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Identification of DNA-binding protein target sequences by physical effective energy functions: free energy analysis of lambda repressor-DNA complexes.

Moroni E, Caselle M, Fogolari F - BMC Struct. Biol. (2007)

Bottom Line: In the present work we use physical effective potentials to evaluate the DNA-protein binding affinities for the lambda repressor-DNA complex for which structural and thermodynamic experimental data are available.The effect of conformational sampling by Molecular Dynamics simulations on the computed binding energy is assessed; results show that this effect is in general negative and the reproducibility of the experimental values decreases with the increase of simulation time considered.As a results of these analyses, we propose a protocol for the prediction of DNA-binding target sequences.

View Article: PubMed Central - HTML - PubMed

Affiliation: Dipartimento di Fisica Teorica, Universià di Torino and INFN, Via P, Giuria 1, 10125 Torino, Italy. moroni@to.infn.it

ABSTRACT

Background: Specific binding of proteins to DNA is one of the most common ways gene expression is controlled. Although general rules for the DNA-protein recognition can be derived, the ambiguous and complex nature of this mechanism precludes a simple recognition code, therefore the prediction of DNA target sequences is not straightforward. DNA-protein interactions can be studied using computational methods which can complement the current experimental methods and offer some advantages. In the present work we use physical effective potentials to evaluate the DNA-protein binding affinities for the lambda repressor-DNA complex for which structural and thermodynamic experimental data are available.

Results: The binding free energy of two molecules can be expressed as the sum of an intermolecular energy (evaluated using a molecular mechanics forcefield), a solvation free energy term and an entropic term. Different solvation models are used including distance dependent dielectric constants, solvent accessible surface tension models and the Generalized Born model. The effect of conformational sampling by Molecular Dynamics simulations on the computed binding energy is assessed; results show that this effect is in general negative and the reproducibility of the experimental values decreases with the increase of simulation time considered. The free energy of binding for non-specific complexes, estimated using the best energetic model, agrees with earlier theoretical suggestions. As a results of these analyses, we propose a protocol for the prediction of DNA-binding target sequences. The possibility of searching regulatory elements within the bacteriophage lambda genome using this protocol is explored. Our analysis shows good prediction capabilities, even in absence of any thermodynamic data and information on the naturally recognized sequence.

Conclusion: This study supports the conclusion that physics-based methods can offer a completely complementary methodology to sequence-based methods for the identification of DNA-binding protein target sequences.

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Related in: MedlinePlus

(a) Structure of the complex of λ repressor [58] with operator DNA. The protein was crystallized with a 19-bp duplex of which the central 17 bps are shown. The consensus half is to the left, (b) Relative free energy changes in the binding of λ repressor to OR1 on base substitutions. The figure shows the change in affinity that results from each of the three possible substitution at all 17 sites. The left part represents the consensus half-operator (solid box) and the right half the non-consensus half-operator (redrawn from ref. [61]).
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Figure 7: (a) Structure of the complex of λ repressor [58] with operator DNA. The protein was crystallized with a 19-bp duplex of which the central 17 bps are shown. The consensus half is to the left, (b) Relative free energy changes in the binding of λ repressor to OR1 on base substitutions. The figure shows the change in affinity that results from each of the three possible substitution at all 17 sites. The left part represents the consensus half-operator (solid box) and the right half the non-consensus half-operator (redrawn from ref. [61]).

Mentions: Atomic coordinates of the λ repressor dimer bound to OL1 DNA operator were taken from the 1.8 Å resolution X-ray crystal structure deposited in the Protein Data Bank [77] (PDB code 1LMB). The operator is 17 base-pairs in length and is composed by two approximately symmetric parts, the "consensus half" (maintaining the notation of the PDB file, base-pairs A19-T23 to G11-C31) and the "non-consensus half" (base-pairs T3-A39 to G10-C32) (see Figure 7). Since the coordinates of the NH2-terminal arm of the repressor bound to the non-consensus half operator were not available, the lacking amminoacids were added using the protein bound to the consensus half operator. Using the program ProFit V2.2 [78], the Cα carbons of the proteins have been superimposed and afterward the amino acids of the rotated structure have been added to the other one. Since the detailed X-ray crystal structure is made up of λ repressor dimer and OL1 operator DNA while the experimental data concern the OR1 site, the WHATIF [73] program was used to substitute the base-pair at position 5 to obtain the wild-type OR1 operator. All possible single base-pair substitutions within the DNA sequence were generated using the program WHATIF [73].


Identification of DNA-binding protein target sequences by physical effective energy functions: free energy analysis of lambda repressor-DNA complexes.

Moroni E, Caselle M, Fogolari F - BMC Struct. Biol. (2007)

(a) Structure of the complex of λ repressor [58] with operator DNA. The protein was crystallized with a 19-bp duplex of which the central 17 bps are shown. The consensus half is to the left, (b) Relative free energy changes in the binding of λ repressor to OR1 on base substitutions. The figure shows the change in affinity that results from each of the three possible substitution at all 17 sites. The left part represents the consensus half-operator (solid box) and the right half the non-consensus half-operator (redrawn from ref. [61]).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2194778&req=5

Figure 7: (a) Structure of the complex of λ repressor [58] with operator DNA. The protein was crystallized with a 19-bp duplex of which the central 17 bps are shown. The consensus half is to the left, (b) Relative free energy changes in the binding of λ repressor to OR1 on base substitutions. The figure shows the change in affinity that results from each of the three possible substitution at all 17 sites. The left part represents the consensus half-operator (solid box) and the right half the non-consensus half-operator (redrawn from ref. [61]).
Mentions: Atomic coordinates of the λ repressor dimer bound to OL1 DNA operator were taken from the 1.8 Å resolution X-ray crystal structure deposited in the Protein Data Bank [77] (PDB code 1LMB). The operator is 17 base-pairs in length and is composed by two approximately symmetric parts, the "consensus half" (maintaining the notation of the PDB file, base-pairs A19-T23 to G11-C31) and the "non-consensus half" (base-pairs T3-A39 to G10-C32) (see Figure 7). Since the coordinates of the NH2-terminal arm of the repressor bound to the non-consensus half operator were not available, the lacking amminoacids were added using the protein bound to the consensus half operator. Using the program ProFit V2.2 [78], the Cα carbons of the proteins have been superimposed and afterward the amino acids of the rotated structure have been added to the other one. Since the detailed X-ray crystal structure is made up of λ repressor dimer and OL1 operator DNA while the experimental data concern the OR1 site, the WHATIF [73] program was used to substitute the base-pair at position 5 to obtain the wild-type OR1 operator. All possible single base-pair substitutions within the DNA sequence were generated using the program WHATIF [73].

Bottom Line: In the present work we use physical effective potentials to evaluate the DNA-protein binding affinities for the lambda repressor-DNA complex for which structural and thermodynamic experimental data are available.The effect of conformational sampling by Molecular Dynamics simulations on the computed binding energy is assessed; results show that this effect is in general negative and the reproducibility of the experimental values decreases with the increase of simulation time considered.As a results of these analyses, we propose a protocol for the prediction of DNA-binding target sequences.

View Article: PubMed Central - HTML - PubMed

Affiliation: Dipartimento di Fisica Teorica, Universià di Torino and INFN, Via P, Giuria 1, 10125 Torino, Italy. moroni@to.infn.it

ABSTRACT

Background: Specific binding of proteins to DNA is one of the most common ways gene expression is controlled. Although general rules for the DNA-protein recognition can be derived, the ambiguous and complex nature of this mechanism precludes a simple recognition code, therefore the prediction of DNA target sequences is not straightforward. DNA-protein interactions can be studied using computational methods which can complement the current experimental methods and offer some advantages. In the present work we use physical effective potentials to evaluate the DNA-protein binding affinities for the lambda repressor-DNA complex for which structural and thermodynamic experimental data are available.

Results: The binding free energy of two molecules can be expressed as the sum of an intermolecular energy (evaluated using a molecular mechanics forcefield), a solvation free energy term and an entropic term. Different solvation models are used including distance dependent dielectric constants, solvent accessible surface tension models and the Generalized Born model. The effect of conformational sampling by Molecular Dynamics simulations on the computed binding energy is assessed; results show that this effect is in general negative and the reproducibility of the experimental values decreases with the increase of simulation time considered. The free energy of binding for non-specific complexes, estimated using the best energetic model, agrees with earlier theoretical suggestions. As a results of these analyses, we propose a protocol for the prediction of DNA-binding target sequences. The possibility of searching regulatory elements within the bacteriophage lambda genome using this protocol is explored. Our analysis shows good prediction capabilities, even in absence of any thermodynamic data and information on the naturally recognized sequence.

Conclusion: This study supports the conclusion that physics-based methods can offer a completely complementary methodology to sequence-based methods for the identification of DNA-binding protein target sequences.

Show MeSH
Related in: MedlinePlus