Re: [ccp4bb] Weakest protein-protein complex crystallised
Continuation about this competition between crystal contacts and biologically-relevant contacts Your example is quite interesting because you were able to make the comparison with different ligand affinities, which is exactly what we would have like to test... I just want to add a comment about a possible kinetic, rather than purely thermodynamic, effect. It may be possible that one cannot really compare the affinity of the ligand with that of the molecule-molecule interaction in the lattice because once a molecule-ligand complex has been incorporated into a growing crystal it may be very rapidly protected against the loss of its ligand (and in our case against the binding of a second ligand). Philippe Dumas IBMC-CNRS, UPR9002 15, rue René Descartes 67084 Strasbourg cedex tel: +33 (0)3 88 41 70 02 [EMAIL PROTECTED] [Philippe DUMAS] -Message d'origine- De : Filip Van Petegem [mailto:[EMAIL PROTECTED] Envoyé : Monday, June 30, 2008 9:01 PM À : Philippe DUMAS Cc : CCP4BB@jiscmail.ac.uk Objet : Re: [ccp4bb] Weakest protein-protein complex crystallised 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
Re: [ccp4bb] Weakest protein-protein complex crystallised
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]
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 /
Re: [ccp4bb] Weakest protein-protein complex crystallised
There are quite a number of structures homodimers and homotetramers in the PDB where the dissociation constant is known to be in the millimolar range. For example the dimerizaion of a humainized antibody VHH domain that mimicks a VH-VL complex (Conrath et al. J. Mol. Biol. (2005) 350, 112125). Remy Loris Vriej universiteit Brussel 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, 923942) 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
Re: [ccp4bb] Weakest protein-protein complex crystallised
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
Re: [ccp4bb] Weakest protein-protein complex crystallised
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
Re: [ccp4bb] Weakest protein-protein complex crystallised
... 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/
Re: [ccp4bb] Weakest protein-protein complex crystallised
Hello John, No, they're not. Crystals were obtained at pH8.0, 200mM NaCl; 10% PEG4000. Calorimetric experiments were done at pH7.4, 150mM KCl. We found the interaction to be driven mainly by hydrophobic contacts (mutants of polar/charged residues have no significant effect on the affinity). I'd only expect a minor effect of pH in this case, but this would have to be tested. So yes, as I've mentioned before, both crystal contacts and crystallization conditions could together reduce the affinity and break the 100nM Kd interaction. The effect of pH on the affinity could be tested directly in an ITC experiment - the effect of 10% PEG4000 would be harder to assess due to insolubility (crystallizability) of the protein... Cheers Filip Van Petegem On Mon, Jun 30, 2008 at 4:47 PM, John A. Newitt [EMAIL PROTECTED] wrote: At 3:28 PM -0700 6/30/08, Filip Van Petegem wrote: 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. Filip: Are you calorimetric binding measurements performed under similar conditions (especially pH) as your crystallization condition for the mutant proteins? We have determined in some cases that apo crystals are due to the fact that a ligand had reduced affinity at the non-neutral pH of crystallization, whereas initial positive binding studies were performed at pH ~7. - John -- http://xri.net/=john.newitt -- 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/
