Protein/Protein and Protein/Ligand interactions in large and dynamically disordered systems studied by NMR in solution

65 Å

SubstrateSubstrate

Protein/Protein and Protein/Ligandinteractions in large and dynamically

disordered systems studied by NMR insolution

Prof. K. Pervushin, BioNMR group , LPC, D-CHAB, ETH Zürich

An overview

-Construction of optimal polarization transfer schemes for 220 kDa complex, CR1(SCR 15-17)/C3b

- 54 kDa dimeric chaperone FkpA and FkpA/substrate complexes

The primate erythrocyte/immune complex clearing mechanism

Human complement receptor type 1 (CR1)

INEPT-based HSQC of 220 kDa CR1/C3b complex

2 (1H) [ppm]

1 (15N) [ppm]

Fundamental bounds associated with polarization/coherence transfer imposed by qunatum spin dynamics

C

1. Maximum transfer bound,

U

2. Minimal spin-evolution time required for the transfer, min

3. Suppression of spurious transfers, Q

4. Combined use of more source operators, C

Differential driving of the manifolds Iand I by

selective rf-pulse

Iz = Iz+ I z → Iz

I z = 2Iz Sz

Ii= Ii(1/2E +Sz)

Ii= Ii(1/2E Sz) Iz

I z

Excitation profile of polychomatic pulse

Polychomatic pulse wave-form and spin trajectory

Polarization transfer using polychromatic irradiation

2 (1H) [ppm]

1 (15N) [ppm]

CRINEPTPOLY-C

PC-SPI spectra of free CR1 and CR1/C3b complex

CR1/C3b complex

CR122 kDa

CR1/C3b complex220 kDa

54 kDa dimeric chaperone FkpA and FkpA/substrate complexes

54 kDa „moonlight“ chaperone with PPIase activity

65 Å

SubstrateSubstrate

54 kDa „moonlight“ chaperone with PPIase activity

15N relaxation measurements of free FkpA at 600 MHz

R1(1/s)

0

0.5

1

1.5

2

0 50 100 150 200 250

R2(1/s)

0

20

40

60

80

100

0 50 100 150 200 250

R1,R2 rates of (15N) is function of local (as well as global) mobility

15N relaxation measurements with FkpA at 600 MHz

1H-15N RDCs measurements in the presence of Pf1 phages

Histogramm of RDCs values in two media

C12E5 / hexanol/H2OLn-Alkyl-poly(ethylene glycol)/n-alkyl alcohol and glucopone/n-hexanol mixtures

Phages Pf1

RDCs values in 2 alignment media

y = 0.4234x - 5.498

R2 = 0.4133

-40

-20

0

20

40

-40 -20 0 20 40

RDCexp

RD

Cth

eo

y = 0.3991x - 3.3923

R2 = 0.3668

-40

-20

0

20

40

-40 -20 0 20 40

RDCexp

RD

Cth

eo

y = 0.9177x - 0.1621

R2 = 0.9058

-40

-20

0

20

40

-40 -20 0 20 40

RDCexp

RD

Cth

eo

y = 0.9024x - 1.8318

R2 = 0.902

-30

-20

-10

0

10

20

30

40

-30 -20 -10 0 10 20 30 40RDCexp

RD

Cth

eo

y = 0.7514x - 1.205

R2 = 0.7349

-40

-20

0

20

40

-40 -20 0 20 40

RDCexp

RD

Cth

eo

y = 1.1474x - 4.2329

R2 = 0.5872

-40

-20

0

20

40

-30 -20 -10 0 10 20 30 40

RDCexp

RD

Cth

eo

C-domainin dimer

C-domainin monomer

N-domainin monomer

A schematic model of intramolecular dynamics in FkpA

Chemical shift changes by complex formation with (1) reduced and carboxymethylated bovine -lactalbumin, (2) RNAse AS

Mapping of chemical shift changes induced by interactions with substrate

15N relaxation measurements of FkpA in free and complex with RNAse AS

R1(1/s)

0

0.5

1

1.5

2

0 50 100 150 200 250

R2(1/s)

0

20

40

60

80

100

0 50 100 150 200 250

R1,R2 rates of (15N) is function of local (as well as global) mobility

R2

0

20

40

60

80

100

0 50 100 150 200 250

R1

0

0.5

1

1.5

2

0 50 100 150 200 250

Equilibrium binding of FkpA to substrates: (1) reduced and carboxymethylated bovine -lactalbumin, (2) RNAse AS

Kd = 540 m

Protein Quality Control in the ER

Substrates recognized by GT

RNase B RNase BS RNase BS protein

alkylated RNase B

- +-GT:

RNase BS”

S peptide 15-mer

scrambled RNase B

small glyco-peptides

+ - - -

RNase A

Crystal and NMRstructure available

• 124 amino acids• 4 disulfide bonds

RNase A S protein

?Ratnaparkhi, G. S., and Varadarajan, R. (2001) J Biol Chem 276, 28789-98.

Chakshusmathi, G., Ratnaparkhi, G. S., Madhu, P. K., and Varadarajan, R.(1999) PNAS 96, 7899-7904.

“NMR … is not possible due to aggregation at millimolar concentration”

No structural dataavailable

• 124 amino acids• 4 disulfide bonds

RNase A 15N-1H HSQC

RNase A:complete assignmentis available

Assignment of S-Protein

6.007.008.009.0010.00

105.00

110.00

115.00

120.00

125.00

130.00

7498

99

62

94

96 41 91

124

60

61

72

70 68

112

123

77

6597

40?

76

109

124

100

75

7144?

83

120?

6395

111

7964

56

5790

21

69 28?

30?

78 67

113

58

59

`39?

46

110

1H (ppm)

15N (ppm)

RNase S Protein:• Line broadening• Resonance doubling

RNase S:an additional set of resonances is observed

RNase A:complete assignmentis available

S peptide

cleavage

conformational exchange

Chemical Shift Difference between S protein and RNase A

Fast Amide Proton Exchange

Highest: < 2 s-1

(by magnetization transfer)

Lowest: 35 min-1

(by 1H/2D – exchange)

15N-Relaxation measurements

ct-XY-TROSY

CCR

Model Free

Rex by cross-correlated relaxation

red - second resonance set observed

Model free analysis

RNase S Protein - Concentration Scan

1.06 mM

H (ppm)1

15N (ppm)

0.2 mM

RNase S Protein - Concentration Scan

H (ppm)1

15N (ppm)

0.08 mM

RNase S Protein - Concentration Scan

1H (ppm)

15N (ppm)

Ratio between peak volumes correspondingto different oligomerization states of RNAse A S protein

Kd=1.2±0.08 mM

2 S-Prot [S Prot]2

RNase S Protein - Concentration Scan

15N (ppm)

H (ppm)1

Yanshun Liu et al. Protein Sci 2002; 11: 1285-1299

Fig. 2. Ribbon diagrams of the structures of the RNase A monomer (2.0 A, Wlodawer et al)

Yanshun Liu et al. Protein Sci 2002; 11: 1285-1299

Fig. 3. Ribbon representations of hypothetical models of RNase A tetramers

Mapping of “dimer” cross-peaks to monomeric and dimeric RNAse structures

High pressure NMR with S-protein

MD

Lys60

Dilution versus titration with chaperone

Dilution of S protein Titration with FkpA Chaperone

Lys 60

Leu 91

S Protein titration by the chaperone

Conformational dynamics in S Protein

S Protein N S Protein Uku

kf

[S Protein]n

>30ms

~80 Hz

/(2 2) 30Hzck

kc

15N relaxation measurements of FkpA/S-protein complex

R1[ 1/ s]

0

0. 5

1

1. 5

2

0 50 100 150 200 250

R2[1/ s]

0102030405060708090

100

0 50 100 150 200 250

A „mother‘a arms“ model of chaperone activity of FkpA

Thanx a lot!

Alexander Eletski Prof. Donald Hilvert

Beat Vögeli Prof. Linda Thöny-Meier

Dr. Osvaldo Moreira Prof. Andreas Plückthun

Kaifeng Hu Dr. Helena Kovac (Bruker AG)

Alexander Kienhoffer

Dr. Maria Johansson

Simon Alioth

Katherina Vamvaca

Dr. Krystina Bromek

Veniamin Galius

SNF and ETH for financial support

Prof. Paul Barlow

Prof. Ari Helenius Dr. Christiana Ritter