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The role of H293 in Protein Arginine Methyltransferases 1 (PRMT1)

Brittany Boykin Auburn University

Department of Chemistry and Biochemistry Computational Seminar

National Organization for the Professional Organization of Black Chemist and Chemical Engineers 2015 Annual Conference

Orlando, Florida24, September 2015

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OutlineBackground

Overall Goal

• Future Work• Acknowledgments • Q/A

Current Study

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Post-Translational Modification (PTMs)

• Remarkably, there are 200+ types of PTM’s that include kinases, phosphatases, transferases

Walsh C. (2006) Posttranslational Modification of Proteins: Expanding Natures Inventory. Englewood, Colo.: Roberts and Co. Publishers. Xxi, 490 p. p.

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Arginine Methylation

FASEB J. 10, 471-480 (1996)

• Methylation reactions involve methyl group transfers (AdoMet is the methyl donor and this reaction displays a Sn2 type geometry)

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Protein Arginine Methyltransferases (PRMTs)

Bedford, M. T., and Clarke, S. G. (2009) Protein Arginine Methylation in mammals: Who, What, and why. Mol Cell 33, 1-13

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Physiological roles of PRMTs

Transcriptional Activity

RNA splicing and transport DNA repair

Chromatin Remodeling

Signal Transduction

Neuronal Cell Differentiation

Virus-host Biochemistry

Journal of Biological Chemistry Vol. 289, NO. 13, pp. 9320-9327, March 28, (2014)

• PRMTs regulate proteins in cell processes and human diseases

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Target Diseases

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PRMT Human Isoforms

Cell. Mol. Life Sci. 2009 66:2109.

PRMT1

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Human Diseases: PRMT1’s Target

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Structure of PRMT1

Structure 2003 11: 509.

Dimerization - essential for SAM binding and enzymatic activity

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Active Site of PRMT1

Biochemistry 2011, 50, 3332-3345J. Bio. Chem. Vol. 289, NO. 13, pp. 9320-9327 2014

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Overall Goal Product Specificity

Org. Biomol. Chem., 2015, 13, 549-560

Control Sterics & Nucleophilicity

• What use of the product depends on the local conditions?

• We want to gain more insight by dissecting the active site of PRMT1 and identifying the significance of specific

residues in regards to the substrate

• What orientation does the substrate display with the

H293S mutation?

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Importance of H293

Salt-Bridge

Biochemistry 2011 April 26; 50(16): 3332-3345

• This short bond plays a critical role in forming the two-helix boundary that impact cofactor and peptide binding

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Alternative Mechanism

NNHNN

N

OO

H3C

S

H

SAM

His293

Glu144

HH

H H

Arg-substrate

O

O

Glu153

NNHNN

N

OOS

H

SAM

His293

Glu144

H

H

H3C H

dicationintermediate

O

O

Glu153

H

NNHNN

N

OOS

H

SAM

His293

Glu144

H

H3C H

MMA-productO

O

Glu153

H

H

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Current Research Objective Objective • Product Specificity of H293: Mutant H293S;

• How does the protein environment influence product specificity using aMD simulations and QM/MM calculations

• Analyzed systems:• H293S-Arg• H293S-MMA-ADMA• H293S-MMA-SDMA

• With the Ser in place of His:• What orientation does the substrate display• Distribution of the His vs Ser

Question:• Does H293 have more affect on the active site than

proposed?

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Assisted Model Building With Energy Refinement (AMBER)

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Molecular Dynamics (MD)

F = ma

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(QM) | (MM)

• Quantum Mechanical (QM) Electronic Processes Bonding Breaking/formation DFT/ Ab Initio Primary subsystem (PS)

• Molecular Mechanical (MM) Force-field based method Computationally Efficient Secondary subsystem (SS)

• Combined QM/MM Chemical Reaction in macromolecules

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PRMT1 RMSF Analysis

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Preferred Methylation

13.512.010.59.07.56.04.5

1.2

1.0

0.8

0.6

0.4

0.2

0.0

d+0.5*cos(angle) radians

Den

sity

H293S-MMA-Nn2-P1

13.512.010.59.07.56.04.5

1.2

1.0

0.8

0.6

0.4

0.2

0.0

d+0.5*cos(angle) radians_1

Den

sity

H293S-MMA-Nn2-P2

13.0511.6010.158.707.255.804.35

1.2

1.0

0.8

0.6

0.4

0.2

0.0

d+0.5*cos(angle) radians

Den

sity

H293S-MMA-Nn1-P1

13.0511.6010.158.707.255.804.35

1.2

1.0

0.8

0.6

0.4

0.2

0.0

d+0.5*cos(angle) radians

Den

sity

H293S-MMA-Nn1-P2

SDMA

ADMA

ADMASDMA

H293S-MMANη2

H293S-MMANη1

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Future Work

• To continue the QM/MM simulations for all mutant complexes: H293S-Arg, H293S-MMA-proADMA, and H293S-MMA-proSDMA

• Continue to compare to double mutant (H293S-M48F) and WT-enzyme

• To compare to experimental results

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AcknowledgementsoAlabama Super Computer Center oHuntsville Alabama

oCollaborators: Dr. Joan Hevel (Utah State University)oOrlando AcevedooMy Lab members oSymon GathiakaoNicole IppolitooRobel GhebreaboBrian Doherty

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