The bh3 domain of puma structure determination and small molecular inhibitor design
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The Bh3 Domain of Puma: Structure Determination and Small Molecular Inhibitor Design. Nicki Zevola Bahar Lab Lab Meeting March 25, 2009. Intro. What are BCl-2 proteins?. 1. A class of pro-survival and pro-apoptotic proteins Easily distinguishable

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The bh3 domain of puma structure determination and small molecular inhibitor design l.jpg

The Bh3 Domain of Puma:Structure Determination and Small Molecular Inhibitor Design

Nicki Zevola

Bahar Lab

Lab Meeting March 25, 2009

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Intro. What are BCl-2 proteins?


  • A class of pro-survival and pro-apoptotic proteins

    • Easily distinguishable

    • Pro-survival: BclXL, Bcl-w, MCl-1, A1, Bax, Bak

      • 2-4 BH domains

    • Pro-apoptotic: Bim, Bad, Bmf, Bid, Bik, Hrk, Noxa, Puma

      • Only 1 BH domain, Bh3

  • Key interactions occur when pro-apoptotic proteins (e.g., Puma) inhibit pro-survival proteins (e.g., BclXL)



Crystal structure of BCl-xl (mouse)


Crystal structure of Puma (mouse)

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Goal of Project: Develop small molecule mitochondrial-targeted drugs to inhibit the binding of the Puma Bh3 domain to Bcl-2 family proteins.

Normally, Puma Bh3 domain:Bcl-2 protein binding antagonizes anti-apoptotic Bcl-2 proteins, inhibiting Apaf-1, and resulting in caspase release and apoptosis.

Studies with Puma-deficient mice have shown that Puma not only activates, but is required for, apoptosis induced by oncogenes and DNA-damaging agents.1,2

Project Overview

1Qui et. al. PUMA Regulates Intestinal Progenitor Cell Radiosensitivity and Gastrointestinal Syndrome. Cell Stem Cell. 2008. 2(6):576-583.

2 Yu J., Zhang L., Hwang PM, Kinzler K.W., Vogelstein B. Puma induces the rapid apoptosis of colorectal cancer cells. Mol. Cell.2001. 7:3:673-82.

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How We Will Find Small Molecular Inhibitors for Puma (Project Design)

Analyze structure of Bh3 domain of Puma

Determine key conserved interactions between Puma Bh3 domain and Bcl-2 proteins

Develop pharmacophore models using conserved interactions

Screen Zinc 8.0 database for lead-like compounds that match pharmacophore model

Develop Puma models to prioritize hits

In vitro testing

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Day et. al.: How Puma Bh3 Domain Bh3-Mcl1 and Noxa Bh3-Mcl1 Structures were Obtained

  • 1. Two samples made

    • 1. 13C, 15N-labeled peptide + unlabeled Mcl-1

    • 2. Unlabeled peptide + labeled Mcl-1

  • 2. NMR techniques used for resonance assignments

  • 3. Structures evaluated for proper geometry

    • No distance violation > 0.2 Å

    • No angle violation > 5o



  • Why only the Bh3 Domain of Puma, Noxa with Mcl-1?

  • Puma and Noxa have extremely unstable structures, and hence only the Bh3 binding domain structure has been accurately determined. In the Bahar lab, we have used the i-Tasser server (Zhang) to help ascertain the entire Puma structure (see next slide).

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i-Tasser Structures were Obtained

  • Sequence of hPUMA submitted to online server developed by Dr. Yang Zhang, Ph.D. (University of Kansas)

    • Finalist at CASP (Critical Assessment of Structural Predictions) Competition

  • Server uses LOMETS (a form of threading) in structural prediction

  • 5 models generated for hPuma

    • Problem: Best C-score: -3.46

      • Will be addressed later

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Step 2. Determine key conserved interactions Structures were Obtained

between Puma Bh3 domain and BCl-2 proteins

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Day et. al.: Comparison of MCl-1 complexes with other BCl-2 proteins

  • 1. Superimposition of Mcl-1:Puma with:

  • BClXL:Bim,

  • A1:Puma,

  • and BclXL

    • RMSD of backbone BClXL:Bim and Mcl-1: Puma: 1.5 Å

    • Most notable structural difference: BclXLhas a long loop between α1 and α2 not found in Mcl-1 or A1

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Day et. al.: Comparison of Mcl-1 complexes with other Bcl-2 proteins (con’t)

  • 2. Structural Alignment

    • Sequence comparison (excluding BclXL loop residues):

      • BclXL,Mcl-1: 25.6% identity, 40.0% similarity

      • A1, Mcl-1: 25.4% identity, 46.% similarity

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Day et. al.: The Consensus Bh3 Domain Binding Motif proteins (con’t)

  • The 13 residue consensus sequence that defines the Bh3 domain in PUMA is: φ1ΣXXφ2XXφ3ΣDZφ4L

    • where φ1-φ4 are hydrophobic residues

    • Σ are small residues

    • Z is usually an acidic residue

    • L is a hydrophilic residue capable of forming an intermolecular cap

    • D is the conserved aspartate

Bh3 domains of Noxa (in yellow, left) and Puma (in pink,right).

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in situ proteins (con’t)Mutagenesis Reveals Other Key Residues (Yu et. al., 2009)

  • Already known from Day et. al.: conserved interactions with Bcl2 proteins

  • Jian Yu et. al. (Univ. of Pittsburgh): 2 specific point mutations alter binding specificity upstream of the Bh3 binding domain!

    • These are therefore included in our pharmacophore model (next step)

Images courtesy Gabriela Mustata, Ph.D.

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Step 3. Develop pharmacophore models proteins (con’t)

using conserved interactions

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Pharmacophore Model Development proteins (con’t)

  • Our superimposition of our hPuma-A1 (from i-Tasser) with hPUMA-Mcl1 (from 2roc) structures resulted in an RMSD of 0.55Å!

  • Four key interactions of Bcl2 proteins and hPuma (see next slide):

    • Phe251.CB (Bcl2) – Leu141.CD1 (hPuma)

    • Ph395.CD1 (Bcl2) – Leu141.CD1 (hPuma)

    • Asp237.OD2 (Bcl2) – Asp142.NH1 (hPuma)

    • Arg244.NE (Bcl2) – Asp146.OD1 (hPuma)

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Pharmacophore Model Development proteins (con’t)

Slide courtesy Dr. Gabriela Mustata, Ph.D.

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Step 4. Screen Zinc 8.0 database for proteins (con’t)

compounds that match pharmacophore model

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Zinc 8.0 Database Searching proteins (con’t)

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Pharmacophore Search Results Thus Far proteins (con’t)

  • Zinc 7.0 Lead-like Database

  • Found 1 hit

  • Zinc 8.0 Lead-like Database

  • So far…Screened 24/37 of database (about 1.3 x 106 compounds)

  • Found 20 hits thus far (some shown below)

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Future Steps proteins (con’t)

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Current Problem: i-Tasser model proteins (con’t)

  • 5 models of hPuma generated by i-Tasser are not entirely satisfactory, as shown by Fast Contact server (Camacho et. al.)

  • Current solution: Use MD Simulations for energy minimization of i-Tasser models, recalculate energies on Fast Contact server

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Questions? Ideas? Comments? proteins (con’t)

A special thank you: Dr. Ivet Bahar; Dr. Gabriela Mustata; Dr. Jian Yu; Dr. Joel Greenberger; Ahmet Bakan (for teaching MD Simulations); everyone else for listening, 