Investigation of Protein Ligand Interactions by NMR EMBO Course 2007 Fast ko>>(Vee-Vpound） A+B KnAB K得 <<(Vfree -Vbound)
Investigation of Protein Ligand Interactions by NMR Helen Mott University of Cambridge EMBO Course 2007 Slow Fast !"free !"bound A + B AB kon koff koff ~ (!free - !bound ) koff > (!free - !bound ) KD = [A][B] = koff [AB] kon
5N-HSQC:Titration of Unlabelled Peptide into Labelled Protein 1:0 1:01 1:0.3 1:0.5 1:1.0 15N-HSQC:Titration of Unlabelled Peptide into Labelled Protein 1:01 103 1:1.2
1:1.0 1:0.5 1:0.3 1:0.1 1:0 Intermediate Fast/ Intermediate Fast Slow? 15N-HSQC: Titration of Unlabelled Peptide into Labelled Protein 1:1.2 1:1.0 1:0.5 1:0.3 1:0.1 1:0 Intermediate Fast/ Intermediate Fast Intermediate? 15N-HSQC: Titration of Unlabelled Peptide into Labelled Protein
6 kDa domain titrated into 20 kDa labelled G protein Slow exchange case 19.0 1:0 119.5 1:0.2 120.0 1:0.5 1205 1:1.0 121.0 21 10.2 10.0 9.8 9.6 94 8.4 Measure or estimate Ko e.g.fluorescence.ITC.Biacore.NMR Minimize.if.limited proteolysis egmionsheeeethaneegneseouabe0enpealue.fe Add the compo ent in e
10.2 10.0 9.8 9.6 9.4 9.2 9.0 8.8 8.6 8.4 119.0 119.5 120.0 120.5 121.0 121.5 1:1.0 1:0.5 1:0.2 1:0 6 kDa domain titrated into 20 kDa labelled G protein Slow exchange case •Measure or estimate KD e.g. fluorescence, ITC, Biacore, NMR •Minimize interacting region, if necessary e.g. limited proteolysis •Find conditions where both components are stable and the interaction still occurs (e.g. salt, pH dependence) •Find conditions where the exchange regime is favourable (temperature, field strength) •Add the unlabelled component in excess if necessary to saturate the labelled component (except when trying to observe the unlabelled component)
Strategy depends on the affinity Ko 1uM K,~1-100uM K,>~100uM strong medium weak Structures of protein complexes tight(ideally) Mapping interactions e.g.for mutagenesis studies chemical shift mapping H/D exchange cross-saturation tight STD any Extracting distance information: transferred NOEs intermolecular NOEs lon Docking any Review:Carlomagno(2005)Ann.Rev.Biophys.Biomol.Struc.34245-66 Measuring Ka by NMR Small ligand,weak binding P+L K聘 △6=△6(K。+[P1,+L,-{K。+[P1,+L,)2-(4[P,·[,)}Ψ2)/2P1 A is the measured chemical shift change at each ligand concentration t change Lt is the total igand conc [ligand]
tight (ideally) any tight tight any weak tight any Structures of protein complexes Mapping interactions e.g. for mutagenesis studies chemical shift mapping H/D exchange cross-saturation STD Extracting distance information: transferred NOEs intermolecular NOEs Docking Strategy depends on the affinity KD ~100 µM strong medium weak Review: Carlomagno (2005) Ann. Rev. Biophys. Biomol. Struc. 34 245-66 Measuring Kd by NMR Small ligand, weak binding ! "# = "#max (KD + [P]t + [L]t ${(KD + [P]t + [L]t) 2 $ (4 •[P]t •[L]t)}1/ 2 )/2[P]t #$ is the measured chemical shift change at each ligand concentration #$max is the maximum chemical shift change [P]t is the total protein concentration [L]t is the total ligand concentration [ligand] 1.0 0.5 f r a ctio n b o u n d Kd P + L PL kon koff KD = [P] [L] [PL] = Koff Kon Changes in volume must be accounted for!
Li YZhang.Y.and Yan,H.(196)Biol Chem 1,3-804 GMP titration into guanylate kinase 191 1.90 ◇ 0.mM GMP 1.88 187 Repeat for multiple crosspeaks 1.85 184 1.83 010203040 50 GMP(ul) A+B K0之AB Ko=KouKon 上eagmeoeeenteeneon6m Lower limit for K1uM 8eaeaeghieoalshsaeoeogeerndsoweeangessw *This is a big assum especially if there are any significant conformational rearrangements involved. .g.Emerson et al(19)Biochemistry 346911-6918
Li, Y., Zhang, Y., and Yan, H. (1996) J. Biol. Chem. 271, 28038–28044 GMP titration into guanylate kinase 0.58mM GK Adding 13.3 mM GMP 29 µM KD Weak binding Fast exchange Repeat for multiple crosspeaks e.g. Emerson et al (1996) Biochemistry 34 6911-6918 Look for the largest chemical shift difference between the free and the bound forms that still shows fast exchange e.g. it is 100 Hz Koff > 100 sec-1 Kon is diffusion limited: 108 M-1 sec-1 * Lower limit for KD ~ 1 µM So if the free and bound chemical shifts are close together and slow exchange is still observed, the KD must be low * This is a big assumption, especially if there are any significant conformational rearrangements involved. A + B AB kon koff KD = Koff/Kon
119.0 1:0 -0.05 ppm 119.5 1:0.2 120.0 1:0.5 1:1.0 120.5 121.0 12 10.2 10.0 9.8 9.6 94 Ko-250 nM Mapping Binding Contacts
10.2 10.0 9.8 9.6 9.4 9.2 9.0 8.8 8.6 8.4 119.0 119.5 120.0 120.5 121.0 121.5 1:1.0 1:0.5 1:0.2 1:0 ~0.05 ppm KD ~ 250 nM Mapping Binding Contacts
Chemical Shift Perturbation Mapping Slow or fast exchange A=X6NHP+(615N2 x=4,10....or some other number Mott et al (2003)J.Biol Chem.27817053-17 Binding a(larger)protein ligand to a labelled protein gCD2(10 kDa)titrated with CD48(540 kDa-glycosylated A 1:0 B 1:02 11 s52 0s52 S52 0 . o. 117- 90 o moar ratio of D4 McAlister et al(1996)Biochemistry 355982-5991
1:0 1:0.5 1:1.0 Chemical Shift Perturbation Mapping Slow or fast exchange # = [x($NH)2 + ($15N)2] 1/2 x = 4, 10 ….or some other number 40 50 60 70 80 90 100 0 1 2 3 4 5 0 10 20 30 Mott et al (2003) J. Biol Chem. 278 17053-17059 Binding a (larger) protein ligand to a labelled protein Overall broadening due to increase in %c. Selective broadening of some residues e.g. CD2 (10 kDa) titrated with CD48 (~35-40 kDa - glycosylated) McAlister et al (1996) Biochemistry 35 5982-5991 Fractional reduction in peak height #h = (hi -hx)/(hi -hf ) plotted against molar ratio of CD48 1:0 1:0.2 1:1
Comparison between intensity loss and chemical shift changes A Loss of intensity in strands C.C'correlates with shift changes 86epes
Comparison between intensity loss and chemical shift changes Loss of intensity in strands C, C’ correlates with shift changes Free Complex
Hydrogen Exchange (high affinity binding) Patterson et al(1990)Science 249755-759 ncubate inD hr to days HN ChetehgRanneastyangeaerncaiaionntepmesenek小adahenceK all residues-there should be a sharp distinction between protected and Assumes that there are no co-operative unfolding events as a result of binding the other molecule L64 …free form bound form Protection factors (k/ 8湖 598 02 Protection factors3 30 nge time (h ours)
Hydrogen Exchange (high affinity binding) Patterson et al (1990) Science 249 755-759 Incubate in D2O 1hr to days HN HN HN HN O DN HN DN/HN HN O Dissociate at low pH (H/D exchange is slow) and isolate labelled protein DN HN DN/HN HN O HSQC to identify remaining NHs Calculate the rate of intensity change after incubation in the presence (kbound) and absence (kfree) of the binding partner Calculate kfree/kbound for all residues - there should be a sharp distinction between protected and unprotected residues Assumes that there are no co-operative unfolding events as a result of binding the other molecule Exchange time (hours) free form bound form Protection factors (kfree/kbound): K60 >60 L64 >50 E66 8 D93 ~0 Residues divided into 2 groups: Protection factor ! 7 Protection factor " 3
Cross-saturation Takahashi et al(2000)Nat.Struc.Biol.7220-223 Protein Proteir Strong binding case 1 NH -CH cross-saturation etoe2laosmcean子ame23taionsapdyansendbyspndmsonoe Cross-saturation to theNHin the Measure peak vstime of saturation tofind residues in more quickly. 言06 N9 04 Chemical shift Cross-saturatior H0215AN 0.2 ppm
Cross-saturation Takahashi et al (2000) Nat. Struc. Biol. 7 220-223 -NH -CH -15NH -C2H Protein 2 Protein 1 R.F. Band-selective proton saturation, followed by TROSYHSQC Saturate the aliphatics of protein 2 - magnetization is rapidly transferred by spin diffusion to the rest of protein 2 i.e. aromatics and amides. Cross-saturation to the 15NH in the interface on protein 1 Measure peak intensity vs time of saturation to find residues in interface Strong binding case cross-saturation Plot intensity ratio vs saturation time. Residues in the interface get saturated more quickly. X-ray structure - buried residues Chemical shift perturbation (#HN2 + 0.15#N2)1/2 > 0.2 ppm H/D exchange protection factor > 10 Cross-saturation (red> light blue > yellow > green > dark blue)