The Effect of New Parameters and Increased Database Size on the Cysteine Oxidation Prediction

Program

Megan Riddle with Ricardo Sanchez and Dr. Jamil Momand

California State University, Los AngelesAugust 23, 2007

HS CH2 C COOH

H

NH2

Overview• Cysteine Oxidation Prediction Program

(COPP) – Oxidation defined

• Biological Significance of Cysteine Oxidation

– Effects of oxidation on proteins

• Summer 2007 Goals– Increase database size– Add new parameters

• Methods and Results

HS CH2 C COOH

H

NH2

Cysteine Oxidation Prediction Program

• Goal: Create a program that will use physicochemical parameters to predict reactive surface cysteine thiols

• Methods:– Gather examples of proteins susceptible to

cysteine oxidation– Extract parameters from Protein Data Bank – Use computer classifier C4.5 to determine

rules that will predict if cysteine can become oxidized

Oxidation of Cysteines

Sanchez (2007)

Cysteine Prediction

Two types of cysteine oxidation:1. Permanent structural oxidation: cysteines

that form permanent disulfide bonds or bind to metals shortly after translation

– Prediction programs based on sequence already exist 88% accuracy (Martelli et al. 2002)

2. Reactive surface cysteine thiols: cysteines that become oxidized under certain conditions, most reversibly

– No prediction programs exist COPP

Biological Significance: Oxidation and Enzyme Function

• The active sites of glutaredoxin and thioredoxin cycle between reduced and oxidized states

http://www.cs.stedwards.edu/chem/Chemistry/CHEM43/CHEM43/Thioredox/RNA2.GIF

Enzyme Inactivation via Oxidation

• H2O2 inactivates PTEN tumor suppressor protein by causing the formation of a disulfide bond

Lee et al. JBC (2002)

Summer 2007 Goals

1. Increase the size of the COPP database

2. Test new parameters to determine if they affect the rules and accuracy of COPP

HS CH2 C COOH

H

NH2

Increase Database Size

• Previously:– 85 proteins that undergo non-structural

cysteine oxidation– 135 cysteines that undergo oxidation– 225 cysteines that remain reduced under

oxidizing conditions

• To create an accurate, general set of rules for cysteine oxidation requires a large, unbiased database

Methods: Increase Database Size 1. Search Entrez for keywords

• i.e. cysteine and oxidation, sulfenic acid, etc.

2. Look for proteins in Protein Data Bank

Potential Proteins

Increase Database Size

3. Do BLASTALL – eliminate proteins with:• Identity > 35%• E value < 1• Conserved cys

Potential Proteins

Cysteines Oxidize

Increase Database Size

C4.5/ J48

Original Proteins

Rules to Classify Cysteines

New Proteins

Results: New Proteins

S1 DISTANCE <= 6: 1 (88.19/17.0)S1 DISTANCE > 6| ASA (Å2) <= 1: 0 (136.51/6.51)| ASA (Å2) > 1| | N1 DISTANCE <= 5.2: 1 (32.54/9.0)| | N1 DISTANCE > 5.2| | | O1 ASA <= 2: 1 (33.0/15.0)| | | O1 ASA > 2: 0 (71.76/15.76)

6Å

+

5.2Å

-

Sanchez (2007)

• Increased database size caused reduction in rules

• Accuracy decreasedOld Rules New Rules

S1 DISTANCE <= 6.1: 1 (115.44/28.0)S1 DISTANCE > 6.1| ASA (Å2) <= 1.8: 0 (177.51/12.51)| ASA (Å2) > 1.8| | N1 DISTANCE <= 5.4: 1 (46.54/17.0)| | N1 DISTANCE >5.4: 0 (133.51/39.51)

81.8% Accuracy 79.1% Accuracy

Methods: Parameters already used by COPP

• S1 DISTANCE distance to nearest sulfur atom• S1 ASA area exposed to the surface• N1 DISTANCE distance to the nearest +nitrogen

atom• N1 DONOR nitrogen’s parent side chain• N1 ASA area exposed to the surface• O1 DISTANCE distance to the nearest -oxygen• O1 DONOR oxygen’s parent side chain• O1 ASA area exposed to the surface• ASA exposed surface of S in question • CLASS class: 0 if reduced; 1 otherwise

Methods: Parameters already used by COPP

• S1 DISTANCE distance to nearest sulfur atom• S1 ASA area exposed to the surface• N1 DISTANCE distance to the nearest +N atom• N1 DONOR nitrogen’s parent side chain• N1 ASA area exposed to the surface• O1 DISTANCE distance to the nearest -oxygen• O1 DONOR oxygen’s parent side chain• O1 ASA area exposed to the surface• ASA exposed surface of S in question • CLASS class: 0 if reduced; 1 otherwise

New Parameters

• pKa: acid dissociation constant– How easily can the S lose a proton?

• Electrostatic Potential: potential energy per unit charge– How well stabilized is the charged S after the

proton is lost?

-S CH2 C COOH

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H

Methods: New Parameters • PCE: Protein Continuum Electrostatics

– Calculates Electrostatic Potential

Coordinates Electrostatic Potential

7.854 0.668 -0.602 -10.725 4.683 1.223 3.305 -25.413 3.330 8.072 3.708 -19.335 2.256 -11.243 9.879 -21.887 14.014 7.907 3.298 -13.670

Miteva et al. NAR (2005)http://bioserv.rpbs.jussieu.fr/cgi-bin/PCE-Pot

New Parameters• PROPKA

– Calculates pKa

Li et al. Proteins (2005)http://propka.ki.ku.dk/

New Parameters

C4.5/ J48

Electrostatic Potential and pKa Data

Original Data

Rules to Classify Cysteines

Results: New Parameters

• New parameters caused an alteration in the final rule

• The accuracy is similar

Old ParametersS1 DISTANCE <= 6.1: 1 (115.44/28.0)S1 DISTANCE > 6.1| ASA (Å2) <= 1.8: 0 (177.51/12.51)| ASA (Å2) > 1.8| | N1 DISTANCE <= 5.4: 1 (46.54/17.0)| | N1 DISTANCE >5.4: 0 (133.51/39.51)

79.0698% Accuracy

S1 DISTANCE <= 6.1: 1 (115.44/28.0)S1 DISTANCE > 6.1| ASA (Å2) <= 1.8: 0 (177.51/12.51)| ASA (Å2) > 1.8| | pKa of S0 <= 8.75: 1 (74.29/32.0)| | pKa of S0 > 8.75: 0 (105.76/26.76)

New Parameters

78.6469% Accuracy

Conclusions

• New Proteins:– A larger database results in a more general,

but less accurate, set of rules

• New Parameters:– A low pKa value correlates with oxidation, but

does not improve the accuracy of COPP

• Future Goals:– Make COPP publicly available– Modify COPP to predict type of oxidation

With many thanks to. . .

Dr. Jamil Momand, Ricardo Sanchez, and the rest of the Momand lab

SoCalBSI fellow students and mentors

California State University at Los Angeles

Funding from:

LA Orange County Biotechnology Center

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Results: New Proteins

• Increased database size caused reduction in rules

• Accuracy decreased

S1 DISTANCE <= 6: 1 (88.19/17.0)S1 DISTANCE > 6| ASA (Å2) <= 1: 0 (136.51/6.51)| ASA (Å2) > 1| | N1 DISTANCE <= 5.2: 1 (32.54/9.0)| | N1 DISTANCE > 5.2| | | O1 ASA <= 2: 1 (33.0/15.0)| | | O1 ASA > 2: 0 (71.76/15.76)

81.8% Accuracy 79.1% Accuracy

Old Rules New RulesS1 DISTANCE <= 6.1: 1 (115.44/28.0)S1 DISTANCE > 6.1| ASA (Å2) <= 1.8: 0 (177.51/12.51)| ASA (Å2) > 1.8| | N1 DISTANCE <= 5.4: 1 (46.54/17.0)| | N1 DISTANCE >5.4: 0 (133.51/39.51)

Cys-SH HS-Cys