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MMG /BIOC 352
Spring 2006
Protein-DNA Interactions: Kinetics and Thermodynamics
Example: the Bacteriophage System
Scott W. Morricalwith special thanks to
Margaret A. Daugherty
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 Review article of bacteriophage , outdated, but ok for understanding the system in general.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
Designing an efficient DNA binding protein
Equations on board
The rationale for the arguments are taken fromAppendix One in reference 1
Designing an efficient DNA binding protein
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
Koblan, K. and Ackers, G.K., (1992) Biochemistry 31: 57-65.
OR1
OR3
OR2
van’t Hoff plots forcI-OR single-siteinteractions
Koblan, K. and Ackers, G.K., (1992) Biochemistry 31: 57-65.
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
STRUCTURAL COMPARISON OF CRO AND cI BINDING
PNAS 95: 3431-3436 (1998)
STRUCTURAL COMPARISON OF CRO AND cI BINDING
P = sites involved in both cro and cI binding
P = sites required for cI
STRUCTURAL COMPARISON OF CRO AND cI BINDING
Only uninterrupted structural homology is in 2-3 region
STRUCTURAL COMPARISON OF CRO AND cI BINDING
crocI
PNAS 95: 3431-3436 (1998)