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WHY DNA BINDING PROTEINS ?

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Akhil Nair Megha Bhaskar Shinjini Chowdhary

Main Paper: Dynamic proteinDNA recognition: beyond what can be seen Monika Fuxreiter, Istvan Simon2and Sarah Bondos

Motive : Accomplishes; mechanisms of DNA read out and protein DNA interactions and theories for the same. Other papers Discussed: 1. SURVEY AND SUMMARY How do site-specific DNA-binding proteins bind their targets? Stephen E. Halford*and John F. Marko1 ** my Addresses the thermodynamic and energy Motive: inference,* cross reference, TF-Transcription factor, DNAP DNA binding protein aspects and existing models

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Classical logic : static!

DNA protein interaction is

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New hypothesis supported by proof but far from complete theory: Dynamic transient interactions between protein and DNA. Layout of discussion:

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*1 DNA READ OUT MECHANISM THOUGHT TO BE

DIRECT : MEANS OF H BONDING INTERACTIONS INDIRECT: ELECTROSTATIC INTERACTIONS WITH PHOSPHATES Major factor affecting the reading: *2 DNA shape dependent electrostatic potential

This about DNA what in protein effects or decide recognition of target sequence. ID Regions: Intrinsically disordered regions : Characteristic segment of protein which responds with conformational change in response to DNA contact.

Cross reference:
*1 Luscombe, N.M. et al. (2001) Amino acid-base interactions: a threedimensional analysis of protein-DNA interactions at an atomic level.Nucleic Acids Res. 29, 28602874 *2 Gromiha, M. et al. (2004) Intermolecular and intramolecular readout mechanisms in proteinDNA recognition. J. Mol. Biol. 337, 285294

ID regions: *3
Facilitate diffusion along DNA Plays role in transition from non specific to specific complex Modulate selectivity

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What are ID regions ? Basic principle: *4 Change in folding pattern change in conformational **DNAP: Two parts DNA binding domain ID regions ID regions outside the binding context can modulate conformational of residues for DNA binding. Cross reference:
*3 Laity, J.H. et al. (2000) DNA-induced alpha-helix capping in conserved linker sequences is a determinant of binding affinity in Cys(2)-His(2) zinc fingers. J. Mol. Biol. 295, 719727 *4 Spolar, R.S. and Record, M.T., Jr (1994) Coupling of local folding to site specific binding of proteins to DNA. Science 263, 777784

entropy! Improves mobility and binding affinity.

And how do they do that ?

*5
1. 2. 3.

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Influence thermodynamics of the binding process. Adjust the spacing between the protein and DNA. Adjust half- life of DNAP complex.

Example: DNA READ OUT MECHANISMS Non specific to specific transition. **DNAP and P-P: Charged Id tails facilitate target search. **Concentration of protein and diffusion rate DYNAMIC MECHANISMS OF DNA increases substantially. RECOGNITION Lock DNA binding motifs to target sequence by snap Modulation by four mechanisms: lock mechanism.*5 1. Flexibility modulation 2. Conformational modulation 3. Competitive binding 4. Tethering. Cross reference:
*5 Pontius, B.W. (1993) Close encounters: why unstructured, polymeric domains can increase rates of specific macromolecular association. Trends Biochem. Sci. 18, 181186

Conformational modulation : ID regions shift conformational equilibrium in favor of binding. Eg: *6 Max TF, interacts with E-Box (CACGTG), bHLH, LZ

Max free dorm Dimer in which LZ domain unstable : folding unfolding transitions ;N terminal mask destabilizing electrostatic potential at LZ.followed by recognition loops decreases Kd by 100 fold. effects Myc gene expression Flexibility modulation Remote regions interact with dynamism maintaining flexibility at DNAP interface: Eg :

unfold Serine rich region regulates that. Phosphrylation in Srr reduces binding affinity. Removal of Srr region mobility increases due to H1 unfolding.**Id region removal of SRR from that specific region

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*7Ets 1 TF, regulated by auto-inhibitory region. Requires H1 helix to

Cross reference:
*6 Naud, J.F. et al. (2005) Structural and thermodynamical characterization of the complete p21 gene product of Max. Biochemistry 44, 1274612758 *7 Pufall, M.A. et al. (2005)Variable control of Ets-1 DNA binding by multiple phosphates in an unstructured

3. Competitive binding : Rapidly fluctuating ID chain can screen electrostatic attraction in DNAP. Or repulsion resulting in unavailability of binding region. Eg: *7 Human positive factor PC4, recruits TF and stimulates RNAP II

activity regulated by Lys rich region NTD alone lacks affinity to ss or ds DNA .ID region interaction based on unwinding the fuzzy complex with DNA and C terminal competes with binding motif thereby **increasing selectivity of binding. 4. Tethering: Multidomain motif DNAP sets of linker ID region present in between those act to preserve the conformational stability of all domains. Eg: *8Human replication protein(RPA),contains weak and high affinity DNA binding domains DBD, **Initial contact by High affinity followed by contraction of the linkers thereby increasing the local concentration of DBD.

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Cross reference:
*7Jonker, H.R. et al. (2006) The intrinsically unstructured domain of PC4 modulates the activity of the structured core through inter- and intramolecular interactions. Biochemistry 45, 50675081 *8NMR chemical shift and relaxation measurements provide evidence for the coupled folding and binding of the p53 transactivation domain. Nucleic Acids Res. 33, 20612077

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REGULATION OF DNA BINDING VIA ID REGIONS: By means of: 1.Post translational modifications 2.Protein-Protein Interaction 3.Alternative Splicing 1.Post translational modifications: Phosphorylation perturbs the DNA binding interface: Eg: *9 FACT, displaces nucleosomes and facilitates RNAPII uses HMG domain flanked by and acidic and basic ID regions. Cross region: maintains intermolecular attraction between with HMG domains Acidic reference:

*9Tsunaka, Region: competes for DNA contact Basic Y. et al. (2009) Phosphorylated intrinsically disorder edregion of FACT masks its nucleosomal DNA binding elements. J. directly by phosphorylation due to inc in electron cloud acidic region Strengthened Biol. Chem. 284, 2461024621.

2.Protein-Protein Interaction : ID regions competing with Binding motifs for DNA : Eg: *8 p53 tumor suppressor activation depends on interaction with 3.Alternative Splicing :

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hRPA70.Affinity of hRPA70 is higher compared to p53. The interaction controlled by linker ID.ID increases the local concentrations of DBD on signal from hRPA70 to increase affinity of p53.

Modulate the DNA expression by altering the ID residue composition: Eg: **Change in sequence of residues would result in change in interaction with the DNA sequence the target sequence of interaction and the pronounced effect of the same. Alternate translation site in MeCP2 increases the length and flexibility of

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Next

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How do site-specific DNA-binding proteins find their targets?

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Facilitated Diffusion
Some proteins locate their target sites very rapidly, much more rapidly than can seemingly be accounted for by diffusional collisions between the protein and the DNA molecule. Hence, these proteins must find their targets by `facilitated diffusion.

Proposals:

v v

One-dimensional diffusion Hopping through 3-D space by dissociating from one site re-associating elsewhere in the same chain Intersegmental transfer Figure 1. Courtesy: Nucleic Acids Research, 2004, Relevant only to proteins with two DNA 32, No. 10 Vol. binding surfaces. Cross references Berg,O.G. e.g. Blomberg,C. (1976) Association kinetics with coupled diffusional ows. Special application to and Lac repressor or Sfil the lac repressoroperator endonuclease

system. Biophys. Chem., 4, 367381

Berg and Blomberg - theoretical analysis of how 1-D diffusion along a DNA contour can facilitate specific site targeting

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Protein will alternatively undergo 1-D sliding and 3-D diffusive hops Characteristic sliding length covered by 1-D sliding Sliding length determined by the lifetime of a non-specific DNA - protein interaction and by the effective 1-D diffusion constant

Berg et al. experiments on Lac repressor protein

Time required for the repressor to find its target in a large DNA can be changed

Sliding length was originally estimated to be 100 bp under physiological conditions.

Cross References:
Berg,O.G., Winter,R.B. and von Hippel,P.H. (1981) Diffusion-driven mechanisms of protein translocation on nucleic acids. 1. Models and theory. Biochemistry, 20, 6929-6948.

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Three Dimensional Diffusion-Limited Targeting

Diffusion of a protein

A single protein moves through buffer by Brownian motion (diffusion) trajectory is a random walk.

Major feature Average of the distance squared covered by a diffusing particle grows linearly with time.

where D is the diffusion constant

Figure 2. Courtesy: Nucleic Acids Research, 2004, Vol. 32, No. 10

Einsteinss formula diffusion of a protein

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Probability of finding a nearby target by diffusion

The diffusing protein, at an initial distance r from the target, will either collide with the target or diffuse off into bulk solution, never finding this target.

During its random walk within a distance r of the target, the protein visits a fraction a/r of the `voxels' of size a.

Figure 3. Courtesy: Nucleic Acids Research, 2004, thus a/r. This is the exact result for diffusion Vol. 32, No. 10

The probability of encountering the target is

to capture. The diffusion-limited reaction rate

The above result is used to compute the association rate for binding.

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Simple-minded View Of Facilitated Diffusion

Main ideas

Non-specific association of proteins with DNA can reduce the time required for them to find their target sequences, essentially by restricting their motion to along the DNA contour.

The association rate is increased by the non-specific DNA flanking the target, which serves to greatly increase the effective target size.

Facilitated diffusion provides accelerated targeting essentially by increasing the target size, without decreasing the diffusion constant of the protein that is doing the search.

Cross references:
Shimamoto,N. (1999) One-dimensional diffusion of proteins along DNA. J. Biol. Chem., 274, 1529315296.

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The Sliding Length

A protein binds non-specifically to the DNA double helix (left) and then undergoes sliding steps randomly to the left and to the right, exploring the DNA contour through 1-D diffusion.

Eventually, a dissociation event Figure 3. Courtesy: Nucleic Acids Research, 2004, occurs; the characteristic Vol. 32, No. 10 distance explored between The sliding length can be controlled association and dissociation events is the sliding length. by adjustment of the non-specific binding affinity. Due to the random nature of 1D diffusion, the same DNA sites will be sampled repeatedly.

Processivity As A Probe For Facilitated Diffusion

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Processivity in this context is defined as the number of reactions in which the enzyme acts at both sites during one DNA-binding event relative to the total number of reactions by the enzyme. It is a unitless parameter, a ratio of rates.

In a processive reaction on a DNA with two sites separated by a known distance, the distance travelled by a protein from an initial non-specific site to its final specific site is indeterminate.

The relationships between processivity and the length of DNA between the sites are much simpler.

In a processivity experiment, one considers a protein that has just acted at one site (here acting means binding, catalysis and dissociation) and then measures the probability of it acting again at a second site in cis. The targeting distance concept permits a simple

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Conclusions and Outlook

v

Fundamental Problem: How DNA-binding proteins find specific sites amongst huge amounts of non-specific (chromosomal) DNA

Association rate measurements and facilitated diffusion

Association rate measurements on Lac repressor protein remain to this time one of the main supports for the facilitated diffusion theory. Information about a spatial reaction pathway can only be extracted from total reaction rate measurements by the use of a model.

Processivity experiments

Direct information about the spatial pathway that is followed by a DNAbinding protein to its target site can be obtained.

Processivity experiments on catenated DNA molecules have Cross provided further evidence for transfer of proteins through space references: Terry,B.J., Jack,W.E. and Modrich,P. (1985) Facilitated diffusion during catalysis by EcoRI endonuclease. from one DNA segment to a nearby one, presumably via diffusion Nonspecic interactions in EcoRI catalysis. J. Biol. Chem., 260, 1313013137. in three dimensions.

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Engineering of repressor protein into site-specific cutters

-an application of DNA-binding proteins
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Converting the protein into sitespecific scission reagent

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Generating TrpR mutants using PCR-based site directed mutagenesis.

Fig 1 and 2 :courtesy Branden, C. and Tooze, J. (1991) Introduction to Protein Structure, Garland Publishing, New York Chapter 7

 Iodoacetamide

chemical modification of cysteine residue of mutant protein. with 5-iodoacetamido-1,10phenanthroline.(IAAOP)

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alkylation

Fig 3 :courtesy Branden, C. and Tooze, J. (1991) Introduction to Protein Structure, Garland Publishing, New York Chapter 7

 The

amino acid residue X must be in close proximity with the minor groove of DNA.

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Binding

of repressor to DNA takes place through helixturn-helix motif .

Fig 3: courtesy Branden, C. and Tooze, J. (1991) Introduction to Protein Structure, Garland Publishing, New York Chapter 7

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trpR

E49C-OP chimeras scission a scission reagent- promotes positioning of trpR on trpEDCBA. which affect their nuclease activity : amino acid position the affinity of binding pH of the cleavage reaction

Factors

Cross reference: Lavoie.T.A. and CaryJ. (1994) In Bokstein.F. and Lilley.D.M.J. (eds). Nucleic Acids and Molecular Biology. Vol. 8. Springer-Verlag, Berlin, Heidelberg,184-188

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Optimizing the orientation of reaction center

Positioning

of OP-Cu on the dyad axis

TrpRE49C-OP,

places two OP-Cu residues right at the centre of the dyad axis within the dyad axis in the minor groove

constrained

Cross reference: Lavoie.T.A. and CaryJ. (1994) In Bokstein.F. and Lilley.D.M.J. (eds). Nucleic Acids and Molecular Biology. Vol. 8. Springer-Verlag, Berlin, Heidelberg,184-188

Fig 4. A Strathclyde minor groove binder in the double helix of DNA showing the antiparallel 2:1 binding mode and the potential for hydrophobic interactions (grey patches). Courtesy: http://www.chem.strath.ac.uk/people/academic/colin_j_suckling/research/minor_groove_binders

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Gel-retardation

assay was used to study DNA-protein interactions. DNA. footprinting- protein binding site on

DNAse

Fig 5:Tryptophan-dependent gel shift of a trpEDCBA containing end-labeled fragment by TrpR E49C-OP. Lanes I and 2, no protein (duplicated for scission purposes); lane 3, retardation by TrpR, gel-shift conditions as described Courtesy: Protein Engineering vol.9 no.7 pp.603-610. 1996

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Optimization of scission yield

Two

methods have been determined:

carrying

out the reaction between pH 7.2 and 7.5 rather than pH 8.0. high-affinity mutants such as A77S.

using

Cross reference: Lavoie.T.A. and CaryJ. (1994) In Bokstein.F. and Lilley.D.M.J. (eds). Nucleic Acids and Molecular Biology. Vol. 8. Springer-Verlag, Berlin, Heidelberg,184-188

CLEAVAGE OF LINEARISED PLASMID DNA

Promotes

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specific background scission.

increases the affinity of the repressor for the trp operator. the binding of the Trp repressor for 'nonspecificprotein-DNA complexes'

destabilizes

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PROPOSED APPLICATIONS OF CHIMERAS

treatment identifying

of disease a specific binding site in the DNA mapping

chromosome Cloning probes

of macromolecular structure

Cross reference: Lavoie.T.A. and CaryJ. (1994) In Bokstein.F. and Lilley.D.M.J. (eds). Nucleic Acids and Molecular Biology. Vol. 8. Springer-Verlag, Berlin, Heidelberg,184-188

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CONCLUSIONS

MAIN REFERENCES:
1. Monika Fuxreiter Istvan Simon and Sarah Bondos 2011; Dynamic proteinDNA recognition: beyond what can be seen; Trends in Biochemical Sciences, August 2011, Vol. 36, No. 8 Cell press 2. Stephen E. Halford* and John F. Marko1 SURVEY AND SUMMARY How do site-specific DNA-binding proteins and their targets?Nucleic Acids Research, 2004, Vol. 32, No. 10 3. Ralf Landgraf, Clark Pan, Christopher Sutton, Lori Pearson and David S.Sigman 1996; Engineering of DNA binding proteins into site-specific cutters:reactivity of Trp repressor-l,10-phenanthroline chimeras Protein Engineering vol.9 no.7 pp.603-610.

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