MMG /BIOC 352 Spring 2006 Protein-DNA Interactions: Kinetics and Thermodynamics Example: the...

MMG /BIOC 352

Spring 2006

Protein-DNA Interactions: Kinetics and Thermodynamics

Example: the Bacteriophage System

Scott W. Morricalwith special thanks to

Margaret A. Daugherty

Contact Information

Scott W. MorricalGiven B407656-8260

[email protected]

Lecture outline:

Introduction to the system Bacteriophage lambda Lysogeny vs. lysis

The molecular switch PR, PRM, cI repressor, cro

Specific vs. Non-specific Interactions What makes a good DNA binding protein?

Thermodynamic “Primer” G = H - TS: importance Intrinsic Free Energy Cooperativity

Techniques Quantitative DNAse Footprinting

cI repressor protein Structure Dimerization Data

Cro protein Structure Dimerization & DNA Binding An example of induced fit Data

Kinetic Aspects of cI and cro binding Facilitated diffusion cro-DNA interactions

Structure Analysis? cro-DNA vs. cI-DNA interactions

Reference list for this topic:Ref 1: Ptashne, M. (1992) A Genetic Switch, 2nd ed., Cell Press & Blackwell Scientific Publications, Cambridge, MA. **This an excellent general review of bacteriophage with simple descriptions of thermodynamics and regulation.

Ref 2: Johnson, A.D., Poteete, A.R., Lauer, G., Sauer, R.T., Ackers, G.K. & Ptashne, M. (1981)

Repressor and cro - components of an efficient molecular switch. Nature 294:

217-223. Review article of bacteriophage , outdated, but ok for understanding the system in general.

Ref 3: Chattophadhyay, R. & Ghosh, K. (2003) A comparative three-dimensional model of

the carboxy-terminal domain of the lambda repressor and its use to build intact

repressor tetramer models bound to adjacent operator sites. J. Struct. Biol. 141:

103-114.

Ref 4: Oda, M. & Nakamura, H. (2000) Thermodynamic and kinetic analyses for understanding sequence-specific DNA recognition. Genes to Cells 5: 319-

326.Just one of many reviews on thermo & kinetic aspects of DNA binding.

Ref 5: Brenowitz, M., Senear, D.F., Shea, M.A. & Ackers, G.K. (1986) “Footprint” titrations yield valid thermodynamic isotherms. P.N.A.S. USA 83: 8462-

8466

Reference list - continuedRef 6: Koblan, K.S. & Ackers, G.K. (1992) Site-Specific Regulation of DNA Transcription at Bacteriophage OR, Biochemistry 31: 57-67.

Ref 7: Darling, P.J., Holt, J.M. & Ackers, G.K. (2000) Coupled Energetics of cro Repressor Self-assembly and Site-specific DNA Operator Binding II: Cooperative

Interactions of cro Dimers. J. Mol. Biol. 302: 625-638.

Ref 8: Albright, R.A. & Matthews, B.W. (1998) Crystal structure of -cro bound to a Consensus Operator at 3.0 Å Resolution, J. Mol. Biol. 280: 137-151.

Ref 9: Spolar, R.S. and Record, M.T. (1994) Coupling of Local Folding to Site-Specific Binding of Proteins to DNA. Science 263: 777 - 784 a classic “must know” paper!Ref 10: von Hippel (1994) Protein - DNA Recognition : New Perspectives and Underlying Themes. Science 263: 769-770. (a review of Spolar & Record)

Ref 11: Frankel, A.D. & Kim, P.S. (1991) Modular Structure of Transcription Factors: Implications for Gene Regulation. Cell 65: 717-719 (quick reading -

introduces notion of induced fit)

Ref 12: Takeda, Y., Ross.P.D. & Mudd,C.P. (1992) Thermodynamics of Cro-protein DNA

interactions. Proc. Natl. Acad. Sci. USA 89: 8180-8184.

Reference list - continuedRef 13: von Hippel, P.H. & Berg, O.G. (1989) Facilitated Target Location in Biological

Systems. J. Biol. Chem. 264: 675-678. Nice mini-review.

Ref 14: Kim, J.G., Takeda, Y., Matthews, B.W. & Anderson, W.F. (1987) Kinetic Studies of Cro-Repressor Operator DNA Interaction, J. Mol. Biol. 196: 149-158

Ref 15: Albright, R.A. and Matthews, B.W. (1998) How Cro and -repressor distinguish between operators: The structural basis underlying a genetic switch. Proc. Natl.

Acad. Sci. USA 95: 3431-3436.

Bacteriophage : an obligate parasite

100,000x

8,000x

Ref 1: Ptashne (1992) A Genetic Switch, 2nd ed., Cell Press & Blackwell Scientific Publications, Cambridge, MA.

Lambda, lysogeny and lysis

R R RRR

Ref 1

lysogenylysis

prophageR R RR

R

infect

inject

An overview of growth: Patterns of gene expression

2

3

10

10 12

PRPL

chromosome pattern of gene expressionRef 1

N cro

cI repressor

int

The molecular switch: Lysogeny to Lysis

Polymerase can bind to PRM or PR

ORi = right operator sites where i = 1,2 3PR = right promoter; polymerase transcribes cro proteinPRM = promoter of repressor maintenance; polymerase bound

here transcribes cI repressor protein.cI repressor protein = maintains bacteria in lysogenic statecro protein = “control of repressor and other genes”; causes switch to lytic lifestyle

repressor vs. cro

Key points: same operator sites; “reverse” affinities; cooperativity; bind as dimers

Ref 1

cI repressor: keeps cro turned off!

X

cro: turns off production of cI!

X

UV irradiation activates RecA cleavage of cI monomers.

P-DNA complex is reversible. cI dimerization is reversible. When cI dimers fall off, they attempt to reestablish equilibrium; monomers get cleaved. Decrease in [cI dimers], hence DNA opens up for cro binding!

The switch: completing the story

Ref 1

Designing an efficient DNA binding protein

Purpose: To understand the factors that influence how efficiently a repressor protein occupies its operator in the cell.

Given: the fraction of time that an operator is bound by repressor is determined by two factors

i). Affinity of repressor for operatorii). Concentration of free repressor

Problem: Non-specific binding!

Goal: Understand how we can increase efficiencyleads us to idea of cooperativity


Equations on board

The rationale for the arguments are taken fromAppendix One in reference 1


How can we increase specificity?

1). Increase protein concentration

2). Improve specificity directly*play with the KD/KOP ratio

hold KD constant; improve KOP

*increase number of contacts by increasing repressor

twice the contacts, twice the energy!KOP = 10-20 M; KD = 10-8 M

good idea, but…. Affinities become a problem, which

give rise to kinetic problems!

Increased efficiency with cooperativity

cI binding to PR

Ref 1

Cooperativity>99% occupied

Nocooperativity

10% 90%

cI binding to PR: OR1-OR2 species predominates

cI binds strongly to OR1 and OR2; weakly to OR3; cooperative interactions enhance interactions at OR2

Ref 1

“fast switch” for gene expression

Biological advantage of cooperativity

Ref 1

cI repressor structure: low resolution

Kd ~ 6 nM

Ref 1

Dimerization & regulation

cI repressor structure: low resolution

N-terminus: major groove interactionsLinker region: flexibleC-terminus: protein-protein interactions that give rise to cooperativity

Ref 1

cI repressor structure: “high resolution”

(pdb1j5g) J. Struct. Biol. 141, 103-114; 2003

REVIEW OF TUESDAY’S LECTURE

lysislysogeny

Designing an efficient DNA-binding proteinnon-specific interactions “mess up” specific

binding!KD/KOP and [R]T/[D]T determine binding efficienciesBest way to improve binding - COOPERATIVITY!Cooperativity gives rise to “faster” biological

responses

Cooperativity and Free Energy

How do we determine that there is cooperativity?

OR3 OR1OR2OR+

G3 G1G2

G1

OR2OR2+

OR3 OR1OR2-

G3

G2

G1+G2+G3 > G1+G2+G3

?

Thermodynamic Primer: Gibbs Free Energy

P + DNA P•DNAKeq

Keq =[P•DNA][P] [DNA]

Go = -RTln Keq

Remember: more negative, more favorable reaction!

van’t Hoff equation: temperature dependence of Keq

A <--> B

Keq = [B]/[A]

Measure K as a function of T

H = +533 kJ/mol

Linear: no Cp changeCurvature: Cp change

= - dlnKeq

d(1/T)H

R

G = H - TS

62C 48C

Thermodynamics and biological reactionsG = H - TS

G = criteria for spontaneitynegative - reaction is favorable

H = direction of energy flownegative - exothermicinformation about chemical interactions

S = tells us about system organizationpositive - increase disorder can reflect conformational entropy or H20 entropy

Cp = proportional to a change in hydrophobic surface area

-- see Spolar reference - very important reference!

e.g., negative - organization of protein structure upon

DNA binding

Constraint: cooperativity can only occur between adjacent operators

Microscopic binding configurations

} Intrinsic binding G

Brenowitz et al., (1986) P.N.A.S. 83: 8462-8466

Autoradiogram of a “footprint”: false color image

standard

OR3

OR1

OR2

standard

Individual site binding isotherms

Fract

ional sa

tura

tion

OR3

Langmuir isotherm-- single site

interactions:

Y = K1[X] / (1 + K1[X])

K1 = 1/[X] at Y = 0.5

For 2-site cooperative interaction:

Y1 = (K1[X] + K1K2K12[X]2) / B

Y2 = (K2[X] + K1K2K12[X]2) / B

where B = (1 + (K1 + K2)[X] + K1K2K12[X]2)

K1 and K2 are intrinsic binding constants for sites 1 & 2, and K12 is the interaction (or cooperativity) constant.

K12 defines the extra free energy of binding 2 sites simultaneously compared to sum of individual free energies, i.e. G12 = Gtotal - (G1 + G2) where G = RTlnK.

Individual site binding isotherms for cI - OR interactions

OR2

OR3 OR1OR2

OR3 OR1

OR3 OR2

Koblan, K. and Ackers, G.K., (1992) Biochemistry 31: 57-65.

1

2 3

1

3

Temperature dependence of G values for cI-OR


OR1

OR3

OR2

van’t Hoff plots forcI-OR single-siteinteractions


cI-OR Interactions are Enthalpically Driven

Cro repressor structure & induced fit

Albright & Matthews, J. Mol. Biol. (1998) 280, 137-151

Cro: helix-turn-helix (like cI and CAP)

Dimer subunits rotate 53o wrt each other upon binding to consensus OR

Creation of extensive H-bond networkplus van der Waals contacts along protein-DNA interface.

DNA is bent 40o through 19 bp.

Recognition helices of Cro dimer makeextensive contacts with bp edges inmajor groove.

Induced Fit (ref 9 -12)Observed in many specific protein-DNA recognition processes, and much less frequently in non-specific binding.

Key thermodynamic feature of specific interactions: large negative Cp.

hydrophobic effect: occlusion of hydrophobic surface area from water -- protein foldingAlso can arise from cation release into H20

Non-specific interactions occur with little or no change in Cp.largely electrostatically stabilized: hydration properties of

individual components retained, driven by displacement of

condensed monovalent cations from DNA.

DNA: linear B-DNA to: smooth bends, kinks that disrupt base-pairing

Protein: Quaternary rearrangement of domains or subunits ordering of disordered loops or N-termini Formation of -helices from unfolded loops

Binding isotherms for Cro repressor

OR+

OR1, OR2 & OR3 templates

31

2

Non-specific Specific (OR3)

Cro-DNA Interactions are Entropically Driven

Specific protein-DNA interactions by induced-fit:large negative Cp

Negative entropic contribution from local or global protein folding must be driven by binding free energy (i.e. formation of more extensive complementary interface, burying more macromolecular surface, releasing more water and ions)

Comparison of cro vs. cI energetics

Kd = 324 nM

Kd = 6.2 nM

Kinetics of protein-DNA interactions

For function, regulatory proteins must reach target DNA.

Problem: regulatory proteins show affinity to non-specific DNAresult: competition, potential slowing down of interactions

Early results on lac repressor show rates were increasedabove simple diffusion rates: 100 - 1000x faster!

What limits biological reaction rates?

In principle, limited by the rates at which diffusion can bring two molecules together.

kencounter = 4(DA+DB)(rA+rB)N0/1000

DA+DB = diffusion constants for A & B

A + B AB

rA & rB = hydrodynamic radii for A & B

Smoluchowski’s equation

Kinetics of biological reactions are complex!

surfaces of macromolecules not uniformly reactiveelectrostatic forces may be attractive or repulsiveasymmetric molecules (rate of diffusion decreases)complex interaction distance

Modified Smoluchowski equation lets us calculate kassoc for protein & DNA (lac-DNA) at 108 M-1 sec-1. In reality, kassoc= 5 x 1010 M-1 sec-1.

kassoc = 4af(DA+DB)N0/1000

represents fraction of A & B that interact a is the interaction distancef reflects attractive/repulsive electrostatic forces

Forward rate constants for macromolecular association

Biological molecules are not small inert spheres!

Small diffusion constantsGeneral stickiness -- van der Waals

Biological molecules are slow to drift apart!

A classically defined “collision” can consist of manymini-collisions. Rotational rearrangements overcome steric factors

Biological molecules are charged!

Macromolecules set up electrostatic fields that guide substrates to functional sites; sometimes “shaped”

Special Features of Protein-DNA Interactions

Two macromolecules - hence inelastic collisions!

Rotational diffusions allows protein to “hop” (4-8 bp)Most proteins have positively charged active site

Initial interactions are with non-specific DNA

More non-specific sites than specific sitesLet N = number of base pairs in the DNA; we could expect the protein to form ~ N transient non-specific complexes before reaching specific site!

Logically, in the non-specific sites are thought of as competitive targets, then increasing N should decrease rate. Experimentally, it increases rate! How?!

Initial interactions are with non-specific DNA

R+D+O <-> RD+O <-> RO + D

diffusion facilitated

k1 k2

k-2k-1

Non-specific binding speeds target location

Two methods:SlidingIntersegment transfer

Similarity: both involve diffusion

while bound to non-specific DNA

Net result: decrease in volume of solution that needs to be searched!

SLIDING: “one-dimensional diffusion”

Question: How does non-specific binding differ from site-specific binding, such that we can keep the protein associated with the DNA, but still be able to slide?

Specific binding: Discrete hydrogen bond donors and acceptors between protein & DNA; energetically favorable.

Non-specific binding: requires more of a delocalized type of interaction; charge-charge interactions

Sliding - continued

++

+++ +++ ++

++

+ +++ ++ ++

+

+

+

+ ++ ++++

+

+

Sliding occurs by displacement of counterions

++

+ ++ ++

++

+ + + +

+

+

+

+ + +++

+

Decrease salt concentration:rate increases!

DIRECT INTERSEGMENT TRANSFER

Protein interacts directly with two double-stranded loops of DNA.

Loops are brought together by diffusion; eventually they diffuse apart.

50% of time, protein goes with new loop.

Question: What will happen to intersegment transfer rates if you increase the duration of the non-specifically bound complex?

Diagnostics for sliding and other modes of facilitated diffusion

1). kassoc > kdiffusion

small increases: electrostatic fields or dockinglarge increases: sliding or intersegment transfer

2). Larger DNA fragments give rise to larger kassoc.

3). Decreased salt concentrations give rise to larger kassoc.

Kinetics of Cro - Operator Interaction

Filter binding assay:

Nitrocellulose binds protein or protein-DNA complexes.

Free duplex DNA flows through

Advantage:sensitive, quantitative

Disadvantage:retention of non-

specificDNA-protein complexes.

Kim et al., J.M.B. 196 (1987): 149-158

Kinetic Analysis of 6 operator sites and a consensus sequence

Association kinetics of cro with OR3

Cro + DNA -------------------------> Cro-DNAIncubate, time, filter

Dissociation kinetics of cro-DNA complex

Cro + *DNA ---------> cro-*DNA + DNA100x -------->cro-DNA + *DNAIncubate 10’ incubate, time, filterFr

act

ion

boun

d

Kinetic parameters for the operators

Kd: OR3 > OR1 > OR2

ka ~ 3 x 108 M-1 sec-1

kd: OR2 > OR1 > OR3

Quick analysis of the association rate

Experimental ka ~ 4 x 108 M-1 sec-1

Theoretical ka ~ 5 x 107 M-1 sec-1

10x larger

On average, electrostatic attraction accelerates 5X

These are 21 bp fragments - no sliding

Kinetics of cro binding to variable-length OR fragments

Kinetic parameters for OR DNA fragments

Kd for larger fragments ~ 1.5 x 10-11 M

kd

KA

ka

DNA length (bp)

Summary of kinetic data for OR-DNA fragments

KA decreases 7 fold

kd increases 100 fold

ka increases 10 fold

700 bp is break point

Theoretical test of the sliding mechanism

R + D + O <-----> RD + O <-----> RO + Dk1

k-1

k2

k-2

kd well described by sliding mechanism;ka described by a sliding mechanism enhanced by electrostatic effects

STRUCTURAL COMPARISON OF CRO AND cI BINDING

Kd = 324 nM

Kd = 6.2 nM


PNAS 95: 3431-3436 (1998)


P = sites involved in both cro and cI binding

P = sites required for cI


Only uninterrupted structural homology is in 2-3 region


crocI

PNAS 95: 3431-3436 (1998)

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