Dear Jens, There is an overwhelming amount of evidence that our mutants still binds to the same site in solution and in vivo - there is a correlation between decrease in binding affinity (calorimetric measurements) and function (electrophysiological measurements) for more than 20 mutants based on the structure. The native binding site was found in 3 different crystal structures from independent groups (all different crystallization conditions and different variants). A crystal artefact of the observed WT binding is therefore excluded.
The crystal artefact is that we don't observe any binding in the crystal structures of a set of mutants (neither to the native site, nor to any other), whereas both calorimetric and electrophysiological data suggest there should be binding in the 100-200nM range. The binding is abolished because of crystal contacts (+ crystallization conditions) for 100nM and weaker binders, but not for 10nM and stronger binders. What you were saying is the other way around (tight binders not binding). Cheers Filip Van Petegem On Mon, Jun 30, 2008 at 2:22 PM, Jens T. Kaiser <[EMAIL PROTECTED]> wrote: > Dear Filip and others, > To play Devils advocate, this could also (in the absence of strongly > supportive biochemical data) be interpreted as a crystal artifact, with the > weakly binding ligand not forming a physiologically relevant contact but > merely occupying the - haphazardly - empty space in the crystal of the > bigger > protein. When tighter binders bind to the 'real' interaction side, they > inhibit the crystal packing, thus yielding only crystals of the free > partner. > I'm not saying this is the case in your example, but this should be > strongly > considered for weak interactors (see for example the HLSV/U debate a few > years ago, where, as far as I can remember, the regulator bound 'its' > protease not in the 'regulating' conformation, but in a 'substrate' like > fashion.) > > Cheers, > > Jens > > > On Monday 30 June 2008 12:01:12 Filip Van Petegem wrote: > > Hi, > > > > we've had a similar situation: a protein-peptide complex with a Kd in > the > > nM range crystallized in the same condition as the protein alone, and > > yielded a structure of a complex (voltage-gated calcium channel beta > > subunit). The exact crystal contacts turned out to be a bit different, as > > the peptide would clash with a neighbouring molecule in the lattice. > > > > However, a mutant protein that increased the Kd to ca 160nM (as > confirmed > > by ITC), using the same peptide crystallized in the same conditions, but > > this time not as a complex. This effect was reproducible: the WT > > consistently crystallized as complex, whereas relatively mild mutants (Kd > > in 100nM range and worse) only yielded crystals of the apo-protein. > > > > Conclusion would be that crystal contacts can break relatively tight > > protein-protein interactions in the ~100nM range, and that crystal > contacts > > are not always that weak. However, the crystallization conditions > > themselves (PEGs, non-neutral pH) are likely to affect the binding as > well. > > > > Cheers > > > > Filip Van Petegem > > > > On Mon, Jun 30, 2008 at 10:42 AM, Philippe DUMAS > > <[EMAIL PROTECTED]> > > > > wrote: > > > Hello > > > > > > We have had an interesting example where the crystal packing seems to > > > have won against the "biological interaction". This is about a "sliding > > > clamp" made of a very symmetric homodimer having the shape of a ring > > > (encircling DNA during its replication). > > > This "beta-ring" had been crystallized alone by the Kuriyan group in P1 > > > (thus there was NCS). In our case, we crystallized it with an > additional > > > peptide mimicking the C-term of a polymerase binding to the beta-ring > > > [Burnouf et al, JMB 335(2004) 1187]. We expected a symmetric binding of > > > two peptides/ring (one peptide for each protein in the dimer). However, > > > we observed only one peptide/ring. It turns out that we had obtained > > > exactly the same packing in P1 and that one of the two possible binding > > > sites was engaged in crystal contacts. We estimated the Kd of > > > peptide-ring interaction as being in the µmolar range and that there > was > > > only a few percent of beta-rings in crystallization drops being singly > > > occupied. Yet the crystallization process selected this minor species > to > > > build crystals with (supposedly) a good crystal packing, rather than > > > "finding" another crystal packing accomodating the doubly-occupied > > > species present in large excess. Our conclusion was that a very modest > > > gain of ca. 2 kcal/mol in the free energy of interaction of > > > singly-occupied beta-rings was sufficient to account for their > selection > > > to build crystals against a great majority of doubly-occupied > > > "contaminants". This is exactly the order of magnitude mentioned by Ed > > > Pozharski: a single additional H-bond is enough to account for 2 > kcal/mol > > > ! And apparently this may be enough to win against "biological > > > interactions". Let us not forget that there are many processes > comparable > > > to crystallization in living cell... > > > > > > I hope this story makes sense in the frame of this discussion. > > > > > > Philippe Dumas > > > IBMC-CNRS, UPR9002 > > > 15, rue René Descartes 67084 Strasbourg cedex > > > tel: +33 (0)3 88 41 70 02 > > > [EMAIL PROTECTED] > > > > > > -----Message d'origine----- > > > De : CCP4 bulletin board [mailto:[EMAIL PROTECTED] la part de > Ed > > > Pozharski > > > Envoyé : Monday, June 30, 2008 4:50 PM > > > À : CCP4BB@JISCMAIL.AC.UK > > > Objet : Re: [ccp4bb] Weakest protein-protein complex crystallised > > > > > > > > > The word "weak" is, of course, relative. Free energy of > crystallization > > > is roughly 1-2 kcal/mole of crystal contacts (I think I carried this > > > number from Sir Blundell's book, but quick look at papers by Peter > > > Vekilov's group seems to confirm it - am I wrong on this?). I think > > > that crystal contacts are still much weaker than any interaction of > > > biological importance (perhaps I am wrong on this one too and there are > > > important biological protein-protein interaction with 10mM affinity, > but > > > I doubt that they are many). > > > > > > On Mon, 2008-06-30 at 10:09 -0400, Patrick Loll wrote: > > > > I hope this isn't too much of a foray into philosophy and semantics, > > > > but can't you argue that the crystals themselves are weak complexes? > > > > And since the energies of crystal contacts are typically very weak, I > > > > would further argue that you should be able to crystallize ANY > complex > > > > with an association constant corresponding to energies as low as > those > > > > associated with crystal contacts. Of course, it's not guaranteed, any > > > > more than getting a crystal is guaranteed--you need some luck. > > > > > > > > > > > > Of course, it's Monday AM, and I haven't approached my asymptote for > > > > caffeination. Am I talking through my hat? > > > > > > > > > > > > Pat > > > > > > > > On 29 Jun 2008, at 3:36 PM, Derek Logan wrote: > > > > > Hi, > > > > > > > > > > > > > > > Can anyone advise me what is currently the weakest protein-protein > > > > > complex yet crystallised? Google searching turned up a paper from > > > > > the Tromsø crystallography group (Helland et al. 1999, JMB 287, > 923– > > > > > 942) in which a complex between beta-trypsin and a P1 mutant of > BPTI > > > > > with a Kd of 68 uM was described as belonging to the weakest > > > > > complexes solved to date, but this article was from 1999 and much > > > > > water has passed under the bridge since then. > > > > > > > > > > > > > > > Thanks > > > > > Derek > > > > > _________________________________________________________________ > > > > > Derek Logan tel: +46 46 222 1443 > > > > > Associate professor fax: +46 46 222 4692 > > > > > Molecular Biophysics mob: +46 76 8585 707 > > > > > > > > > > Centre for Molecular Protein Science > > > > > Lund University, Box 124, 221 00 Lund, Sweden > > > > > > > ------------------------------------------------------------------------- > > >-------------- > > > > > > > Patrick J. Loll, Ph. D. > > > > > > > > Professor of Biochemistry & Molecular Biology > > > > > > > > Director, Biochemistry Graduate Program > > > > > > > > Drexel University College of Medicine > > > > > > > > Room 10-102 New College Building > > > > > > > > 245 N. 15th St., Mailstop 497 > > > > > > > > Philadelphia, PA 19102-1192 USA > > > > > > > > > > > > (215) 762-7706 > > > > > > > > [EMAIL PROTECTED] > > > > > > -- > > > Edwin Pozharski, PhD, Assistant Professor > > > University of Maryland, Baltimore > > > ---------------------------------------------- > > > When the Way is forgotten duty and justice appear; > > > Then knowledge and wisdom are born along with hypocrisy. > > > When harmonious relationships dissolve then respect and devotion arise; > > > When a nation falls to chaos then loyalty and patriotism are born. > > > ------------------------------ / Lao Tse / > > > -- Filip Van Petegem, PhD Assistant Professor The University of British Columbia Dept. of Biochemistry and Molecular Biology 2350 Health Sciences Mall - Rm 2.356 Vancouver, V6T 1Z3 phone: +1 604 827 4267 email: [EMAIL PROTECTED] http://crg.ubc.ca/VanPetegem/
