Investigations on HAT catalyst
mechanisms and its site-specificity

2016.02.13
B4 Yamaji Kyohei

Contents

1. Introduction of chromatin modifications
2. Concept of Catalysis medicine
3. HAT introduction
4. HAT catalytic mechanisms
5. HAT site-specificity
6. SummaryEpigenetics
The same genes, but different phenotypes
Cell differentiation from the embryo

Production of a clone frog

Differentiated cells have all theinformation to produce anindividual.

Change in gene expression
induces different phenotypes.
https://searchforbetterhealth.wikispaces.com/Genes+%26+Health
http://learn.genetics.utah.edu/content/cloning/clonezone/3

Epigenetics ; chemical modifications
DNA/Histone modifications can control its gene expression patterns.
DNA methylation pattern is different between stem cells and differentiated cells

stem cells

Differentiated cells

methylation

Dirk Schübeler
Nature 462, 296-297

differentiatedundifferentiatedExpression of some genes is suppressed in differentiated cells with reduced
global Histone acetylation.
Expression ofDifferentiated cells
gene A and B

Histone proteinsDNA
stem cells

Expression ofgene A and B 4
Loring, J F, et al. Cell Research (2014) 24:143–160

Various Histone modifications

Kouzarides T, Cell (2007) 128, 4, 693–705

Histone acetylation is reported to play vital roles in many aspects.

5Interactions between Histone and DNA
tailNegative charge of DNA

globularDNA
H3, H4, H2A, H2B

Histone rich in positive charge
Luger, K. et al. Nature 389, 252 (1997)

interaction
6

Interactions between Histone tail and acidic patch
Highly electronegative
acidic patch of Histone

tailglobularinteractionDNA
H3, H4, H2A, H2B

Acidic patch

Santos, G M, et al. Trends in Pharmacological Sciences, 36, 5, 263–269, (2015)

7Structural changes induced by Histone acetylation
Usual state

Histone acetylation neutralizes the positive charge
acetylated

89The function depends on acetylation site
Histone acetylated Lysine residues
Lysine
H2A
H2B
H3
H4all
1320
1311130126136103

Acetylation at Histone tailAcetylation at Histone globular
Acetylation

Acetylation

Work as a scaffold for effector protein

Directly induces structural changeNat. Struct. Mol. Biol. 2013, 20, 657.

Contents

1. Introduction of chromatin modifications
2. Concept of Catalysis medicine
3. HAT introduction
4. HAT catalytic mechanisms
5. HAT site-specificity
6. Summary

11

Cataｌysis medicine
In some cancer cells, tumor suppressor genes are suppressedwith decreased Histone acetylation.
Inducing Histone acetylation can be a hopeful anti-cancer strategy.

tumor suppressor gene(e.g., p53)

anti-cancer
agentstumor suppressor gene
Manel Esteller (2007)

12

Cataysis medicine
Histone acetylation level is regulated on the balance of two
catalysts called HAT and HDAC.
What if HAT is defected...?
HAT (histone acetyltransferase)+ Acetyl-CoA (donor)

AcAcAcAc

HDAC (histone deacetylase)
OH
NO

NH

OH

SAHA/Vorinostat/Zolinza

HDAC inhibitor is actually used as an anti-cancer drug.

13Cataysis medicine
Mutations of HAT are frequently found in B-cell lymphoma.

CBP(HAT)

Inactivating mutations of acetyltransferase genes in B-cell lymphoma
Pasqualucci, L. et al. Nature 2011, 471, 189.

HAT-independent Histone acetylation can be the solution?

HAT-independent histone acetylationby artificial catalyst system
Artificial Catalyst System
for histone acetylation
NEWdrug?

AcAcAcAc

HDAC (histone deacetylase)transcription
OFFtranscription
ONSAHA/Vorinostat/Zolinza
14

Catalysis medicine
Requirements for the rational Histone acetylation catalyst.
1.
2.
3.
4.
5.

Ability to transfer acetyl group to Lysine residues of proteinsfrom a donor.
Substrate specificity (Histone↔other proteins)
Site specificity (ability to target residues in interest)
DNA sequence dependent targeting (expression of targetedgenes)(Specifically works in or be delivered to cancer cells)

Histone acetylation in cell is regulated to satisfy theserequirements.
So, there may be something to learn from them.

15

Contents

1. Introduction of chromatin modifications
2. Concept of Catalysis medicine
3. HAT introduction
4. HAT catalytic mechanisms
5. HAT site-specificity
6. Summary

HAT (Histone acetyltransferase)familyGNAT
p300/CBP

MYST

GNAT
Rtt10917

C. D. Allis, et al. Cell 2007 131, 633

HAT (Histone acetyltransferase)
Some HAT proteins acetylate non-Histone substrates.

p300 acetylates 70 other proteins than Histone proteins→HAT employs rigorous regulations as to what substrate to acetylate.
E. Verdin, et al. Chemistry & Biology, 17, 5, 471-482 (2005)

18

HAT (Histone acetyltransferase)
HAT proteins have various domains to interact with otherproteins and DNA modifications.

Interact with over 400 protein
HAT domain : catalysis

Other domains : substrate recognition, scaffold and so on.
Cole P A, et al. Chem. Rev., 2015, 115 (6), pp 2419–2452

19

Catalysis medicine

Requirements for the rational Histone acetylation catalyst.
1.
2.
3.
4.

5.

Ability to transfer acetyl group to Lysine residues of proteinsfrom a donor. (HAT domain)
Substrate specificity (Histone↔other proteins) (co-factor)
Site specificity (ability to target residues in interest) (HATdomain)
DNA sequence dependent targeting (expression of targetedgenes) (co-factor)(Specifically works in or be delivered to cancer cells)

20

Contents

1. Introduction of chromatin modifications
2. Concept of Catalysis medicine
3. HAT introduction
4. HAT catalytic mechanisms
5. HAT site-specificity
6. Summary

Reaction mechanisms proposed forHistone acetylation 1
Proposed for all the HAT subfamilies
1. The deprotonation of ε-amino group of the lysine substrate
LysineLysine2. Activation of Ac-CoA

Acetyl-Lys22

Reaction mechanisms proposed forHistone acetylation 2
Proposed for Esa1 of MYST family (denied? afterwards)① Thiol-thioester exchange

Acetyl-Lys23② Acetyl transfer reaction Marmorstein, R. et al. Nat. Struct. Biol. 2002, 9,862.

Investigation on catalysis mechanisms
1. Obtain crystal structure of the catalyst and its substrate
2. Observe possible interactions between the catalyst and its
substrate
3. Mutational analysis to investigate the importance of aresidue
4. Kinetics analysis (especially for bi-substrate catalyst)
Interactions

residues
Co-Crystal with substrate
Kinetics

Mutation→activity?
24

Lys-CoA : bisubstrate analogue inhibitor
Lys-CoA : The 1st and most potent bisubstrate p300 inhibitor

Ac-CoA mimic
Acetyl-Lysmimic25

Co-crystal of p300 HAT domain and Lys-CoA
HAT consists of several domains
D Panne, et al. Nature Structural & Molecula Biology 20, 1040–1046 (2013)

N-subdomain(28kDa)

Catalytic coreCo-crystal of p300 HAT domain with Lys-CoA

C-subdomain(10kDa)Lys-CoA
HAT domain
N-, C-subdomain (inactive, but makecrystallization easy)
Cole PA, et al. Nature 451, 846-850 (2008)26

Interactions between Lys-CoA and Lys-CoA
Ac-CoA binding

Acetyl-Lys

27

R1410 : intensive interaction with phosphate moiety

28

Confirmation of the importance of R1410
R1410A mutation shows reduced affinity for Ac-CoA

Hydrogen bond between R1410 and 3’-phosphate is essential for binding

HActivity (%)

WTp300 vs Lys-CoA

3’-dephospho-Lys-CoA

80%
noinhibition Inhibition29

Important residues interacting with Lysine moiety
Residues essential for catalysis shown by mutational analysis

30

Detailed proton transfer mechanism
No general base in enough proximity for proton transfer.
LysineLysine

Proposed mechanisms for proton transfer.
Chen L et al. J. Phys. Chem. B, 2014, 118 (8), pp 2009–2019

Tyr1394

LysineLysinewater
Trp1436

Tyr1394

water
Asp1507
Trp1436

Asp150731

Calculations supporting the mechanism
QM/MM calculations : simulation for large molecule
・QM/MM methods are the combination of QM(quantum mechanics) and MM(molecular mechanics)
・QM : accurate but high computational complexity (applied only to catalytic core)・MM : low content but low computational complexity (applied to all the molecules)

proposed mechanisms for proton transfer was energetically rational
Chen L et al. J. Phys. Chem. B, 2014, 118 (8), pp 2009–2019

0.65
10.15

32

catalytic mechanisms proposed for
p300
Proposed for all the HAT subfamilies
1. The deprotonation of ε-amino group of the lysine substrate
Tyr1394

LysineLysineTyr1394

water
Trp1436

2. Activation of Ac-CoA

water
Asp1507

Asp1507

Trp1436
Tyr1467
Tyr1467

Acetyl-Lys
Trp1436

Trp1436

33

Possibility of other mechanism?
Why not this mechanisms?

① Thiol-thioester exchange

Acetyl-Lys

34② Acetyl transfer reaction Marmorstein, R. et al. Nat. Struct. Biol. 2002, 9,862.

Enzyme kinetics classification
Classifications of kinetics for bisubstrate catalysts
Sequential mechanisms
Complex formation of all the substrates before the product

E : HAT, S1 : Ac-CoA, S2 : Lysine, P1 : CoA-SH, P2 : Ac-Lys

Ping-pong mechanism
Intermediate formation and no complex of all the substrates

E : HAT, S1 : Ac-CoA, S2 : Lysine,
P1 : CoA-SH, P2 : Ac-Lys, E* :Ac-HAT
35

Detailed classification of sequential mechanisms
Sequential mechanism is classified into three detailed ones
Random sequential mechanisms
S1 and S2 bind to E randomly (no order)

E : HAT, S1 : Ac-CoA, S2 : Lysine, P1 : CoA-SH, P2 : Ac-Lys

Ordered sequential mechanisms
S1 binding to E is necessary before S2 binds to E
E : HAT, S1 : Ac-CoA, S2 : Lysine, P1 : CoA-SH, P2 : Ac-Lys

Theorell-Chance (hit-and-run) mechanisms
Ordered but no accumulation of ternary complex

E : HAT, S1 : Ac-CoA, S2 : Lysine, P1 : CoA-SH, P2 : Ac-Lys

Fairly fast36

Fundamentals for enzymology
Kinetics analysis of bisubstrate enzymes
At a fixed concentration of one substrate, the kinetics fits MichaelisMenten.

Michaelis-Menten equation and Lineweaver-Burk plot
The reaction

can be expressed aswithThis equals

37

Kinetics investigation
By changing the conc. of fixed substrate, ineweaver-burk plotshows different characteristics depending on the mechanisms
Conc. of fixed S2Conc. of fixed S2Ordered or Randomcrossed line patterns

Ping pong
酵素キネティクス 中村隆雄(1993).

Parallel line patterns

Theorell-chance : depends on enzymes

38

Kinetics investigation
Why ordered mechanisms show crossed line patterns

Both slope and intercept
depends on [S2][S2]

1/[S1]39

kinetics investigation
Why ping-pong mechanisms show parallel line patterns

Only intercept depends on [S2][S2]

1/[S1]

40

P300 kinetics
P300 showed parallel line patterns[S2]

1/[S1]

Characteristics of ping pong mechanisms, but sometimes ofTheorell-chance mechanisms
41

Qualifications for Theorell-chance to show parallelline patterns
Theorell-chance mechanisms can show parallel line patterns if k-1<<k1

If k-1<<k,Ki S1=k-1/k1≓0Then, only intercept
depends on [S2]→how strong is the affinityfor AcCoA?

42

Affinity of p300 for AcCoA
p300 shows strong affinity for Ac-CoA and weak affinity for CoA-SH

Kd=7.3±1.4uM

Kd=740±60nM→k-1<<k1, supporting Theorell-Chance
43Investigations on product inhibition
Product inhibition patterns differs among different mechanisms

Productinhibitor

Productinhibitor

44

P300 fits Theorell-Chanace mechnisms
product inhibition of p300 is consistent with Theorell-Chance
S1

S2

p300
P2

1. mixed

S1

1.

P2

2.

2. competitive
P2

S245

Other HATs kinetics and affinity for inhibitors
Theorell-chance mechanism is unique to p300 among HATs
Gcn5/PCAF(GNAT) : sequential (random or ordered)Esa1(MYST) : ping-pong or sequential (random or ordered)
Rtt109 : unknown (none of the 4 classifications applied)

The relation between HAT inhibitors and catalytic mechanisms

Zheng, Y. G. Et al. Bioorg. Med. Chem. 2009, 17, 1381.

46

Grounds and meaning of different mechanisms
L1 loop unique to p300 realizes the strong affinity for Ac-CoA
Residues of L1

p300
Gcn5/PCAF(GNAT)

R Marmorstein et al. Nature 401, 93-98 (1999)47

Contents

1. Introduction of chromatin modifications
2. Concept of Catalysis medicine
3. HAT introduction
4. HAT catalytic mechanisms
5. HAT site-specificity
6. Summary

P300 site specificty
Substrate and site specificity of p300
P2 and groove

Lysine pocket (P1)49

Essential residues for site specificity
Mutational analysis

All p300 substrates havepositive residues 10Å downor up-stream of the substrate
residue

Mutations in P2
Mutations in G

50

Important interactions between substrate and p300
residues
Electrostatic interaction between the nearby residues andD1625/D1628 is importantMutations that maintain electrostatic interactions
between peptide and p300 lower the drop in V/K.

51

Contents

1. Introduction of chromatin modifications
2. Concept of Catalysis medicine
3. HAT introduction
4. HAT catalytic mechanisms
5. HAT site-specificity
6. Summary

Summary
・All HAT proteins acetylate Histone by two-step reactions; protontransfer and activation of Ac-CoA by sequential mechanisms.
・Only Esa1 is reported to catalyze the reaction by ping-pongmechanisms with enzyme nucleophile.
・ p300 employs unique Theorell-chance mechanisms with thehelp of L1 binding loop, realizing its broad substrate recognition.
・p300 recognizes acetylation site with its acidic pocket near thecatalytic pocket.

53

Appendix; P300 kinetics
Product inhibition in ordered mechanisms
Equations of ordered mechanisms considering product inhibition

When only P1 is added (when [P2] is 0,)

For a fixed [S2], [S1] inhibition of [P1] is mixed-type(both slope and intercept depends on [P1])54