, when we took those crystals and soaked them with ATPgammaS
(nonhydrolyzable ATP analogue) we found that one of two sites in the
asymmetric unit was completely substituted by ATPgS whereas the second
site
contained 50% sulfate and 50% ATPgammaS despite the fact that solvent
accessibility is equal for both sites.  In our case the partial
occupancy in
one site has some mechanistic implications.
So my answer is yes, you can substitute sulfate with a substrate
analogue.
I would suggest you use higher concentrations of ATP (we had used 5mM
for
ATPgammaS over three days) and also try the ATPgammaS and AMPPNP
(another
nonhydrolyzable analogue).  Also prolonging the soaking time might help
dramatically.

Savvas

>
Yes, the sulfate is probably competing with ATP for the binding to the
phoshate groups binding sites. Actually, i would guess that the map
shows clearly a sulfate ion bound in the nucleotide-binding site. You
may try to do soaking experiments with a really high concentration of
ATP (about 10, 20, 50 mM ...). May be you can also decrease the sulfate
concentration in your soaking solution (to 0.1 M or 0.05 M). The other
alternative is to replace the ammonium sulfate by something else in the
a soaking solution (NH4Cl or NaCl).
If you don't see the sulfate ion bound in the nucleotide-binding site,
then the problem is different: the nucleotide-binding-site is not
accessible or the affinity for ATP is very low ! May be you have need to
add Mg2+ (if your protein use Mg2+ to hydrolyse ATP, in this case of
course
you will get ATP-hydrolysis, ...). Then you could try with AMPPNP-Mg2+
or AMPPCP-Mg2+ !!!
Have you tried to soak with ADP-Mg2+ ?
Gregory Verdon



>
A protocol has been worked out in our lab in the late eighties,
where crystals grown in high ammoniumsulphate concentration
2.4 M) were transferred to various other precipitant solutions
with the same water activity to keep them stable. After
transferring to PEG 6000 the crystals could be used successfully
for soaking studies, because the sulphate ion previously bound
in the active site was now absent.
See :

Appl. Cryst. (1988). 21, 426-429    [DOI 10.1107/S0021889888004108]
The transfer of protein crystals from their original mother liquor to a
=
solution with a completely different precipitant
H. A. Schreuder, H. Groendijk, J. M. van der Laan and R. K. Wierenga
Good luck,

Tjaard Pijning

>
Since your crystallization conditions are ammonium sulphate, you may
want to
try crystallizing in Na Malonate. Na Malonate is a good substitute for
ammonium sulfate, and it may help with the sulfate competition of the
binding
site. I would recomend screening similar concetrations of Na Malonate as
you
used with NH4SO4. You can buy Na Malonate in solution from Hampton at
various
pH's as well.

Jason Yano, Ph.D.