Poster template
Towards Manipulation and Comprehensive Understanding of Ribonucleotide Reductase Specificity Andrew Knappenberger (Biochemistry, Case Western Reserve University), Md. Faiz Ahmad (Pharmacology, Case Western Reserve University), Chris Dealwis (Pharmacology, Case Western Reserve University), Michael Harris (Biochemistry, Case Western Reserve University)
• Ribonucleotide reductase (RR) generates all deoxynucleotides found in Nature • RR has significance for studies of cancer chemotherapy, specificity, allostery, and dynamics • RR specificity changes with binding of effectors at an allosteric site (the specificity site) (Figure 1)
Design and validate experimental system
Measure native RR specificity
6000
1. Relative velocity should increase linearly with relative [S].
5000 4000
Perturb specificity with molecules or mutations
Measure new specificity; generate new hypotheses
dGDP dCDP dTDP dUDP
1000
dADP
min
15.0
12.5
2. Relative second-order rate constant should remain constant.
Black: absorbance at 259nm Red: absorbance at 271nm
• We want to gain an understanding of the mechanistic basis for specificity in RR
1.1
0.6
0.1 -0.9
-0.4
0.1
0.6
-0.4
1.1
1.6
y = 0.8576x + 0.6272 R² = 0.984
-0.9
log([ADP]/[CDP]) 5 4
-1.2
3 2 1 0 -1
-0.8
-0.6
-0.4
-0.2
-1 -2 -3
0
0.2
0.4
0.6
0.8
1
y = -0.1363x + 0.6215 R² = 0.6169
2. Measure native RR specificity under all physiologically relevant conditions.
H. sapiens and S. cerevisiae RR specificity: AMPPNP/UDP
dTTP-bound human RR
dGTP-bound human RR
ATP-bound human RR
Physiological human RR
1 0.1
Relative V/K
Relative V/K
Relative V/K
Relative V/K
1 0.1
0.1
0.1
Specificity sites
0.01
0.01
0.01
0.001
0.001
ADP CDP GDP UDP
dGTP/ADP
• Quantitative readouts of specificity largely recapitulate qualitative accounts in previous decades, except that UDP is always disfavored
1
1
0.01
ADP CDP GDP UDP
ADP CDP GDP UDP
ADP CDP GDP UDP
dTTP/GDP 10
10
10
Physiological yeast RR
dTTP-bound yeast RR
dGTP-bound yeast RR
ATP-bound yeast RR
10
0.01
Relative V/K
Figure 1: H. sapiens R1 homodimer. Fairman et al., Nat Struct Mol Biol. 2011
0.1
Relative V/K
Relative V/K
Loop 2
0.1
0.01
0.001
0.0001
ADP CDP GDP UDP
ADP CDP GDP UDP
ADP
CDP
GDP
UDP
3c. In silico screen for novel chemotherapeutics:
3b. Interrogation of site-directed loop 2 mutants:
• Future studies will incorporate mutagenesis and exogenous small molecules to engineer altered in specificity Preliminary Results:
ATP-bound RR processing of CDP
3a. Mechanistic interrogation through comprehensive chemical mutagenesis:
1E-11
• The energetic contribution of the exocyclic amine of GDP to processing by dTTPbound hRR is ~10-fold
dGTP-bound wild-type RR 10
uV
1
2250
IDP; rV/K=1.5
2000
• P294K hRR has 200-fold decreased activity but so far shows wild type specificity
1E-12 velocity (mol dCDP/s)
A typical activity assay consists of R1, R2, substrates, effectors, DTT, gly-gly pH 7.7, and MgCl2.Physiological [effectors] are taken from Traut (1994) and McElhinny (2010), respectively. Relative rates of formation for each dNDP are measured by borate chromatography, ion exchange chromatography, and UV absorbance essentially as in Hendricks (8). Each substrate acts as a competitive inhibitor for the other three, so determining the relative rates of formation at known substrate concentrations is sufficient for the determination of relative second-order rate constants (7). R1 and R2 are recombinantly expressed in E. coli. R1 is purified by peptide affinity chromatography, while R2 is purified using an N-terminal His-tag and nickel resin. Purification methods are similar for human and yeast RR, with the exception that yeast R2 requires association with a similar protein called R4 which must also be purified. Site-directed mutagenesis was conducted using the Thermo site-directed mutagenesis kit. All in silico experiments were conducted using Schrodinger’s small molecule drug discovery suite, the ZINC database and PDB entry 3HND.
0.1
0.001
3. Preliminary results:
Materials and Methods
1
0.01
0.001
ADP CDP GDP UDP
0.1
• Human and yeast RR specificity are identical within error, consistent with their identical loop 2 sequences • This is an integral first step to understanding the mechanistic basis for specificity
1
1
Relative V/K
1
• RR specificity across all four cognate substrates simultaneously and quantitatively determined via internal competition kinetics
10
10
10
10
Specificity Assays:
-4
log([ADP]/[CDP])
AMPPNP/CDP
• The ratio of observed velocities for two substrates increases as the ratio of the substrates’ concentration increases • Observed rV/K remains constant (within 2-fold) across a 100-fold range of substrate concentrations
-5
• We developed a method that allows for simultaneous measurement of V/K values for multiple substrates • We comprehensively and quantitatively determined the specificity of human RR • We screened for artificial effectors in silico using the Schrodinger software
Internal Competition Validation:
-1.4
[ADP]/[CDP] vs rV/K(ADP/CDP)
0
10.0
1.6
-1.4
3000 2000
Conclusions
[ADP]/[CDP] vs vobs(ADP/CDP)
uV
7.5
Active sites
νobs1 /νobs 2 = [(V / K )1 /(V / K ) 2]( S 1 / S 2)
1. Develop an experimental system for measuring RR specificity using alternative substrate kinetics.
log(vobs(ADP)/vobs(CDP))
Results
log(rV/K (ADP))
Introduction
1750
GDP; rV/K=14
CDP; rV/K=1
1500 1250 1000
0.1
ADP
CDP
dGTP-bound P294K RR 1E-13
• Schrodinger has revealed ~10 hits which may be capable of acting as artificial effectors
10
750 500
1
250 7.5
10.0
12.5
References: 1. Wijerathna, S.R.; Ahmad, M.F.; Xu, H.; Fairman, J.W.; Zhang, A.; Kaushal, P.S.; Wan, Q.; Kiser, J.; Dealwis, C.G. Pharmaceuticals 2011, 4, 1328-1354. 2. Burstein, Harold J. Journal of Clinical Oncology 18.3 (2000): 693-693. 3. Galmarini, Carlos M., John R. Mackey, and Charles Dumontet. The lancet oncology 3.7 (2002): 415-424. 4. Shewach, Donna S., and Theodore S. Lawrence. Investigational new drugs 14.3 (1996): 257-263.
15.0
17.5
min
0.1
1E-14
wild-type
5. Oliver, F. J., M. K. L. Collins, and A. Lopez-Rivas. Experientia 52.10-11 (1996): 995-1000. 6. Xu, Hai, et al. Proceedings of the National Academy of Sciences of the United States of America 103.11 (2006): 4022-4027. 7. Cornish-Bowden, Athel. Journal of theoretical biology 108.3 (1984): 451-457. 8. Hendricks, Stephen P., and Christopher K. Mathews. "Regulation of T4 Phage Aerobic Ribonucleotide Reductase." Journal of Biological Chemistry 272.5 (1997): 2861-2865.
P294K
ADP
CDP
Acknowledgments
Further Information
AJK was supported in part by NIGMS T32 GM008056.
Email: [email protected]
We thank the members of the Dealwis lab, especially Chris, Faiz, Tessianna and Sarah, for experimental advice and comments on the results.
Please email me with any questions, comments, or concerns,
Template downloaded from: http://colinpurrington.com/tips/academic/posterdesign.
• Ribonucleotide reductase (RR) generates all deoxynucleotides found in Nature • RR has significance for studies of cancer chemotherapy, specificity, allostery, and dynamics • RR specificity changes with binding of effectors at an allosteric site (the specificity site) (Figure 1)
Design and validate experimental system
Measure native RR specificity
6000
1. Relative velocity should increase linearly with relative [S].
5000 4000
Perturb specificity with molecules or mutations
Measure new specificity; generate new hypotheses
dGDP dCDP dTDP dUDP
1000
dADP
min
15.0
12.5
2. Relative second-order rate constant should remain constant.
Black: absorbance at 259nm Red: absorbance at 271nm
• We want to gain an understanding of the mechanistic basis for specificity in RR
1.1
0.6
0.1 -0.9
-0.4
0.1
0.6
-0.4
1.1
1.6
y = 0.8576x + 0.6272 R² = 0.984
-0.9
log([ADP]/[CDP]) 5 4
-1.2
3 2 1 0 -1
-0.8
-0.6
-0.4
-0.2
-1 -2 -3
0
0.2
0.4
0.6
0.8
1
y = -0.1363x + 0.6215 R² = 0.6169
2. Measure native RR specificity under all physiologically relevant conditions.
H. sapiens and S. cerevisiae RR specificity: AMPPNP/UDP
dTTP-bound human RR
dGTP-bound human RR
ATP-bound human RR
Physiological human RR
1 0.1
Relative V/K
Relative V/K
Relative V/K
Relative V/K
1 0.1
0.1
0.1
Specificity sites
0.01
0.01
0.01
0.001
0.001
ADP CDP GDP UDP
dGTP/ADP
• Quantitative readouts of specificity largely recapitulate qualitative accounts in previous decades, except that UDP is always disfavored
1
1
0.01
ADP CDP GDP UDP
ADP CDP GDP UDP
ADP CDP GDP UDP
dTTP/GDP 10
10
10
Physiological yeast RR
dTTP-bound yeast RR
dGTP-bound yeast RR
ATP-bound yeast RR
10
0.01
Relative V/K
Figure 1: H. sapiens R1 homodimer. Fairman et al., Nat Struct Mol Biol. 2011
0.1
Relative V/K
Relative V/K
Loop 2
0.1
0.01
0.001
0.0001
ADP CDP GDP UDP
ADP CDP GDP UDP
ADP
CDP
GDP
UDP
3c. In silico screen for novel chemotherapeutics:
3b. Interrogation of site-directed loop 2 mutants:
• Future studies will incorporate mutagenesis and exogenous small molecules to engineer altered in specificity Preliminary Results:
ATP-bound RR processing of CDP
3a. Mechanistic interrogation through comprehensive chemical mutagenesis:
1E-11
• The energetic contribution of the exocyclic amine of GDP to processing by dTTPbound hRR is ~10-fold
dGTP-bound wild-type RR 10
uV
1
2250
IDP; rV/K=1.5
2000
• P294K hRR has 200-fold decreased activity but so far shows wild type specificity
1E-12 velocity (mol dCDP/s)
A typical activity assay consists of R1, R2, substrates, effectors, DTT, gly-gly pH 7.7, and MgCl2.Physiological [effectors] are taken from Traut (1994) and McElhinny (2010), respectively. Relative rates of formation for each dNDP are measured by borate chromatography, ion exchange chromatography, and UV absorbance essentially as in Hendricks (8). Each substrate acts as a competitive inhibitor for the other three, so determining the relative rates of formation at known substrate concentrations is sufficient for the determination of relative second-order rate constants (7). R1 and R2 are recombinantly expressed in E. coli. R1 is purified by peptide affinity chromatography, while R2 is purified using an N-terminal His-tag and nickel resin. Purification methods are similar for human and yeast RR, with the exception that yeast R2 requires association with a similar protein called R4 which must also be purified. Site-directed mutagenesis was conducted using the Thermo site-directed mutagenesis kit. All in silico experiments were conducted using Schrodinger’s small molecule drug discovery suite, the ZINC database and PDB entry 3HND.
0.1
0.001
3. Preliminary results:
Materials and Methods
1
0.01
0.001
ADP CDP GDP UDP
0.1
• Human and yeast RR specificity are identical within error, consistent with their identical loop 2 sequences • This is an integral first step to understanding the mechanistic basis for specificity
1
1
Relative V/K
1
• RR specificity across all four cognate substrates simultaneously and quantitatively determined via internal competition kinetics
10
10
10
10
Specificity Assays:
-4
log([ADP]/[CDP])
AMPPNP/CDP
• The ratio of observed velocities for two substrates increases as the ratio of the substrates’ concentration increases • Observed rV/K remains constant (within 2-fold) across a 100-fold range of substrate concentrations
-5
• We developed a method that allows for simultaneous measurement of V/K values for multiple substrates • We comprehensively and quantitatively determined the specificity of human RR • We screened for artificial effectors in silico using the Schrodinger software
Internal Competition Validation:
-1.4
[ADP]/[CDP] vs rV/K(ADP/CDP)
0
10.0
1.6
-1.4
3000 2000
Conclusions
[ADP]/[CDP] vs vobs(ADP/CDP)
uV
7.5
Active sites
νobs1 /νobs 2 = [(V / K )1 /(V / K ) 2]( S 1 / S 2)
1. Develop an experimental system for measuring RR specificity using alternative substrate kinetics.
log(vobs(ADP)/vobs(CDP))
Results
log(rV/K (ADP))
Introduction
1750
GDP; rV/K=14
CDP; rV/K=1
1500 1250 1000
0.1
ADP
CDP
dGTP-bound P294K RR 1E-13
• Schrodinger has revealed ~10 hits which may be capable of acting as artificial effectors
10
750 500
1
250 7.5
10.0
12.5
References: 1. Wijerathna, S.R.; Ahmad, M.F.; Xu, H.; Fairman, J.W.; Zhang, A.; Kaushal, P.S.; Wan, Q.; Kiser, J.; Dealwis, C.G. Pharmaceuticals 2011, 4, 1328-1354. 2. Burstein, Harold J. Journal of Clinical Oncology 18.3 (2000): 693-693. 3. Galmarini, Carlos M., John R. Mackey, and Charles Dumontet. The lancet oncology 3.7 (2002): 415-424. 4. Shewach, Donna S., and Theodore S. Lawrence. Investigational new drugs 14.3 (1996): 257-263.
15.0
17.5
min
0.1
1E-14
wild-type
5. Oliver, F. J., M. K. L. Collins, and A. Lopez-Rivas. Experientia 52.10-11 (1996): 995-1000. 6. Xu, Hai, et al. Proceedings of the National Academy of Sciences of the United States of America 103.11 (2006): 4022-4027. 7. Cornish-Bowden, Athel. Journal of theoretical biology 108.3 (1984): 451-457. 8. Hendricks, Stephen P., and Christopher K. Mathews. "Regulation of T4 Phage Aerobic Ribonucleotide Reductase." Journal of Biological Chemistry 272.5 (1997): 2861-2865.
P294K
ADP
CDP
Acknowledgments
Further Information
AJK was supported in part by NIGMS T32 GM008056.
Email: [email protected]
We thank the members of the Dealwis lab, especially Chris, Faiz, Tessianna and Sarah, for experimental advice and comments on the results.
Please email me with any questions, comments, or concerns,
Template downloaded from: http://colinpurrington.com/tips/academic/posterdesign.
