Soaking Kinase Crystals with ATP analogues

From: Dianfan Li
Date: 1 February 2012 19:17


Dear all,

Sorry about a non-crystallographic question here.

I am working on a kinase and would like to get an ATP analogue into
the crystals. When soaked with AMP-PCP, the kinase crystals crack in
about 15 min at 4 C.

I could try other analogues like AMP-PNP etc, but those would probably
behavour in a same way as AMP-PCP. Is it a good idea of trying quick
soaks at high concentrations of AMP-PCP? Co-crystallization is another
option I have but AMP-PCP is a substrate of the kinase (with low
rate).

What are other ways of getting ATP analogues into a crystal?

Thanks for suggestions,

Dianfan

Dianfan Li, PhD

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From: Nat Echols

On Wed, Feb 1, 2012 at 11:17 AM, Dianfan Li  wrote:
> I am working on a kinase and would like to get an ATP analogue into
> the crystals. When soaked with AMP-PCP, the kinase crystals crack in
> about 15 min at 4 C.

This isn't too surprising; most kinases undergo global conformational
changes (domain closure) when binding ATP.

> I could try other analogues like AMP-PNP etc, but those would probably
> behavour in a same way as AMP-PCP. Is it a good idea of trying quick
> soaks at high concentrations of AMP-PCP? Co-crystallization is another
> option I have but AMP-PCP is a substrate of the kinase (with low
> rate).
>
> What are other ways of getting ATP analogues into a crystal?

I'd recommend trying ATP-gammaS - it could also be a substrate, but
it's worth a look.  (Is there any reason to believe that AMP-PNP is a
substrate?)  I've noticed that the various analogues have been known
to result in different conformations in the crystal structure, so it
may be a good idea to try more than one anyway.

-Nat

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From: Fischmann, Thierry

Dianfan

Some kinases have such conformation in non-activated apo form that the ATP binding site is partially obstructed. Soaking an ATP analog may then have 3 outcomes: 1) successfully open up the binding site without damage to the crystal, 2) fail to open up the active site and the compound cannot diffuse to the active site, or 3) induce conformational changes which lead to serious disorder in the crystals (which then loose their diffraction) or even crack.

Hence my question: is the ATP binding site unoccluded in the apo structure?

If you're in situation #3 then soaks at low concentrations may get you to #1 as a more "gentle" diffusion may be better accommodated by a crystal. Or you may stay in #3, or you may have lowered the concentration so much that the crystals don't crack and you're end up in situation #2. Still a worthwhile experiment.

If the ATP binding site is unoccluded then another possibility would be that the kinase-ATP analog may be more soluble than the apo kinase, in which case increasing the precipitant concentration in your soaking buffer may help.

Good luck
Thierry

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From: Francis E Reyes

On Feb 1, 2012, at 12:17 PM, Dianfan Li wrote:

> I am working on a kinase and would like to get an ATP analogue into
> the crystals. When soaked with AMP-PCP, the kinase crystals crack in
> about 15 min at 4 C.

15 minutes is a long time. Scoop crystals during that time period.


Do the cracked crystals diffract? Do you see the analogue?


F




---------------------------------------------
Francis E. Reyes M.Sc.

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From: Artem Evdokimov

I assume that cocrystallization has failed? What you are experiencing is likely the effect of conformational transition caused by ligand. You can try very slow adition (even microdialysis) or if your ligand is fairly insoluble then you can just add a tiny solid particle of inhibitor to your drop and hope it dissolves slowly and saturates the crystal slowly. Hope it helps :-)
Artem

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From: Boaz Shaanan


Hi,

First, it's  very much a crystallographic question. Second, the success or failure in soaking in ligands/cofactors depends quite often also to the crystal packing. Some packing forms (and the spacegroups that go with it) will tolerate the soaking even if it's accompanied with a conformational change, whereas others won't (like the spacegroup you currently have). So you could try, among the other good suggestions that you were given, more crystallization conditions, perhaps you might get lucky and come across crystals in a different spacegroup  which will behave more nicely vis-a-vis soaking.

 Good luck,

                 Boaz

Boaz Shaanan, Ph.D.

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From: <Sofia Caria>


Dear Dianfan,

In some cases the ATP-lid of the kinase is blocking the active site in the crystal form. In those cases the only option is to try co-crystallisation.

Besides ATP and the homologs you mention you can also try ADP that as you will see in the PDB has been heavily used for kinases.

Best of luck

Sofia
Sent via BlackBerry® from Vodafone

--

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From: Yuri Pompeu

Maybe someone has suggested this already... If so, I am re-enforcing it.
If the cracking is coming from actual molecular movement induced by binding (and not other reason like differing ionic strength in your soaking conditions) you could try setting up some co-crystallization and (hopefully)
grow some enzyme-substrate complex crystals...
hth
yuri

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From: Ed Pozharski

Consider cross-linking crystals with glutaraldehyde.  The caveat here is
that you may end up with the protein conformation that is forced by
lattice, but if the issue is just the fragility, you should be fine.  I
assume that crystals simply crack but do not dissolve?

Certainly, as others have said, slower inhibitor addition should be
tried.

--
"Hurry up before we all come back to our senses!"
                          Julian, King of Lemurs