The interaction analysis oflinker histone and nucleosome
for developing new nucleosome ligand
2023/08/04
Literature Seminar2
M1 Takeuchi

1Contents

Contents◆Introduction:◆Main:➢Mouse, drosophila: Off-dyad binding mode (model)➢Chicken: On-dyad binding mode (crystal structure)➢Human: Distinct Structures of chromatosomeswith different human linker histone isoforms (cryo-EM)◆Summary2

Contents

Contents◆Introduction:◆Main:➢Mouse, drosophila: Off-dyad binding mode (model)➢Chicken: On-dyad binding mode (crystal structure)➢Human: Distinct Structures of chromatosomeswith different human linker histone isoforms (cryo-EM)◆Summary3

Introduction

Histone post-translational modification (PTMs)

・PTMs have a significant impact on the structure and function of proteins.
・Histone PTMs can change the structure of chromatin.
・Dysregulation of histone acetylation can result in the development of diseases.
→ Regulation of histone acetylation levels can lead to the development ofnovel therapeutic strategies.

4

Introduction

Chromatin acetylation by an artificial catalyst system

・Chemical catalysts enables various PTMs in various positions ofproteins and histones.
Ishiguro, T. et al., Chem, 2017, 2(6), 840-859.

Amamoto, Y. et al., JACS, 2017, 139(22), 7568-7576.

5

Introduction

Existing strategy: limited acetylation position (near acidic patch)

The use of LANA as the ligand→ H2BK120, H2BK108, H2BK43→ Limited acetylation position (only near histone acidic patch)

6

Introduction

New nucleosome ligand candidate: linker histone H1
Sequence alignment of the linker histone H1 globular domains

mH1 (20-97)(cH5: chicken linker histone H1 (H5), hH1.0: human, dH1: Drosophila, mH1 : mouse)

Structural feature of linker histone・NTD contributes little tonucleosome binding

198

2324197

97

Co-crystal Structure of the GH5 (GD of H5) andthe nucleosome with linker-DNA (3.5Å)

・GD can bind to the nucleosome.
・CTD interacts with linker DNA andis important for higher-affinity
binding of linker histones to thenucleosome.

Zhou, B. R. et. al., Molecular Cell, 2015, 59(4), 628-638

7Introduction

Lysine residues near the dyad: H2AK118, H3K115, H3K122

H2AK118

H1K68

H1K54
H1K51

H1K5490°

90°
H3K122

H2AK118H3K115

H3K115

H1K68
H1K51

H3K122(hH1.0 and chromatosome)(PDB: 7k5x)

8Introduction

PTMs of H2AK118, H2AK119, H3K115, H3K122

H2AK118: acetylation, methylation, ubiquitination, crotonylation,2-hydroxyisobutyrylation, β-hydroxybutyrylation
H2AK119: ubiquitination, crotonylation, β-hydroxybutyrylation
H3K115:acetylation, butyrylation

H3K122:acetylation, methylation, succinylation, crotonylation,malonylation, butyrylation, 2-hydroxyisobutyrylation,β-hydroxybutyrylation
Liu, J. et. al., Journal of Cellular and Molecular Medicine, 2021, 25(15), 7101-7109

9

Contents

Contents◆Introduction:◆Main:➢Mouse, drosophila: Off-dyad binding mode (model)➢Chicken: On-dyad binding mode (crystal structure)➢Human: Distinct Structures of chromatosomeswith different human linker histone isoforms (cryo-EM)◆Summary10Off-dyad mode (model)

FRAP (Fluorescence Recovery After Photobleaching)mouseThe concept of FRAP experiment

Shorter recovery half time (t50) → weaker binding affinity
Sequence alignment of the mGH1 (GD of mouse H1) (20-97)

FRAP (WT: mH1-GFP)(mouse BALB/c 3T3 cells)

Brown, D. T. et. al., Nature Structural and Molecular Biology, 2006, 13(3), 250-255 11

Off-dyad mode (model)

Definition of two distinct binding sites of mouse GH1
mouseSequence alignment of the mGH1 (GD of mouse H1) (20-97)

FRAP (WT: mH1-GFP) (mouse BALB/c 3T3 cells)
Map of the interaction surface of mGH1
Site 2 (flexible domain, linker DNA)

Site 1(inflexible domain, dyad DNA)

Nine positively charged residues mutants:
t50 < 20 s and t80 < 90 s(exception: K55)

Brown, D. T. et. al., Nature Structural and Molecular Biology, 2006, 13(3), 250-255 12

Off-dyad mode (model)

Definition of two distinct binding sites of drosophila GH1
Sequence alignment of the dGH1 (GD of drosophila H1)

drosophila

ITC (Isothermal Titration Calorimetry)
Map of the interaction surface of dGH1
Site 2Site 1Zhou, B. R. et. al., PNAS, 2013, 110(48), 19390-19395

13

Off-dyad mode (model)

Proposed binding mode model (Off-dyad binding mode)
Sequence alignment of the GH1 (GD of drosophila H1)

drosophila

Map of the interaction surface of dGH1
Site 2• K91 and K95 were forced tointeract with the nucleosomal DNA
near the dyad. (①)• K58, K102, K107, and K116 wereSite 1

Off-dyad binding mode (model)

forced to interact with the nearby
linker DNA. (②)• ①, ② → docking calculation• It is possible that GH1 may interact
with both DNA linkers,

one strongly and the other weakly.
(using HADDOCK program)Zhou, B. R. et. al., PNAS, 2013, 110(48), 19390-19395

14

Contents

Contents◆Introduction:◆Main:➢Mouse, drosophila: Off-dyad binding mode (model)➢Chicken: On-dyad binding mode (crystal structure)➢Human: Distinct Structures of chromatosomeswith different human linker histone isoforms (cryo-EM)◆Summary15

On-dyad mode (crystal structure)

Detailed interactions between GH5 and linker-L1chicken• Crystal structure is consistent withon-dyad binding mode.
• R94 has stable interaction with thelinker-L1.
• K40, R42, and K97 have substantial(3.5 Å resolution)

local dynamic motions.

GH5: GD of chicken linker histone H1 (H5)Zhou, B. R. et. al., Molecular Cell, 2015, 59(4), 628-638

16On-dyad mode (crystal structure)Detailed interactions between GH5 and the DNA at the dyad
chicken• R47 has stable interaction with thedyad.

• K85 has substantial local dynamicmotions.
Zhou, B. R. et. al., Molecular Cell, 2015, 59(4), 628-638

17

On-dyad mode (crystal structure)

Detailed interactions between GH5 and the DNA at the dyad
chicken• The charge-neutral residues Q48,
V87, and S90 insert into the minor

groove of the DNA at the dyad

Zhou, B. R. et. al., Molecular Cell, 2015, 59(4), 628-638

18

On-dyad mode (crystal structure)

Detailed interactions between GH5 and linker-α3
chicken• H25, Y28, Q67 are in close contact
with the linker-α3Zhou, B. R. et. al., Molecular Cell, 2015, 59(4), 628-638

19

On-dyad mode (crystal structure)

Detailed interactions between GH5 and DNA near the dyad and linker-α3chicken• R73, R74 have stable interaction
with the dyad and linker-α3.
• K69 has substantial local dynamic

motions.

Zhou, B. R. et. al., Molecular Cell, 2015, 59(4), 628-638

20

On-dyad mode (crystal structure)

Short summary of Detailed interactions between GH5 and nucleosome
Positively charged GD residues chicken

Non-charged GD residues
Interactions between
GH5 residues and nucleosome
Linker-L1: K40(L1), R42(L1), R94(β2), K97(β2-CTD)
Dyad: R47(α2), Q48(α2), K85(L3), V87(L3), S90(L3)
Linker-α3: H25(NTD-α1), Y28(α1), Q67(α3)
Dyad and linker-α3: K69(α3), R73(α3), R74(α3)
Zhou, B. R. et. al., Molecular Cell, 2015, 59(4), 628-638

21

On-dyad mode (crystal structure)

Effects of mutations in the GD on the binding affinity of H5 to the nucleosome
ITCWT(H5_22-142)

Positively charged GD residueschicken(high expression yield)

Non-charged GD residues

Zhou, B. R. et. al., Molecular Cell, 2015, 59(4), 628-638

22

On-dyad mode (crystal structure)

Short summary of Detailed interactions between GH5 and nucleosome
Positively charged GD residues chicken

Non-charged GD residues
Interactions between
GH5 residues and nucleosome
Linker-L1: K40(L1), R42(L1), R94(β2), K97(β2-CTD)
Dyad: R47(α2), Q48(α2), K85(L3), V87(L3), S90(L3)
Linker-α3: H25(NTD-α1), Y28(α1), Q67(α3)
Dyad and linker-α3: K69(α3), R73(α3), R74(α3)
Zhou, B. R. et. al., Molecular Cell, 2015, 59(4), 628-638

23

Contents

Contents◆Introduction:◆Main:➢Mouse, drosophila: Off-dyad binding mode (model)➢Chicken: On-dyad binding mode (crystal structure)➢Human: Distinct Structures of chromatosomeswith different human linker histone isoforms (cryo-EM)◆Summary24

Human linker histone isoforms

Cryo-EM structures are consistent with on-dyad binding mode.
Sequence alignment of the linker histone globular domains

human(A, C) Cryo-EM (2.8 Å to 3.1 Å), (B, D) low-pass-filtered (6 Å) density map

Zhou, B. R. et. al., Molecular Cell, 2021, 81(1), 166-182

25

Human linker histone isoforms

Interactions between DNA and the GDs of isoforms
humanEven though thestructures of the H1
GDs were similar,
interactions between H1
GDs and nucleosome aredifferent (→ next page)

Zhou, B. R. et. al., Molecular Cell, 2021, 81(1), 166-182

26

Human linker histone isoforms

Variant GDs redistribute interactions with the nucleosomal and linker DNA.
The number of contacts between the GD and DNA averaged over MD trajectories

human

・the majority of contacts occurred between the residues from the NT of the α2 helix
and the CT of the L3 loop in the GH1 and the DNA near the dyad.
・H1.10 interacts mostly with the nucleosomal DNA near the dyad region,

whereas H1.0 and H1.4 globular domains have more pronounced interactions
with the linker strands.
Zhou, B. R. et. al., Molecular Cell, 2021, 81(1), 166-182

27

Contents

Contents◆Introduction:◆Main:➢Mouse, drosophila: Off-dyad binding mode (model)➢Chicken: On-dyad binding mode (crystal structure)➢Human: Distinct Structures of chromatosomeswith different human linker histone isoforms (cryo-EM)◆Summary28

Summary & Short proposal

Summary
chickenhuman

Positively charged GD residues (chicken)

Drosophila

・Different linker histone GH1 isoforms havedifferent binding modes.

Non-charged GD residues (chicken)

・GH1 bind to the dyad DNA and linker-DNA.
・GH1 can interact with both DNA linkers,

one strongly and the other weakly.
・In addition to positively charged GD residues,
Non-charged GD residues are also important tothe interaction with GD and nucleosome
Zhou, B. R. et. al., Molecular Cell, 2015, 59(4), 628-63829