Thermal stability of I-AniI variants

Y2 : F13Y / S111Y

M5 : F13Y / I55V / F91I / S92T / S111Y

M5 I-AniI is less stable than WT I-AniI and Y2 variant.

In vitro cleavage assay

WT AniIsite

LIB4site

I-AniI variants displayed higher cleavage activity against WT AniI site than WT enzyme,

but showed the same cleavage efficiency against LIB4 site.

Binding assay by ITC

Enzyme Targ et site

KD (nM)

H (kcal/mol)

S (cal/mol/deg)

-T S (kcal/mol)

G (kcal/mol)

WT I-AniI WT AniI 96 ± 10 11.2 ± 1.1 69.3 ± 3 - 21.0 - 9.8 WT I-AniI LIB4 8 ± 1 6.6 ± 0.7 59.0 ± 3 - 17.9 - 11.3 Y2 I-AniI WT AniI 11.6 ± 3.0 17.1 ± 1.0 93.0 ± 3.1 - 28.2 - 11.0 Y2 I-AniI LIB4 13.9 ± 2.0 14.8 ± 2.4 84.7 ± 7.6 - 25.7 - 10.9

WT I-AniI Y2 I-AniI

Substrate : WT AniI site

Binding specificity

WT I-AniI Y2 I-AniI

-0.4

-0.2

0

0.2

0.4

0.6

0.8

1

1.2

-10 -9 -8 -7 -6 -5 -4 -3 -2 -1 1 2 3 4 5 6 7 8 9 10

A C G T

-0.4

-0.2

0

0.2

0.4

0.6

0.8

1

1.2

-10 -9 -8 -7 -6 -5 -4 -3 -2 -1 1 2 3 4 5 6 7 8 9 10

T G A G G A G G T T T C T C T G T A A A T G A G G A G G T T T C T C T G T A A A

Re

lativ

e b

ind

ing

aff

inity

WT I-AniI and Y2 variant showed similar binding profile against the target site with single

mismatches, except for the central 4 base positions.

WT I-AniI Y2 I-AniI M5 I-AniI

Thermal stability + + ±

Cleavage activity+ (WT)

++ (LIB4)++ (WT)++ (LIB4)

++ (WT)++ (LIB4)

Binding affinity+ (WT)

++ (LIB4)++ (WT)++ (LIB4)

N/A

Binding specificity + + N/A

Cleavage efficiency in bacteria- (WT)

- (LIB4)++ (WT) ++ (WT)

Another property might contribute to the in vivo cleavage activity.

Cleavage activity at various temperatures

I-AniI variants showed better cleavage efficiency below 37 °C than WT I-AniI.

Why do I-AniI variants possess stronger activity?

F13Y might influence hydrophibic

network to stabilize the protein core

fold.

This would confer the flexibility with the

instability to the first LAGLIDADG motif.

S111Y would make a novel contact with a DNA backbone.

S111Y111

Why do I-AniI variants possess stronger activity?

I55V might make a new a contact with a

DNA backbone (?).

F91I might influence the hydrophobic interactions with residues on the second LAGLIDADG motif.

S92T might cause a steric clash between C of T92 and L177.

L177

Crystallization of Y2 I-Ani with WT AniI site

Hopefully, coming soon.