Tumor Suppressor p53-Mediated Structural Reorganization of the

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Tumor suppressor p53-mediated structural reorganization of the transcriptional coactivator p300 Raka Ghosh, Stephanie Kaypee, Manidip Shasmal, Tapas Kumar Kundu, Siddhartha Roy, and JAYATI SENGUPTA Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.9b00333 • Publication Date (Web): 17 Jul 2019 Downloaded from pubs.acs.org on July 18, 2019

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Biochemistry

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Tumor suppressor p53-mediated structural reorganization of the transcriptional

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coactivator p300

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Raka Ghosh1, Stephanie Kaypee2, Manidip Shasmal1,4, Tapas K. Kundu3,*, Siddhartha Roy1,*,

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Jayati Sengupta3,*

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1

Department of Biophysics, Bose Institute, Kolkata, India

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2

Transcription and Disease Laboratory, Molecular Biology and Genetics Unit, Jawaharlal

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Nehru Centre for Advanced Scientific Research, Bangalore, Karnataka, India

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Biology, Kolkata, India

Division of Structural Biology and Bioinformatics, CSIR-Indian Institute of Chemical

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*

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Tapas K. Kundu: [email protected]

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Siddhartha Roy: [email protected]

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Jayati Sengupta: [email protected]

Corresponding Authors:

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Service, West Bengal, India

Current Address: Government General Degree College, Keshiary West Bengal Education

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ACS Paragon Plus Environment

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Abstract

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Transcriptional coactivator p300, a critical player in eukaryotic gene regulation, primarily

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functions as a histone acetyltransferase (HAT). It is also an important player in acetylation of

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a number of non-histone proteins, p53 being the most prominent one. Recruitment of p300 to

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p53 is pivotal in the regulation of p53-dependent genes. Emerging evidence suggest that

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p300 adopts an active conformation upon binding to the tetrameric p53, resulting in its

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enhanced acetylation activity. As a modular protein, p300 consists of multiple well-defined

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domains where the structured domains are interlinked with unstructured linker regions. A

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crystal structure of the central domain of p300 encompassing Bromo, RING, PHD and

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histone acetyltransferases (HAT) domains demonstrates a compact module where the HAT

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active site stays occluded by the RING domain. However, although p300 has a significant

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role in mediating the transcriptional activity of p53, little structural details on the complex of

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these two full-length proteins are available.

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Here, we present a cryo-electron microscopy (cryo-EM) study on the p300-p53 complex.

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The 3D cryo-EM density map of the p300-p53 complex, when compared to the cryo-EM map

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of free p300, revealed that substantial change in the relative arrangement of Bromo and HAT

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domains occurs upon complex formation which is likely required for exposing HAT active

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site and subsequent acetyltransferases activity. Our observation correlates well with previous

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studies showing that the presence of Bromo-domain is obligatory for effective

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acetyltransferase activity of HAT. Thus, our result sheds new light on the mechanism

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whereby p300, following binding with p53, gets activated.

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.

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Biochemistry

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Introduction

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Histone acetyltransferases p300 and its paralog CBP are important transcription coactivators

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in eukaryotes. p300 is a 2414 amino acids long, multi-domain protein that interacts with a

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large number of regulatory proteins1-2 and other coactivators3. As a histone acetyltransferase

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(HAT),1 it regulates transcription of genes via acetylation of histones leading to chromatin

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remodeling4. It also plays important roles in acetylation of a large number of other non-

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histone proteins5, one of the most prominent non-histone substrate being the tumor

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suppressor p536.

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As a modular protein, p300 has several well-defined functional domains, interspersed

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with intrinsically disordered segments7, 8. Each domain has specific functional roles and

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domain-specific probes have been developed to explore these functional roles7. It plays

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crucial roles in the pathogenesis of many diseases, and the relation of individual domains to

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pathogenesis has become better understood only recently9. Despite its immense importance,

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only a low-resolution cryo-EM structure of this regulatory protein is available to date10, and

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how all these domains are organized in the folded tertiary structure of p300 is still unclear.

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Nevertheless, a crystal structure has defined the architecture of the central (core) domain

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(subdomains Bromo-RING-PHD-HAT) of p30011. A characteristic feature of this structure is

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a sharp contortion (‘kink’) between the Bromo and HAT domains where RING domain

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occludes the active site of HAT forming an ‘autoinhibited’ conformation.

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One of the most important non-histone substrates of p300 is p53 which is termed the

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‘guardian of the genome’ as it plays one of the most important roles in maintaining the

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integrity of the genome12. Its role in stress response is now well documented13. Beyond

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these well-defined canonical roles, it is now becoming clear that p53 also plays crucial roles

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in controlling other non-canonical processes, such as metabolism and homeostasis14, 15. It

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normally adopts a tetrameric form in the cell16. It has four major domains, namely, the 3


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natively unfolded N-terminal transactivation region (TAD1 and TAD2), the DNA-binding

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domain, the tetramerization domain, and the C-terminal region. Although structural

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information is available for these individual regions, little is known about the tetrameric

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structure, except at a low-resolution17, 18, and even then, conflicting reports on the tetrameric

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structure have been reported19.

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Acetylation activates p53, and p300 is one of the key acetyltransferases that acetylates

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important lysine residues at the N-terminal transactivation domains of p53 and, thus, primes

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it for its specific functions. In spite of their importance, little is known about structures of

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p300, p53 tetramer or their complex at a high enough resolution to derive meaningful

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information about structure-function relationships of these proteins. Only a few structural

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studies on isolated domain-wise interactions of the p300-p53 complex have been reported20-

12

22

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terminal transactivation domains (TAD) of p53 tetramer23-25.

. It has been suggested that various N- and C-terminal domains of p300 interact with N-

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In this study, we have determined the structure of the complex of full-length p300 and

15

p53 using cryo-electron microscopy (cryo-EM) and compared it with that of the free p300. A

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significant opening of the ‘kink’ angle between Bromo and HAT domains of p300 upon

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binding to p53 has been identified suggesting that, while various structured modules of the

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central domain of p300 remain closely packed in an autoinhibited conformation11, the central

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domain opens up through rearrangements of its sub-domains following interaction with p53.

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Based on our analysis we propose that, opening of the ‘kink’ between Bromo and HAT

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domains plays a pivotal role in p53-mediated activation of p300. As a consequence, the

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active site of p300 HAT domain which is hemmed in by other domains in inactive p30011

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gets exposed in the activated form. Previous reports showing the presence of Bromo-domain

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is crucial for effective substrate specificity as well as the transcriptional activity of the HAT

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domain26, 27 support our proposition. 4


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Biochemistry

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Experimental procedures:

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Antibodies:

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Anti-p300 (N-15) was procured from Santa Cruz (Catalog no. sc-584),anti-p53 (DO-1) and

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anti-α-tubulin (DM1A) was procured from Merck Millipore (Catalog nos.OP43 and 05-829,

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respectively). Rabbit polyclonal antibodies against autoacetylated p300 (K1499ac

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p300/acp300), H3K18ac, and H3 were raised in-house.

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Recombinant Protein expression and Purification:

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His6-tagged full-length p300 was expressed by transfection of the recombinant baculovirus

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into Sf21 (Thermo Fisher Scientific) insect ovary cells. The sf21 cells were grown in Graces’

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Media (Gibco, Thermo Fisher Scientific, USA) containing an antibiotic solution (penicillin,

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streptomycin, amphotericin) and 10% FBS. 60 hours post-infection the cells were harvested.

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The His6-tagged p300 protein was purified as described previously28. FLAG-tagged p53 was

14

expressed and purified from BL21(DE3) pLysS E. coli strain. The recombinant FLAG-tagged

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p53 protein was purified through M2-agarose (Sigma Aldrich) based affinity chromatography

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as described previously29. The purification protocols were described in details elsewhere30.

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Cell Culture:

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Human non-small cell lung carcinoma H1299 cells (ATCC® CRL-5803™, American Type

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Culture Collection (ATCC), USA) were cultured in RPMI-1640 media (HiMedia,

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India)supplemented with 2 mM glutamine, 10% fetal bovine serum (FBS (Life Technologies

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India, India))and antibiotic solution (100 U/ml penicillin, 0.1 mg streptomycin, 0.25 μg

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amphotericin (HiMedia, India)). The pEBTetD p53 H1299 cells (doxycycline-inducible p53-

24

expressing H1299 cell line) were cultured in supplemented RPMI-1640 under 1.2 μg/ml

25

Puromycin antibiotic selection. Human hepatocellular carcinoma HepG2 cells (ATCC® HB5


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8065™, ATCC, USA) were cultured in supplemented MEM media (HiMedia, India). The

2

cells were grown at 37 °C in 5% CO2in a humified incubator.

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Acetyltransferase Assays:

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In the autoacetylation assay, 20 nMp300enzyme was incubated in HAT assay buffer (50 mM

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Tris–HCl, pH 7.5, 1 mM PMSF, 0.1 mM EDTA, and 10% v/v glycerol) and 100 mM sodium

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butyrate at 30 °C for 30 min with or without p53 in the presence of 1 µl of 4.7 Ci/mmol [3H]-

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acetyl-CoA (NEN-PerkinElmer). After the autoacetyltransferase assay, the radiolabeled

9

proteins were separated on a 8% SDS-PAGE and processed for fluorography.

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Small Molecule Inhibitor or Doxycycline treatment:

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HepG2 cells were grown on poly-lysine coated cover slips. When the cells were 70%

13

confluent, they were treated with the small molecule inhibitor Nutlin-3a (5 μM) for 6 hours.

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The cells were then processed for immunofluorescence as described previously31. The

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pEBTetD p53 H1299 cells were treated with 1 mg/ml doxycycline for 24 hours. The cells

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were harvested and lysed in Laemmli buffer (2% SDS, 62.5 mM Tris-HCl, pH 6.8, 10%

17

glycerol). The lysates were separated on a 10% SDS-PAGE and processed for western

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blotting analysis as described previously32.

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Sample for cryo-grid preparation:

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T18E p53, prepared in a buffer containing 10 mM Tris-Cl, 150 mM NaCl, 5 mM MgCl2, 20

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µM ZnCl2, 2 mM PMSF, 1mM DTT, pH-7.5, was mixed with 200 nM p300 at a molar ratio

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(p300: p53 tetramer = 1:1) and incubated at room temperature for 5 min.

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The affinity between p300 and N-terminal domain of p53, as well as some mutants, has

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been reported previously33 indicating that p300 preparation (obtained following very similar 6


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Biochemistry

1

methods) is biologically active with respect to p53 binding. One molecule of p300 per p53

2

tetramer has been established earlier by Alan Fersht’s group34. In support of Alan Fersht’s

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2007 PNAS paper, biochemical data presented in Kaypee et al35 show that only when the

4

molar ratios of p300:p53 is 1:4 (or 1:1 p300:p53 tetramer) a significant increase in the levels

5

of p300 autoacetylation is observed suggesting that one molecule of p300 per p53 tetramer

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form a distinct complex.

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For cryo-EM, 4 μl of homogeneous p300-p53 complex was applied on glow-discharged

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Quantifoil® holey carbon TEM grids (R2/2, Quantifoil, Micro Tools GmbH, Jena, Germany),

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which were coated with a home-made continuous thin layer of carbon, followed by blotting

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and vitrification with a Vitrobot™ (FEI Inc, Hillsboro, Or, USA) and then frozen in liquid

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ethane36.

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Purified p300, diluted to a final concentration of 200 nM in a buffer containing 10 mM

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Tris-Cl, 150 mM NaCl, 1 mM DTT, 2 mM PMSF pH 8.0, was used for sample preparation.

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Grids were prepared and sample imaged similarly as p300-p53 complex.

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Cryo-EM data collection and 3D image processing:

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Data collection was performed on a Tecnai POLARA microscope (FEI, USA) equipped with

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a FEG (Field Emission Gun) operating at 300 kV. Images were collected with 4K Χ 4K

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‘Eagle’ charge-coupled device (CCD) camera (FEI, USA) at ~79000X magnification,

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resulting in a pixel size of 1.89 Å at the specimen level with defocus values ranging from 1.5

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to 4.5μm. All images were acquired using low-dose procedures with an estimated dose of

22

~20 electrons per Å

2 37

.

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Micrographs screening and particle picking were done separately with EMAN238 and

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SPIDER39. Micrographs are selected on the basis of their visual quality, Thon rings in the

25

power spectra and defocus values. Initially, for the p300-p53 complex sample, 5400 particles 7


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Page 8 of 39

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were picked manually according to their sizes, using the BOXER program from the EMAN2

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package with a pixel size of 1.89Å/pixel. Particle screening was done by visual inspection

3

with utmost care so that only complex particles (~ 10-12 nm) were kept and particles of

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smaller sizes were rejected. 2D and 3D classification and refinement were performed using

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EMAN2. The raw particle images were first subjected to multivariate statistical analysis and

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classification. The starting model was generated by the common line technique from selected

7

2D class averages.

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In SPIDER, defocus of each micrograph was estimated on the basis of its 2D power spectrum and then micrographs were divided into groups of similar defocus. Image

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processing was done using the reference-based alignment method, where the final refined

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volume of the p300-p53 complex obtained from EMAN2 was used as the reference. Using

12

projections of this model as a template, a total of 10,088 particles were selected semi

13

automatically from all the micrographs. The 3D reconstruction was then done following the

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standard SPIDER protocols for reference-based reconstruction39. The overall resolution,

15

estimated by comparing the FSC of the two half maps, was 15.7 Å using the 0.5 cutoff

16

criteria and 10.7Å using the 0.143 cutoff criteria (Figure S2C).

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For free p300 sample, data were processed essentially the same way as for the complex

18

described above. Briefly, micrograph screening, particle selection, 3D reconstruction and

19

refinement were done in EMAN2 to generate an initial model. Coordinates of a total of

20

16,152 particles, picked manually in EMAN2, were imported in SPIDER for further

21

reference-based particle alignment, classification, 3D reconstruction and refinement. The

22

resolution of the final map was 13.5 Å using the 0.5 cutoff criteria and 9.8 Å using the 0.143

23

cutoff criteria (Figure S2D). For display, resolution guided amplitude enhancement with

24

reference was done.

8


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Biochemistry

1

Surface rendering, docking of crystal structures, and segmentation of the 3D maps were

2

performed in the program UCSF Chimera40 and Pymol41. Cross-Correlation coefficients

3

(CCC) were calculated using Chimera (https://www.cgl.ucsf.edu/chimera/docs/UsersGuide/).

4

Simulated density map was created first for each fitted model at the appropriate resolution

5

(FSC 0.5 cut-off) using ‘molmap’ command available in Chimera

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(https://www.rbvi.ucsf.edu/chimera/docs/UsersGuide/midas/molmap.html), where the sigma

7

factor was chosen critically by comparing the features of the resulting simulated map and

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cryo EM map (used 0.187 and 0.225 for p300-p53 complex and p300 maps, respectively).

9

The local cross-correlation was then calculated between the simulated map and the target

10

map using the ‘measure correlation’ command in Chimera at respective contour levels

11

(appropriate contour levels of the resulting simulated maps were chosen to accommodate the

12

model within the densities).

13 14

Validation of initial models

15

Initial models were generated in EMAN2 from reference free 2D class averages for p300 and

16

p300-p53 complex. Validation of the 3D models was done in multiple ways.

17

The crystal structure of the Bromo-HAT shows close resemblance with the core structures of

18

p300 in both maps (Figure S4). Reliability of the initial model of the complex obtained in

19

EMAN2 was examined by comparing different views of the reference-free 2D class averages

20

generated by different softwares, e.g., EMAN238, Xmipp42, RELION43 and FREALIGN 44, 45

21

as well as reference-based SPIDER39. Further, it was shown that the 2D re-projections of the

22

3D maps were consistent with the 2D class averages (Figure S2). Further, we have generated

23

initial models of both free p300 and the complex using FREALIGN (CisTEM)44, 45 which

24

were very similar to EMAN2 generated initial models. Final 3D maps reconstructed in

25

SPIDER using these initial models were also very similar to the structures reported here. 9


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Page 10 of 39

We also manually produced another starting model from the crystal structure of the

2

central domain (4BHW) by low pass filtering. 3D Image processing of the p300 dataset

3

using two different starting models resulted in similar 3D maps. Reprocessing of the image

4

data of the complex using RELION43, an alternative image processing software, resulted in a

5

reconstruction fairly similar in overall structural features.

6

Furthermore, we acquired two sets of images (grid preparation and data collection in

7

different days) for both p300 and p300-p53 complex. Independently processed data sets

8

resulted in similar maps for both samples (CCC > 0.8).

9 10

Results:

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Cryo-EM structure of p300 in complex with p53:

12

The tumor suppressor p53 can modulate the activity of the master epigenetic enzyme p300 by

13

inducing its ability to trans-autoacetylate itself46. The in vitro autoacetylation assay reveals

14

that the levels of autoacetylated p300 (acp300) increase in a dose-dependent manner in the

15

presence of increasing concentrations of p53 (Figure 1A).

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Biochemistry

Figure 1. p53-induced p300 autoacetylation. (A) An in vitro autoacetylation assay was performed to determine the levels of p300 autoacetylation in the presence of increasing concentrations of p53 as indicated. The autoradiogram panels depict the acetylated proteins and Coomassie panels are the loading control. (B) The protein levels of p53, alpha-tubulin, autoacetylated p300 (ac-p300), p300, H3K18ac, and H3 were determined through western blotting analysis in the presence and absence of 1 μg/ml of doxycycline (Dox) in the p53-expressing dox-inducible H1299 cells. (C) Immunofluorescence to determine the levels of ac-p300(green) and p53 (red) in HepG2 cells treated with 5 μM Nutlin3a or vehicle control (DMSO) for 6 hrs. The cells were counterstained with Hoechst. Scale bar, 10 μm. Quantification of the data is shown in the left panels of A and C. All statistical analysis was performed using unpaired two-tailed Student's t test.

14

In doxycycline-inducible p53 expressing H1299 cells (p53 null cells) acp300 levels

15

increased upon the induction of p53 expression and a concomitant increase in p300-mediated

16

histone acetylation mark, H3K18ac was also observed (Figure 1B). When the levels of p53

17

were stabilized in HepG2 cells (wild-type p53 expressing cells) using the MDM2-inhibitor,

18

Nutlin3a, the levels of acp300 were found to be elevated in comparison to the vehicle control

19

(DMSO) (Figure 1C)31. However, the precise molecular underpinnings of p300 catalytic

20

activation by p53 are still unknown.

21

A plausible hypothesis is that p53 binding stimulates p300 activity through a structural

22

switch, resulting in an active p300 conformation. Various previous studies have also

23

proposed that p53-mediated structural reorganization results in an active p300 conformation6,

24

47

25

employed cryo-electron microscopy (cryo-EM) and single particle reconstruction technique

26

for visualizing the structures of the complex of full-length proteins. Purified proteins (Figure

27

S1A,B) were incubated together to form the p300-p53 complex and grids were prepared for

28

acquiring cryo-EM images. We employed primarily EMAN238 and SPIDER39 image

29

processing softwares (see Methods) to generate a cryo-EM map (Figure 2) of p300 in

. To gain mechanistic insights into p300 activation that follows its autoacetylation, we

11


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Page 12 of 39

complex with p53 (resolution ~15.7Ả using the 0.5 cutoff criteria, Figure S2).

2 3 4 5 6 7 8 9 10 11 12 13 14 15

Figure 2. Cryo-EM 3D reconstructions of p300-p53 complex. (A) Front and (B) back views of the 3D cryo-EM map of the p300-p53 complex map (surface representation in purple, contour level 2.0). Landmarks: SP, spout; H, handle (Deposited to EMDB database, ID: EMD-6791). (C) Surface representation of the densities encompassing p300 (blue, contour level 0.95) and p53 (brick red, contour level 1.4) in the complex following segmentation. (D) Superimposition of the density map of the free p300 protein (dark cyan surface, contour level 0.02) on the p300 density isolated from the p300p53 complex map (blue mesh, contour level 2.0). It is clearly seen that the central part of the protein superimposes well, whereas, peripheral regions are bloated considerably in the p53bound form of p300. The asterisk (*) indicates the absence of density in the p300 map, whereas, in the the p300-p53 complex, density can be clearly seen in this region. Landmarks in p300: F, fist; Th, thumb. (EMDB id of the deposited p300 density map: EMD-6792.).

16

To ensure the reliability of the maps, we further used several validation strategies (see

17

Methods; Figure S2A-B).

12


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Biochemistry

1

The p300-p53 complex map adopted a shape similar to that of a kettle, with a protruding

2

density (termed here as ‘spout’) in the middle and a ‘handle’ at the side (Figure 2A-B). The

3

centrally located, protruded ‘spout’ was the structural hallmark in the density map of p300 in

4

complex with p53 which resembles the helical bundle structure of the Bromo-domain (Figure

5

S3A,B). A bi-lobed density attributable to the p53 tetramer was visible within the density

6

map of the complex (Figure 2A). On the basis of shape, densities corresponding to p300 and

7

p53 were computationally separated by using a cluster segmentation procedure (Figure 2C).

8 9

The yield of p53-p300 complex was low, and therefore, molecular weight of the monodispersed complex was difficult to determine by standard biophysical method. However,

10

estimated sizes of the proteins in the complex are consistent with the dimensions of p300

11

monomer and p53 tetramer reported in previous studies48-50.

12 13

The cryo-EM density map of p300 (resolution ~13.5Ả using the 0.5 cutoff criteria, Figure S2), on the other hand, showed a ‘fist-like’ appearance with a ‘thumb’ feature (Figure 3C).

14

13


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Figure 3. Interpretation of the densities in molecular terms. (A) Domain organization of p300 in schematic representation is shown. The subdomains of central (core) domain are color-coded with residue numbers. (B) Cartoon representation of the published crystal structure of the Bromo-HAT domain (PDB ID: 4BHW). Bromodomain, RING, PHD and HAT domains are shown in yellow, green, orange and red, respectively. A rotated view of the same is also shown where the N- and C-terminal ends are marked. Stereo views of the (C) p300 map (dark cyan mesh, contour level 0.02) and (D) p300-p53 complex map (p300, blue mesh, contour level 0.95.; p53, red mesh, contour level 1.4) are shown (IDs of the deposited maps are: p300-p53 complex: EMD-6791 and p300: EMD-6792). The maps are fitted with the crystal structure of central domain of p300 (PDB ID: 4BHW). Different subdomains are represented in the same colors as mentioned in (A, B). (PDB id of the fitted central domain models are: p300-p53 complex: 5XZC and p300: 6K4N) Additional empty densities are marked which can be attributed to other N- and C-terminal domains (N and C-terminals could not be unambiguously determined). Less amount of extra densities are visible in free p300 than that of in the p300 segment of the complex indicating that some parts of the N- and C-terminal domains likely get stabilized upon complex formation. Landmarks are same as in Figure 2.

20 14


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Biochemistry

1

On the basis of the size of the enclosed volume, the molecular mass of the p300 density

2

map (~230 kDa estimated from the occupied volume, with 0.82 D/Å3 as the protein density)

3

appeared somewhat smaller than the known molecular mass of p300 (~264 kDa). Evidently,

4

significant parts of the unstructured regions at the N- and C-termini (~650 amino acids,

5

Figure 3A) were not visible in our reconstruction, likely because of the dynamic nature of

6

those regions. Considering that the p300 map encompassed the structured core domain and

7

partly other N- and C-terminal structured domains, the effective molecular mass was

8

adequate. We concluded that in free form, the central (core) domain of p300 stays in a closely

9

compact conformation as seen in the crystal structure, while unstructured linkers and part of

10 11

N- and C-terminal domains remains in an ensemble of assorted dynamic conformations. Overall, the cryo-EM structure of p300 is in agreement with the prolate ellipsoidal shape

12

of the central region of p300 described in a previous AFM study30. Moreover, the overall

13

shape of the crystal structure of the central domain (PDB code: 4BHW, Figure 3B), when

14

converted to density map and filtered to low resolution, showed visually identifiable

15

resemblance (CCC: 0.88) with the central region of the cryo-EM map (Figure S4) giving us

16

confidence about the reliability of the map. Extra densities observed at either side of the

17

central region of the p300 density map were attributable to parts of the N- and C-terminals of

18

the protein (Figure 3C).

19

When free p300 density and the p300 segmented part of the complex density map were

20

juxtaposed, the resemblance of the topology of the central region could be identified (Figure

21

2D). However, compared with the free p300, the central core of the protein in complex with

22

p53 appeared to be relatively relaxed. On the other hand, in line with previous results8, some

23

dynamic parts of the N- and C-terminal domains of p300 seemed to get stabilized upon

24

binding to p53. The native PAGE (Figure S1C) also supports this observation. Figure S1C

25

showed, while the isolaterd proteins were not resolved, the complex band migrated down 15


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Page 16 of 39

1

suggesting that, in the free forms, both the proteins are in much larger overall sizes, whereas,

2

they are more compact upon complex formation.

3 4

Conformational switching in p300 central domain induced by p53 binding

5

In order to describe the density maps of the p300 protein and p300-p53 complex in molecular

6

terms, we attempted to dock existing crystal structures of the protein. The protein p300

7

comprises (Figure 3A) the central domain (combining sub-domains Bromo-RING-PHD-

8

HAT, PDB code: 4BHW, Figure 3B), preceded by the N-terminal (KIX and TAZ1) and

9

followed by the C-terminal domains (TAZ2, IBiD). The largest piece of the p300 protein that

10

could be crystallized is the central domain which shows an ‘autoinhibited’ conformation,

11

where RING domain is loosely packed on the HAT domain11.

12

The crystal structure of the combined central domain shows a ‘kink’ (angle ~90o) between

13

the Bromo-domain and the HAT domain (Figure 3B)11. Docking of this structure (PDB code:

14

4BHW) in p300 map (CCC: 0.88) as a rigid body using Chimera40 placed the Bromo-HAT at

15

the central region of the density envelope (Figure 3C, 4A) where the Bromo-domain occupied

16

in the ‘fist’ region.

16


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Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

Figure 4. Conformational changes in the central domain of p300 upon complex formation with p53. Fitting of different subdomains (Bromo (yellow), RING (green), PHD (orange) and HAT (red)) of the central domain of p300 (PDB code: 4BHW) in (A) p300 (dark cyan mesh, contour level 0.02) and (B) p300-p53 complex (p300, blue mesh, contour level 0.95; p53, red mesh, contour level 1.4) maps. Subdomains are shown in cartoon representations. Opening up of the kink between the Bromo and HAT domains is clearly detected. Close-up views of the superposed cryo-EM maps of p300 and p300-p53 complex (dark cayan mesh, p300 map (EMDB-6792); purple mesh, p300-p53 complex map (EMDB-6791)) with the corresponding fitted structures (p300-p53 complex: 5XZC and free p300: 6K4N) showing (C) reorientation of the Bromo-domain (yellow), (D) loop-like density seen on the back of p300 density in the complex (marked as L in Figure 2) that can accommodate a model of the autoinhibitory loop, and (E) RING (green) and PHD (orange) sub-domains of p300 central domain are displaced following complex formation.

18 19 20

The RING domain remained slightly outside the density. However, unoccupied density could clearly be identified in adjacent region. We have docked this domain into density 17


Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 18 of 39

1

available at the closest neighborhood (Figure 4A). Recently demonstrated51 positional

2

variations of the RING domain in different Acetyl-CoA derivative bound structures support a

3

slight displacement of the RING domain observed in our map. The fact that Bromo-HAT

4

domain could be docked into the p300 density as a rigid body (CCC: 0.85) where the ‘kink’

5

was maintained, as seen in the crystal structure, gave us confidence about the reliability of the

6

map.

7

In contrast, the Bromo-HAT domain could no longer be fitted as a rigid piece into the

8

p300 segment of complex density map. Interestingly, the protruded trunk-like density

9

(sprout) showed a striking resemblance with the Bromo-domain feature (helical bundle)

10

(Figure S3A, B). When these two domains were fitted separately (CCC: 0.95), the Bromo-

11

domain occupied the spout while HAT resided in the body (Figure 3D and 4B, C). It was

12

clearly seen that the ‘kink’ between HAT and Bromo-domains widened (kink angle ~113o)

13

(Figure 4B). It appeared that densities corresponding to other central domains (RING and

14

PHD) adjacent to Bromo (as seen in the p300 map) were missing in the complex density map.

15

Instead, additional densities were seen at the top of the Bromo-domain (Figure 3D and 4E)

16

indicating that the PHD and RING domains were displaced (Figure 4E), as proposed in a

17

previous study11(CCC for the central domain (PHD-RING-Bromo-HAT): 0.92). The

18

densities corresponding to these domains, however, were much weaker, likely due to inherent

19

flexibility of the linker regions.

20

Fitting of the central domain into the p300 density isolated from the density map of the

21

p300-p53 complex left substantial empty density which could be attributed to the other

22

domains (Figure 3D). Although the current resolutions of the maps did not allow us to mold

23

the crystal structures of N- and C-terminal domains into the density maps, a loop-like density

24

is seen at the back of p300 density isolated from the complex map (Figure 2C). A model of

18


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Biochemistry

1

the autoinhibitory loop was made (using PHYRE252) which could be accommodated in this

2

loop density (Figure 4D).

3

Overall, the compact nature of the central domain of p300 as seen in the crystal structure

4

clearly opened up upon interaction with p53 (Movie S1). Thus, our structural analyses

5

illustrated that, while conformation of the p300 central (core) domain in free form can be

6

considered as a ‘closed’ configuration, in the complex with p53, it transformed to an ‘open’

7

conformation. As opposed to the central domain, the intrinsically dynamic N- and C- terminal

8

regions of free 300 appeared to become partly ordered upon complex formation.

9 10

Structural organization of p53 tetramer in the p53- p300 complex

11

A distinct bi-lobed feature of the p53 tetramer could be recognized in the cryo-EM map of the

12

p300-p53 complex. In the complex density map (~12 nm), the p53 tetramer apparently

13

adopted a distorted shape (Figure 5), with two side-lobes exhibiting a centrally connected

14

density.

19


Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 20 of 39

1 2

Figure 5. Interpretation of p53 densities in molecular terms and comparison with other

3

reported cryo-EM structures of p53 tetramer.

4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

(A) Schematic representation of domain organization of p53 in different color codes. (B) Stereo representation of segmented p53 density (red mesh, contour level 1.4) in close-up view with the fitted crystal structures of the DNA-binding domains (DBD) (published crystal structure of DBD tetramer PDB code: 3KMD in yellowish green color) and tetramerization domains (published crystal structure, PDB code: 4D1M in dark green). Dimer of DBD can be fitted as a rigid module in each lobe. (C) Qualitative docking of a piece of DNA (pink surface) shows that it can be accommodated in the groove of p53 lobes (red surface). (D) Fitting of tetramerization domain (PDB code: 4D1M in dark green) at the central region of the density attributed to p53 (red surface). (E) Schematic representations of different cryoEM structures available for p53 tetramer. Cryo-EM maps of the p53 tetramer published by different groups (upper panel) form1:18; form2: 49. The lower panel shows the DNA bound conformation of p53 tetramer reported by different groups (form1:53; form2:17). Middle panel (highlighted) shows the conformation adopted by p53 following complex formation with p300 (current study). Landmarks: N-term: N-terminal domain, DBD: DNA binding domain, Tet domain: tetramerization domain, C-term: C-terminal domain.

20


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Biochemistry

1

Calculated molecular mass corresponding to the segmented p53 density (estimated from the

2

enclosed volume at the viewing threshold) is comparable to the molecular mass equivalent to

3

p53 tetramer (~ 200 kDa).

4

Crystal structure (PDB code: 3KMD) of a dimer of p53 DNA-binding domains (DBD,

5

Figure 5A) could nicely be accommodated as a rigid body into each of the side lobes of p53

6

density (Figure 5B). Following fitting of the DBDs, substantial empty density remained in

7

each lobe that could be attributed to the other domains (Figure 5A). A segment of DNA

8

could be placed in the cleft created by the two side lobes (Figure 5C). Incurved nature of the

9

middle part of p53 density showed similarity to the concave nature of the crystal structure of

10

the packed tetramerization domain with a β-strand-turn-α-helix architecture (Figure S3C,D),

11

and the crystal coordinates (PDB code: 4D1M) could be fitted into the indented middle

12

density (Figure 5D). A model structure of the p53 C-terminal end (containing residues that

13

get acetylated by p300) was built using PHYRE252. Apparently, in the p300-bound form,

14

mostly unstructured extreme C-termini of p53 tetramer can easily access the active site of

15

p300 (Figure 6).

16 21


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Page 22 of 39

1 2 3 4 5 6 7 8 9 10 11 12

Figure 6. The active site of the p300 HAT domain in the p300-p53 complex and its closeup view. A model of flexible C-terminal end part (residues 357-393, magenta) of p53, generated using PHYRE2, is qualitatively docked at the end of tetramerization domain (green), fitted into the density map (EMDB-6791) of p300-p53 complex (p300 in blue mesh, contour level 0.95; p53 in red mesh, contour level 1.4). The close-up view (left panel) shows that the distal Cterminal (substrate for acetylation) can access the active site of the HAT domain (red) easily. Acetyl-CoA residue (blue sticks) marks the active site cleft of the HAT domain. C-terminal end of each tetramerization domain is indicated by an asterisk (*). The distances from the ends of each C-terminal of tetramerization domain to the active site of the HAT domain vary within the range of ~30-50 Ǻ.

13

There are some discrepancies among previously published p53 tetramer structures (with or

14

without DNA, approximately 6-8 nm). However, in all the structures the four DNA-binding

15

domains remain closely packed54, while the tetramerization domains are displaced out of the

16

plane in some structures (schematic representation in Figure 5E). In contrast, the p300-bound

17

form of p53 reported here, demonstrated a distorted organization where the dimers of DNA

18

binding domains (DBDs) were widely spaced (highlighted in Figure 5E), and tetramerization

19

domains occupied the central region. Intriguingly, similar p53 architecture, as we observed

20

in p300-p53 complex, has been proposed from a SAXS-based open cross-shaped model

21

where centrally-located tetramerization domains in p53 tetramer are placed in the plane of the

22

DBDs in the absence of DNA17, 55.

23 24

Discussion:

25

We present here cryo-EM reconstructed 3D structures of free p300 and p300 in complex with

26

p53 tetramer to gain insights into molecular mechanisms of p53-induced activation of p300.

27

Despite the resolution limitation of the maps, a global conformational change in the p300

28

structure upon interaction with p53 was clearly identifiable. Interpretation of the density

29

maps in molecular terms showed that the crystal structure (PDB code: 4BHW) of the central 22


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Biochemistry

1

domain could directly be accommodated into the density as a rigid piece keeping the ‘kink’

2

between Bromo and HAT domains intact, whereas, opening of the ‘kink’ angle is obligatory

3

to accommodate the bromo and HAT domains into the p300 density of the complex.

4

Consequently, the RING domain, which remains packed against the histone acetyltransferase

5

active site in inactive p300, is displaced away from the substrate-binding pocket of the HAT

6

domain resulting in a conformation that may be characterized as ‘open’ and ‘activated’.

7

Evidently, the opening of the kink between Bromo and HAT is a vital step in HAT activation.

8

Notably, there are reports showing that the presence of the Bromo-domain is essential for

9

effective substrate acetylation activity of histone acetyltransferases (HAT) domain26 which

10

support our observation. We speculate that, while changing into an ‘open’ conformation

11

following interaction with p53, the HAT domain of p300 rotates and exposes the auto-

12

inhibitory loop (Figure 4D).

13

Overall structural arrangements of the p300-p53 complex presented here support a

14

previous model proposing that the p300 protein embraces p53 tetramer with its N- and C-

15

terminal domains8, 34. Based on our observation we propose a model where the intrinsically

16

disordered N-terminal domains of p53 tetramer bind first to the N- and C-terminal domains of

17

p300 with varying affinities to induce conformational changes in p300 exposing the HAT

18

active site. This conformational change allows the C-terminal domains of the p53 to gain

19

access to HAT domain of p300, indicating that the activation mechanism likely is allosteric in

20

nature. Such an allosteric activation may be an essential component of the specificity of p300

21

action as its catalytic activity will only become functional when bound to the transcription

22

factor, thus creating targeted acetylations in the genome.

23 24 25 23


Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 24 of 39

1

Accession numbers:

2

The raw cryo-EM density maps and coordinates of p300-p53 complex and free p300 were

3

deposited in the Electron Microscopy Data Bank (EMDB) under the accession numbers

4

EMD-6791 and EMD-6792, and deposited in the RCSB Protein Data Bank (PDB) under the

5

accession codes 5XZC and 6K4N, respectively.

6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

Supplementary Information: Figure S1: Gel images of purified p300, p53 proteins, their complex, and TEM image of p53 (in complex with DNA) are shown. Figure S2: Validation of 3D reconstructions of p300-p53 complex using various imageprocessing softwares. Figure S3: Feature similarities of the published crystal structures of p300 Bromo-domain (taken from 4BHW) and p53 tetramerization domain (4DIM) with the topology of the p300 and p53 densities within the p300-p53 complex map (EMDB-6791). Figure S4: Comparison of the shape of the p300 density map (EMDB-6792) with the published crystal structure of the central domain (4BHW). Movie S1: Opening of the kink angle between Bromo and HAT subdomains of the p300 central domain upon p53 binding is displayed.

25 26

Acknowledgements: This work was supported by CSIR Network project ‘UNSEEN’

27

(BSC0113), Sir JC Bose Fellowship (SR/S2/JCB-28/2010), Department of Science and

28

Technology, Government of India, Jawaharlal Nehru Centre for Advanced Scientific

29

Research, Bangalore, and CSIR-Indian Institute of Chemical Biology, Kolkata. TKK and SR

30

are Sir J. C. Bose Fellows. RG and SK are supported by CSIR and University Grant

31

Commission (UGC), Government of India, respectively. We thank Central Instrument

32

Facility of CSIR-IICB for the access of cryo-electron microscope, and Mr. Chiranjit Biswas

33

for cryo-EM data collection and Mr. Sayan Bhakta for troubleshooting during image 24


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Biochemistry

1

processing. We acknowledge Dr. Dirk Gründemann (University of Cologne, Germany) for

2

the pEBTetD SLC22A1 construct.

3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44

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Gu, W., and Roeder, R. G. (1997) Activation of p53 sequence-specific DNA binding by acetylation of the p53 C-terminal domain, Cell 90, 595-606. Banerjee, S., Arif, M., Rakshit, T., Roy, N. S., Kundu, T. K., Roy, S., and Mukhopadhyay, R. (2012) Structural features of human histone acetyltransferase p300 and its complex with p53, FEBS letters 586, 3793-3798. Kaypee, S., Sahadevan, S. A., Sudarshan, D., Halder Sinha, S., Patil, S., Senapati, P., Kodaganur, G. S., Mohiyuddin, A., Dasgupta, D., and Kundu, T. K. (2018) Oligomers of human histone chaperone NPM1 alter p300/KAT3B folding to induce autoacetylation, Biochim Biophys Acta 1862, 1729-1741. Balasubramanyam, K., Altaf, M., Varier, R. A., Swaminathan, V., Ravindran, A., Sadhale, P. P., and Kundu, T. K. (2004) Polyisoprenylated benzophenone, garcinol, a natural histone acetyltransferase inhibitor, represses chromatin transcription and alters global gene expression, The Journal of biological chemistry 279, 33716-33726. Polley, S., Guha, S., Roy, N. S., Kar, S., Sakaguchi, K., Chuman, Y., Swaminathan, V., Kundu, T., and Roy, S. (2008) Differential recognition of phosphorylated transactivation domains of p53 by different p300 domains, J Mol Biol 376, 8-12. Teufel, D. P., Freund, S. M., Bycroft, M., and Fersht, A. R. (2007) Four domains of p300 each bind tightly to a sequence spanning both transactivation subdomains of p53, Proc Natl Acad Sci U S A 104, 7009-7014. Kaypee, S., Sahadevan, S. A., Patil, S., Ghosh, P., Roy, N. S., Roy, S., and Kundu, T. K. (2018) Mutant and Wild-Type Tumor Suppressor p53 Induces p300 Autoacetylation, iScience 4, 260-272. Grassucci, R. A., Taylor, D. J., and Frank, J. (2007) Preparation of macromolecular complexes for cryo-electron microscopy, Nature protocols 2, 3239-3246. Grassucci, R. A., Taylor, D., and Frank, J. (2008) Visualization of macromolecular complexes using cryo-electron microscopy with FEI Tecnai transmission electron microscopes, Nature protocols 3, 330-339. Tang, G., Peng, L., Baldwin, P. R., Mann, D. S., Jiang, W., Rees, I., and Ludtke, S. J. (2007) EMAN2: An extensible image processing suite for electron microscopy, Journal of Structural Biology 157, 38-46. Shaikh, T. R., Gao, H. X., Baxter, W. T., Asturias, F. J., Boisset, N., Leith, A., and Frank, J. (2008) SPIDER image processing for single-particle reconstruction of biological macromolecules from electron micrographs, Nature Protocols 3, 19411974. Pettersen, E. F., Goddard, T. D., Huang, C. C., Couch, G. S., Greenblatt, D. M., Meng, E. C., and Ferrin, T. E. (2004) UCSF chimera - A visualization system for exploratory research and analysis, Journal of Computational Chemistry 25, 16051612. The PyMOL Molecular Graphics System, D. S. L., San Carlos, CA. (2002). Sorzano, C. O. S., Marabini, R., Velazquez-Muriel, J., Bilbao-Castro, J. R., Scheres, S. H. W., Carazo, J. M., and Pascual-Montano, A. (2004) XMIPP: a new generation of an open-source image processing package for electron microscopy, Journal of Structural Biology 148, 194-204. Scheres, S. H. W. (2012) RELION: Implementation of a Bayesian approach to cryoEM structure determination, Journal of Structural Biology 180, 519-530. Grant, T., Rohou, A., and Grigorieff, N. (2018) cisTEM, user-friendly software for single-particle image processing, eLife 7. Grigorieff, N. (2007) FREALIGN: high-resolution refinement of single particle structures, Journal of structural biology 157, 117-125. 27


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Kaypee, S., Sahadevan, S.A., Patil, S., Ghosh, P., Roy, N.S., Roy, S., Kundu, T.K. (2018) Mutant and Wild-Type Tumor Suppressor p53 Induces p300 Autoacetylation, iScience 4, 260–272. Dornan, D., Shimizu, H., Perkins, N. D., and Hupp, T. R. (2003) DNA-dependent acetylation of p53 by the transcription coactivator p300, The Journal of biological chemistry 278, 13431-13441. Okorokov, A. L., Sherman, M. B., Plisson, C., Grinkevich, V., Sigmundsson, K., Selivanova, G., Milner, J., and Orlova, E. V. (2006) The structure of p53 tumour suppressor protein reveals the basis for its functional plasticity, The EMBO journal 25, 5191-5200. Pham, N., Lucumi, A., Cheung, N., and Viadiu, H. (2012) The tetramer of p53 in the absence of DNA forms a relaxed quaternary state, Biochemistry 51, 8053-8055. Vasudevarao, M. D., Mizar, P., Kumari, S., Mandal, S., Siddhanta, S., Swamy, M. M., Kaypee, S., Kodihalli, R. C., Banerjee, A., Naryana, C., Dasgupta, D., and Kundu, T. K. (2014) Naphthoquinone-mediated inhibition of lysine acetyltransferase KAT3B/p300, basis for non-toxic inhibitor synthesis, J Biol Chem 289, 7702-7717. Kaczmarska, Z., Ortega, E., Goudarzi, A., Huang, H., Kim, S., Marquez, J. A., Zhao, Y., Khochbin, S., and Panne, D. (2017) Structure of p300 in complex with acyl-CoA variants, Nat Chem Biol 13, 21-29. Kelley, L. A., Mezulis, S., Yates, C. M., Wass, M. N., and Sternberg, M. J. (2015) The Phyre2 web portal for protein modeling, prediction and analysis, Nat Protoc 10, 845-858. Aramayo, R., Sherman, M. B., Brownless, K., Lurz, R., Okorokov, A. L., and Orlova, E. V. (2011) Quaternary structure of the specific p53-DNA complex reveals the mechanism of p53 mutant dominance, Nucleic acids research 39, 8960-8971. Kitayner, M., Rozenberg, H., Kessler, N., Rabinovich, D., Shaulov, L., Haran, T. E., and Shakked, Z. (2006) Structural basis of DNA recognition by p53 tetramers, Molecular Cell 22, 741-753. Tidow, H., Melero, R., Mylonas, E., Freund, S. M., Grossmann, J. G., Carazo, J. M., Svergun, D. I., Valle, M., and Fersht, A. R. (2007) Quaternary structures of tumor suppressor p53 and a specific p53 DNA complex, Proc Natl Acad Sci U S A 104, 12324-12329.

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1

Biochemistry

Figure Legends:

2 3

Figure 1. p53-induced p300 autoacetylation.

4

(A) An in vitro autoacetylation assay was performed to determine the levels of p300

5

autoacetylation in the presence of increasing concentrations of p53 as indicated. The

6

autoradiogram panels depict the acetylated proteins and Coomassie panels are the loading

7

control. (B) The protein levels of p53, alpha-tubulin, autoacetylated p300 (ac-p300), p300,

8

H3K18ac, and H3 were determined through western blotting analysis in the presence and

9

absence of 1 μg/ml of doxycycline (Dox) in the p53-expressing dox-inducible H1299 cells.

10

(C) Immunofluorescence to determine the levels of ac-p300(green) and p53 (red) in HepG2

11

cells treated with 5 μM Nutlin3a or vehicle control (DMSO) for 6 hrs. The cells were

12

counterstained with Hoechst. Scale bar, 10 μm. Quantification of the data is shown in the left

13

panels of A and C. All statistical analysis was performed using unpaired two-tailed Student's t

14

test.

15 16

Figure 2. Cryo-EM 3D reconstructions of p300-p53 complex.

17

(A) Front and (B) back views of the 3D cryo-EM map of the p300-p53 complex map

18

(surface representation in purple, contour level 2.0). Landmarks: SP, spout; H, handle

19

(Deposited to EMDB database, ID: EMD-6791). (C) Surface representation of the densities

20

encompassing p300 (blue, contour level 0.95) and p53 (brick red, contour level 1.4) in the

21

complex following segmentation. (D) Superimposition of the density map of the free p300

22

protein (dark cyan surface, contour level 0.02) on the p300 density isolated from the p300-

23

p53 complex map (blue mesh, contour level 2.0). It is clearly seen that the central part of the

24

protein superimposes well, whereas, peripheral regions are bloated considerably in the p53-

25

bound form of p300. The asterisk (*) indicates the absence of density in the p300 map, 29


Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 30 of 39

1

whereas, in the the p300-p53 complex, density can be clearly seen in this region. Landmarks

2

in p300: F, fist; Th, thumb. (EMDB id of the deposited p300 density map: EMD-6792.).

3 4

Figure 3. Interpretation of the densities in molecular terms.

5

(A) Domain organization of p300 in schematic representation is shown. The subdomains of

6

central (core) domain are color-coded with residue numbers. (B) Cartoon representation of

7

the published crystal structure of the Bromo-HAT domain (PDB ID: 4BHW). Bromo-

8

domain, RING, PHD and HAT domains are shown in yellow, green, orange and red,

9

respectively. A rotated view of the same is also shown where the N- and C-terminal ends are

10

marked. Stereo views of the (C) p300 map (dark cyan mesh, contour level 0.02) and (D)

11

p300-p53 complex map (p300, blue mesh, contour level 0.95; p53, red mesh, contour level

12

1.4) are shown (IDs of the deposited maps are: p300-p53 complex: EMD-6791 and p300:

13

EMD-6792). The maps are fitted with the crystal structure of central domain of p300 (PDB

14

ID: 4BHW). Different subdomains are represented in the same colors as mentioned in (A, B).

15

(PDB id of the fitted central domain models are: p300-p53 complex: 5XZC and p300: 6K4N)

16

Additional empty densities are marked which can be attributed to other N- and C-terminal

17

domains (N and C-terminals could not be unambiguously determined).

18

Less amount of extra densities are visible in free p300 than that of in the p300 segment of the

19

complex indicating that some parts of the N- and C-terminal domains likely get stabilized

20

upon complex formation. Landmarks are same as in Figure 2.

21 22

Figure 4. Conformational changes in the central domain of p300 upon complex

23

formation with p53.

24

Fitting of different subdomains (Bromo (yellow), RING (green), PHD (orange) and HAT

25

(red)) of the central domain of p300 (PDB code: 4BHW) in (A) p300 (dark cyan mesh, 30


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Biochemistry

1

contour level 0.02) and (B) p300-p53 complex (p300, blue mesh, contour level 0.95; p53, red

2

mesh, contour level 1.4) maps. Subdomains are shown in cartoon representations. Opening up

3

of the kink between the Bromo and HAT domains is clearly detected. Close-up views of the

4

superposed cryo-EM maps of p300 and p300-p53 complex (dark cayan mesh, p300 map

5

(EMDB-6792); purple mesh, p300-p53 complex map (EMDB-6791)) with the corresponding

6

fitted structures (p300-p53 complex: 5XZC and free p300: 6K4N) showing (C) reorientation

7

of the Bromo-domain (yellow), (D) loop-like density seen on the back of p300 density in the

8

complex (marked as L in Figure 2) that can accommodate a model of the autoinhibitory loop,

9

and (E) RING (green) and PHD (orange) sub-domains of p300 central domain are displaced

10

following complex formation.

11 12

Figure 5. Interpretation of p53 densities in molecular terms and comparison with other

13

reported cryo-EM structures of p53 tetramer.

14

(A) Schematic representation of domain organization of p53 in different color codes. (B)

15

Stereo representation of segmented p53 density (red mesh, contour level 1.4) in close-up

16

view with the fitted crystal structures of the DNA-binding domains (DBD) (published crystal

17

structure of DBD tetramer PDB code: 3KMD in yellowish green color) and tetramerization

18

domains (published crystal structure, PDB code: 4D1M in dark green). Dimer of DBD can be

19

fitted as a rigid module in each lobe. (C) Qualitative docking of a piece of DNA (pink

20

surface) shows that it can be accommodated in the groove of p53 lobes (red surface). (D)

21

Fitting of tetramerization domain (PDB code: 4D1M in dark green) at the central region of

22

the density attributed to p53 (red surface). (E) Schematic representations of different cryo-

23

EM structures available for p53 tetramer. Cryo-EM maps of the p53 tetramer published by

24

different groups (upper panel) form1:18; form2: 49. The lower panel shows the DNA bound

25

conformation of p53 tetramer reported by different groups (form1:53; form2:17). Middle panel 31


Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 32 of 39

1

(highlighted) shows the conformation adopted by p53 following complex formation with

2

p300 (current study). Landmarks: N-term: N-terminal domain, DBD: DNA binding domain,

3

Tet domain: tetramerization domain, C-term: C-terminal domain.

4 5

Figure 6. The active site of the p300 HAT domain in the p300-p53 complex and its close-

6

up view.

7

A model of flexible C-terminal end part (residues 357-393, magenta) of p53, generated using

8

PHYRE2, is qualitatively docked at the end of tetramerization domain (green), fitted into the

9

density map (EMDB-6791) of p300-p53 complex (p300 in blue mesh, contour level 0.95; p53

10

in red mesh, contour level 1.4). The close-up view (left panel) shows that the distal C-

11

terminal (substrate for acetylation) can access the active site of the HAT domain (red) easily.

12

Acetyl-CoA residue (blue sticks) marks the active site cleft of the HAT domain. C-terminal

13

end of each tetramerization domain is indicated by an asterisk (*). The distances from the

14

ends of each C-terminal of tetramerization domain to the active site of the HAT domain vary

15

within the range of ~30-50 Ǻ.

16 17

Graphical Abstract: A cartoon representation of the proposed activation mechanism of

18

p300 upon binding with p53.

19

In the free p300, the central domain (Bromo-RING-PHD-HAT) remains in compact

20

‘autoinhibited’ conformation, while the N- and C-terminal domains are in ensemble of

21

dynamic conformations. P53 binding induces opening of the ‘kink’ between Bromo and HAT

22

subdomains of the central domain. On the other hand, the intrinsically dynamic N- and C-

23

terminal domains of p300 get stabilized upon interaction with p53.

24 25 26 32


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1

Biochemistry

For Table of Contents Use Only

2 3

Tumor suppressor p53-mediated structural reorganization of the transcriptional

4

coactivator p300

5 6

Raka Ghosh1, Stephanie Kaypee2, Manidip Shasmal1,4, Tapas K. Kundu3,*, Siddhartha Roy1,*,

7

Jayati Sengupta3,*

8 9

1

Department of Biophysics, Bose Institute, Kolkata, India

10

2

Transcription and Disease Laboratory, Molecular Biology and Genetics Unit, Jawaharlal

11

Nehru Centre for Advanced Scientific Research, Bangalore, Karnataka, India

12

3

13

Biology, Kolkata, India

Division of Structural Biology and Bioinformatics, CSIR-Indian Institute of Chemical

14 15 16

*

17

Tapas K. Kundu: [email protected]

18

Siddhartha Roy: [email protected]

19

Jayati Sengupta: [email protected]

Corresponding Authors:

20 21 22

23 24 33


Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


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Biochemistry


Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Page 36 of 39

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Biochemistry


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Figure 5 171x112mm (300 x 300 DPI)


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Biochemistry


Tumor Suppressor p53-Mediated Structural Reorganization of the

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