Table 2 Estimates of Km and kobs for neddylated CRL2*-mediated poly-ubiquitin chain formation

From: Mechanism of millisecond Lys48-linked poly-ubiquitin chain formation by cullin-RING ligases

UBAa

UBDb

UBE2Rc

RBX1

Kmd (µM)

Fold change (Km)

kobsS1-S2 (s−1)

Fold change (kobs)

WT

K48R

WT (R2)

WT

111.33 ± 7.58

–

45.55 ± 2.88

–

WT

K48R

WT (R1)

WT

310.77 ± 43.37

–

33.60 ± 1.68

–

Interface 1: synergy loop–UBA

H68A

K48R

WT (R2)

WT

815.48 ± 70.88

7.3

64.37 ± 2.71

0.71

R54D

K48R

WT (R2)

WT

>800

>7

17.02 ± 0.96

2.7

N60R

K48R

WT (R2)

WT

200.45 ± 21.71

1.8

–

–

WT

K48R

D143K (R2)

WT

1,261.60 ± 132.41

11.3

27.01 ± 1.03

1.7

WT

K48R

R149D (R2)

WT

>1,200

>10

29.40 ± 1.41

1.5

R54D

K48R

D143K (R2)

WT

334.51 ± 59.99

3

31.68 ± 1.49

1.4

N60R

K48R

R149D (R2)

WT

269.36 ± 28.66

2.4

38.32 ± 2.30

1.2

WT

K48R

D103A (R2)

WT

563.97 ± 99.14

5.1

31.67 ± 1.02

1.4

Interface 2: synergy loop–synergy loop

WT

K48R

D102A (R2)

WT

147.66 ± 8.03

1.3

24.94 ± 1.35

1.8

WT

K48R

R113A (R2)

WT

>1,000

>9

24.71 ± 3.24

1.8

WT

K48R

R113A (R1)

WT

>1,200

>4

22.15 ± 0.87

1.5

Interface 3: synergy loop–UBD

WT

K48R

S106A (R2)

WT

384.62 ± 48.04

3.5

–

–

WT

K48R

S106R (R2)

WT

>1,000

>9

16.84 ± 1.40

2.7

WT

K48R

E112A (R2)

WT

>1,000

>9

11.61 ± 1.01

3.9

WT

K48R/R74E

WT (R2)

WT

1,667.90 ± 180.82

15.0

21.37 ± 0.74

2.1

WT

K48R/R74E

WT (R1)

WT

>1,200

>4

22.91 ± 1.91

1.5

Interface 4: synergy loop–RING domain

WT

K48R

E108A (R2)

WT

>1,000

>9

13.18 ± 1.50

3.5

WT

K48R

E108A (R1)

WT

>1,200

>4

15.29 ± 0.66

2.2

WT

K48R

E108R (R2)

WT

>1,200

>10

5.17 ± 0.21

8.8

WT

K48R

WT (R2)

R91E

>1,680

>15

24.67 ± 1.59

1.8

WT

K48R

WT (R1)

R91E

>1,200

>4

8.92 ± 0.62

3.8

Interface 5: multiple interfaces

WT

K48R

D102A/103A (R2)

WT

924.45 ± 126.37

8.3

16.21 ± 0.73

2.8

WT

K48R

E108/112A (R2)

WT

>1,200

>10

4.23 ± 0.14

10.8

R54D

K48R

E108A (R2)

WT

>800

>7

0.89 ± 0.06

51.2

R54D

K48R

E108A (R1)

WT

>800

>2.5

0.68 ± 0.06

49.4

WT

K48R/R74E

E108A (R2)

WT

>1,200

>10

3.68 ± 0.33

12.4

  1. *Experiments estimating kobs included Elongin B/C–FEM1C and Sil1-ubiquitin substrate; aAcceptor ubiquitin; refers to either unanchored ubiquitin for Km (D77 ubiquitin; see Methods) or conjugated to Sil1 peptide (kobs); bDonor ubiquitin; cUBE2R1 and UBE2R2 paralogs are denoted as (R1) and (R2), respectively; dKm of unanchored acceptor ubiquitin for UBE2R2, ‘>’ denotes the highest concentration of the dilution series in cases for which Km could not be estimated; S1, Sil1-ubiquitin; S2, Sil1-ubiquitin2. The standard error of measurements are shown for all estimates.
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