Biosensors to detect enzyme-ligand andProtein-protein interactions

-Physical parameters in binding studies-principles, techniques and instrumentation-Methods to probe non-covalent macromolecular interaction (stopped-flow, BIAcore, and Microcalorimetry)

Lecturer: Po-Huang Liang 梁博煌 , Associate Research FellowInstitute of Biological Chemistry, Academia SinicaTel: 27855696 ext. 6070

Stopped-flow for measurements of protein-protein and protein-small molecule interaction

A B

Flow CellLight

Stop SyringeFluorescence Signal

Absorbance Signal

Substrate binding kinetics

E ESk1[S] Rate = d[E]/dt = -k1[S][E]

d[E]/[E] = -k1[S]dtln([E]t / [E]o) = -k1[S]t[E]t = [E]o exp (-k1[S]t)[ES] = [E]o-[E]t = [E]o(1-exp (-k1[S]t))kobs = k1 [S]

E ESk1[S]

kobs = k1[S] + k-1

The slope of kobs vs [S] gives kon and intercept gives koff

k-1

3-D structure of E. coli UPPsTwo conformers were found: one (closed form) with Triton bound and the other (open form) has empty active site

Ko, T. P. et al, (2001) J. Biol. Chem 276,47474-47482.

Substrate-binding site

UPPs kcat (s-1) Km (FPP) (M) Km (IPP) (M) relkcat

a

wild type 2.5 0.1 0.4 4.1 0.3 1

akcat relative to that of wild type

¡Ó0.1¡Ó ¡Ó

49

3.0 27

1 x 10-4

¡Ó

¡Ó5

0.7 ¡Ó

0.7 ¡Ó0.1(2.4 0.1) x 10-4¡ÓR194A

R200A (2.5 0.2) x 10-3¡Ó 3 1 x 10-3

W31F 1.1¡Ó0.1 2.0 ¡Ó0.3 5.9 ¡Ó0.8 0.4

R30A 1.2 ¡Ó0.3 1108 ¡Ó250

0.4¡Ó0.1 1.6 ¡Ó0.3 65 ¡Ó10 0.2R39A

(3.0 0.3) x 10-2¡Ó 1 x 10-2

0.7 ¡Ó0.1 16 ¡Ó2N28A (3.0 0.3) x 10-2¡Ó 1 x 10-2

H43A (2.6 0.2) x 10-3¡Ó 3 ¡Ó0.3 63 ¡Ó3.6 1 x 10-3

14.1 1.40.5

0.7 280 20

1.3 x 10-3

1 x 10-2

(3.30 0.03) x 10-3

(2.60 0.02) x 10-2

D26A

E213A ¡Ó

¡Ó0.1

0.1

¡Ó

¡Ó

¡Ó

¡Ó

The amino acids in 1 area are important for catalysis and substrate bindingD26 is located in a P-loop conserved for pyrophosphate binding

Pan et al., (2000) Biochemistry 39, 13856-13861

Large L137 on the bottom controls product chain length

upper: + Tritonbottom: no Triton

Long-lived intermediate C30 formed by A69Land C35 by L67W

A69L

L67W

Active site topography of UPPs

L137

A69

Flexible loop

32

1

O P O P O--O

-O

O O

-O

PO

O-

OP

O

O-

O

chain elongation

E213D26 Mg2+

Mg2+

L67BD

Synthesis of FsPP to Probe UPPs Conformational Change Chen et al.(2002) J. Biol. Chem. 277, 7369-7376.

P

O

MeO OMeOMe

1 equiv Bu4NOHP

O

MeO O-

OMeP

O

MeO OOMe

POMe

S

OMe

5.64 equiv TMSI

24 h, 94%100 oC Acetonitrile

-35 oC, 30 min

1 equiv (OMe)2P(S)Cl

-35 oC rtover 6 h

30~35%

P

O

TMSO OOTMS

PSTMS

O

OTMS

P

O

-O OO-

PS-

O

O-

Bu4NOH/H2OP

O

-O OO-

PS

O

O-

0.45 equiv farnesyl chloride

Acetonitrile25 oC

6 h, 70%

3 NH4+

FsPP

Ki of FsPP as an inhibitor = 0.2 M kcat of FsPP as an alternative substrate = 3 x 10-7 s-1

Stopped-Flow experimentsUPPs-FPP + IPP

UPPs-FsPP + IPP

Binding rates vs. [IPP] gives IPP kon = 2 M-1 s-1

3 phases in 10 sec

2 phases in 0.2 sec

1 phase in 0.2 sec

300 320 340 360 380 400 420 4400

500

1000

1500

2000

2500

3000

3500Fl

uore

scen

ce In

tens

ity (a

.u.)

Wavelength (nm)

300 320 340 360 380 400 420 4400

500

1000

1500

2000

2500

3000

3500

Fluo

resc

ence

Inte

nsity

(a.u

.)

Wavelength (nm)

300 320 340 360 380 400 420 4400

500

1000

1500

2000

2500

3000

3500

Fluo

resc

ence

Inte

nsity

(a.u

.)

Wavelength (nm)

Different level of Trp fluorescence quench by FPP

wild-type W31F has less quench

W91F has almost no quench

FPP binding mainly quenches thefluorescence of W91, a residue inthe 3 helix that moves toward theactive site during substrate binding

FPP binding does not require Mg2+; IPP binding needs Mg2+

FPP (or FsPP) quenches the UPPs Intrinsic fluorescence even in the absence of Mg2+

+ Mg2+

Mg2+ is required fro IPP binding

The role of a flexible loop of residues 71-83

The invisible loop in the E. coli UPPs structure is responsible to bring IPP to the correct position and orientation to react with FPP

UPPs kcat (s-1) Km (FPP) (M) Km (IPP) (M) relkcat

a

wild type 2.5 0.1 0.4 4.1 0.3

133 14

16.2 2.2

1.0

0.4

8

1.6 15.7

1

0.04

0.1

0.01

0.30 0.01

S71A

E73A

N74A

akcat relative to that of wild type

¡Ó

¡Ó

¡Ó

¡Ó

¡Ó

0.1

0.2

0.1

0.6

0.3

¡Ó

¡Ó ¡Ó

¡Ó

¡Ó

¡Ó¡Ó

E81A 0.4 0.1¡Ó 0.4¡Ó0.1 88 10¡Ó 0.2

0.4 ¡Ó0.1(2.20 0.03) x 10-2¡Ó

R77A (1.4 0.1) x 10-4 5 x 10-5

2.5

0.11 0.01¡Ó

W75A 1.1¡Ó0.1 3.2 ¡Ó0.3 46 ¡Ó4 0.5

Ko et al., (2001) J. Biol. Chem. 47474-47482

Model of UPPs conformational change during catalysis

Flexible loop

32

1

O P O P O--O

-O

O O

-O

PO

O-

OP

O

O-

OFlexible loop

32

1

Mg2+

closed-form open-form

Chain elongation

W75W31

binding release

L137

W91

E213 -O

P

O

O-

OP

O

O-

O

OPP

PPOPPO

Inhibitor

Fluorescent probe for ligand interaction and inhibitor binding

Chen et al., (2002) J. Am. Chem. Soc. In press

OH O O O OOH

O OBr

O OOO

R

O

O OHOO

R

O BrOO

R

O OOO

R

P O

OP O-

O

O-O-

1 2 3

4 5a R = CH35b R= CF3

(a) DHP, PPTs, CH2Cl2, 90%; (b) SeO2, t-BuOOH, salicylic acid, CH2Cl2, 32%; (c) NBS, Me2S, CH2Cl2, 92%; (d) 5a. K2CO3, DMF, 7-hydroxy-4,8-dimethylcoumarin, 98%, 5b. K2CO3, DMF,7-hydroxy-8-methyl-4-trifluoromethylcoumarin, 94%; (e) PPTs, EtOH, 6a. 99%, 6b. 92%; (f) CBr4, PPh3, CH2Cl2, 7a. 87%, 7b. 80%; (g) (n-Bu4N)3HP2O7, CH3CN, 8a. 54%, 8b. 46%.

a b

c d

e f

g

6a R = CH36b R= CF3

7a R = CH37b R= CF3

8a R = CH38b R= CF3

3 NH4+

Synthesis of Fluorescent Substrate Analogue

400 450 500 550 6000

200

400

600

800

1000

1200

Fluo

resc

ence

Inte

nsity

(a.u

.)

Wavelength (nm)

0.0 0.5 1.0 1.5 2.0 2.5 3.0

200

300

400

500

600

700

Fluo

resc

ence

Inte

nsity

(a.u

.)

Concentration ()

Characterization of the fluorescent probe

(A) Fluorescence is quenched by UPPs and recovered by replacement with FPP(B) Probe binds to UPPs with 1:1 stoichiometry

(A) (B)

(C) (D)

(C ) Probe binds to UPPs with a kon = 75 M-1 s-1

(D) Probe releases from UPPs (chased by FPP) with a koff = 31 s-1

Substrate and product release rate

FPP is released at 30 s-1 UPP is released at 0.5 s-1

Can this method apply to drug-targeted prenyltransferases to find non-competitive inhibitor?

IPPE + FPP E-FPP

fast

30 s-1E-FPP-IPP E-C20

E-C25 E-C30 E-C35 E-C40

E-C45 E-C50 E-C55 E + C55

2.5 s-1 2 s-1

3.5 s-1 2.5 s-1 3 s-1 3.5 s-1

3 s-13.5 s-1 0.5 s-1

2 M-1 s-1

Reaction: DHF + NADPH THF + NADP+

Association:

Competition experiments to measureDissociation rate constants usingStopped-flow

Rate constant for the pre-steady-state burstmeasured by stopped-flow energy transfer

Uisng NADPH, 450 s-1 is followed by a 12 s-1 steady-state rate.Using NADPD, 150 s-1 is followed by the same rate at pH 6.5, isotope effect kH/kD =3

Pre-steady-state rate is decreased with pH

Observed rate constants for hydride transfer as a function of pHand predictable kinetic behavior

Interaction of colicin E7 and immunity 7

Entrance of colicin E is through the BtuB (vitamin B12 receptor) and TolA

kon = 4 x 109 M-1 s-1

koff = 3.7 x 10-7 s-1

Kd = koff /kon = 7.2 x 10-17 M

Association kinetics of ColE9-Im9 complex (lower) and E9-DNase-Im9 (upper), [ColE9]

= 0.35 M, [Im] = 1.75 – 7 M.

Wallis et al., Protein-protein interactions in colicin E9 Dnase – immunity protein complexes. 1. Diffusion controlled association binding for the cognate complex Biochemistry 1995, 34, 13743-13750

Dissociation kinetics of ColE9-Im9 complex at 0 and 200 mM NaCl. The preincubated E9with [3H]Im (6 M) was mixed with unlabeled Im (54 M)

N + I NI* NIk1

k-1

k2

K-2

BIACORE (Biosensor)

Sensor chip and couplingCM5: couple ligand covalently

NTA: bind His-tagged lignadSA: capture biotinylated biomolecules

HPA: anchor membrane bound ligand

SPR: surface plasmon resonance

Objects of the experiments

•Yes/No binding, ligand fishing•Kinetic rate analysis ka, kd

•Equilibrium analysis, KA, KD

•Concentration analysis, active concentration, solution equilibrium, inhibition

Control of flow rate (l/min) and immobilized level (RU)for different experiments

Definition

•Association rate constant: ka (M-1 s-1)---Range: 103 to 107

---called kon, k1

•Dissociation rate constant: kd (s-1)---Range: 10-5 to 10-2

---called koff, k-1

•Equilibrium constant: KA (M-1), KD (M)---KA = ka/kd = [AB]/[A][B]---KD = kd/ka = [A][B]/[AB]---range: pm to uM

A + B ABka

kd

Association and dissociation rate constant measurements

A + B ABka

kd

In solution at any time t : [A]t = [A]o – [AB]; [B]t = [B]o – [AB]d[AB]/dt = ka[A]t[B]t – kd[AB]tIn BIAcore at any time t: [A]t = C; [AB] = R; [B]o = Rmax thus [B]t = Rmax – Rd[R]/dt = ka*C*(Rmax-Rt) – kd (R)

It

It is easy to mis-interpret the data

Distinguish between fast bindingand bulk effect: use referenceor double reference

Two ways to overcome mass transfer limitation: 1.increase flow rate2. reduce ligand density

Example 2: Lackmann et al., (1996) Purification of a ligand for the EPH-like receptor using a biosensor-based affinity detection approach. PNAS 93, 2523 (ligand fishing)

HEK affinity column

(A) Phenyl-Sepharose(B) Q-Sepharose

Ion-exchangeRP-HPLC

The ligand is Al-1, which is previous found as ligand for EPH-like RTK family

BIAcore analysis of bovine Insulin-like Growth Factor (IGF)-binding protein-2Identifies major IGF binding site determination in both the N- and C-terminal domainsJ. Biol. Chem. (2001) 276, 27120-27128.

IGFBPs contain Cys-rich N- and C-terminal and alinker domains. The truncated bIGFBP-2 weregenerated and their interaction with IGF werestudied.

Lane 2: 1-279 IGFBP-2HisLane 3: 1-132 IGFBP-2Lane 4: 1-185 IGFBP-2Lane 5: 96-279 IGFBP-2HisLane 6: 136-279 IGFBP-2His

MicroCalorimetry System Right: ITC (Isotheromal titration Calorimetry)

Inject “ligand” into “macromolecule”

A small constant power is applied to the reference To make T1 (Ts – Tr) negative. A cell feed-back(CFB) supplies power on a heater on the sample cell to drives the T1 back to zero.

Binding isotherms

Simulated isotherms for different c valuesc = K (binding constant) x macromoleculeconcentrationc should be between 1 and 1000Make 10-20 injections

can be used to obtain binding affinity or binding equilibrium constant (Keq),molecular ration or binding stoichiometry (n),And heat or enthalpy (H).

Signaling pathway of GPCR and RTK

Activation of Ras following binding of a hormone (e.g. EGF)to an RTK

GRB2 binds to a specific phosphotyrosine on the activated RTK and to Sos, which in turn reacts with inactive Ras-GDP. The GEF activity of Sos then promotes theformation of the active Ras-GTP.

Example: O’Brien et al., Alternative modes of binding of proteins with tandem SH2 domains (2000) Protein Sci. 9, 570-579

(A) pY110/112 bisphosphopeptide binds to ZAP70 showing a 1:1 complex

(B) Monophoshorylated pY740 binds to p85 with two binding events

(C) Binding of pY740/751 peptide intop85. The asymmetry of the isotherm shows two distinct binding eventsshowing that an initial 2:1 complex of protein to peptide is formed. As further peptide is titrated, a 1:1 complex is formed.

ITC data for the binding of peptides to ZAP70, p85, NiC, and isolated c-SH2 domain

KB1 and KB2 correspond to the equilibrium binding constants for the first and the second binding events.

Conformational change of two SH2 binding with phosphorylated peptide

(A) Primary sequence NiC(B) a. NiC; b.NiC + bisphosphorylated peptide (C ) a. N-terminal SH2 alone; b.N-terminal SH2 + pY751 peptide; c. C-terminal SH2; .d. C-terminal SH2 + pY751 peptide

Model for binding of bisphosphorylated peptide to the SH2 domain

(A) For AZP70, SH2 protein:peptide = 1:1(B) For p85 (or NiC), initial titration results in peptide: SH2 protein = 0.5:1, adding more peptide to reach 1:1 complex.

Interactions between SH2 domains and tyrosinephosphorylated PDGF – receptor sequences

(A) SH2 protein only binds to Phosphorylated Y751P peptide(B) The inclusion of competing peptide in the buffer yields first-orderdissociation

The N-terminal SH2 domain bound with high affinity to the Y751P peptide but not to the Y740P, whereas C-terminal SH2 interacts strongly with both

Panayotou et al., Molecular and Cellular Biology (1993) 13, 3567-3576

Thomas et al., (2001) Kinetic and thermodynamic analysis of the interactionsOf 23-residue peptide with endotoxin. J. Biol. Chem. 276, 35701-35706.