Lecture 6

Protein-protein interactions

Affinities (cases of simple and cooperative binding)

Examples of Ligand-protein interactions

Antibodies and their generation

0 2 4 6 8 100

0.2

0.4

0.6

0.8

U1 r( )

U2 r( )

U4 r( )

U6 r( )

r

1/r2

1/r6

1/r

Long-range and short-range interactions

Even without NET CHARGES on the molecules, attractive interactions always exist. In the presence of random thermal forces all charge-dipole or dipole-dipole interactions decay steeply (as 1/r4 or 1/r6)

1/r4

Interatomic interaction: Lennard-Jones potential describes both repulsion and attraction

Uo 1

U x( ) Uo x12

2x6

0.6 0.8 1 1.2 1.4 1.6 1.8

1

0

1

2

U x( )

x

600

1200 )/(2)/()( rrErrErEp

r = r0 (attraction=minimum)

r = 0.89r0

r = r0

steric repulsion

Bond stretching is often considered in the harmonic approximation:

202

1 )()( xxxU

desolvdispstericeltot EBAqE

Van der Waals

Here is a typical form in which energy of interactions between two proteins or protein and small molecule can be written Ionic pairs +

H-bondingremoval of waterfrom the contact

What determines affinity and specificity?

Tight stereochemical fitand Van der Waals forces Electrostatic interactionsHydrogen bondingHydrophobic effect

All forces add up giving the total energy of binding:

Gbound– Gfree= RT

lnKd

Simple binding

LRRL

]][[][ RLKLR b

off

onb k

k

RL

LRK

]][[

][

][][][

][][

LRRLR

totalRLR

B

Receptor occupancy:

][1

][

LK

LKB

b

b

bd K

K1

][

][

LK

LB

d

Mass action:

(Langmuir isotherm)kinetic parametersequilibrium parameter

1/koff = residence time

in the bound state

][

][

LK

LB

d

Receptor occupancy is a hyperbolic function of [L ](Langmuir adsorption isotherm)

B1 x(

B2 x(

B3 x(

L0 10 20 30 40 50

0

0.2

0.4

0.6

0.8

1

)

)

)

Kd = 1Kd = 3

Kd = 10

Bmax

Kd has the dimension of concentration and should be measured in the same units as L (M).

Note that for a shallow curve it is hard to say where it saturates

97% of O2 is carried in the form of Oxyhemoglobin (HbO2)

3% - dissolved in plasma

P1/2 = 28 mm Hg

When PO2 changes from 100 to 40 mm Hg, the saturation decreases from 98 to 75%

physiological range

Oxygen and Hemoglobin

From G. Hummer

CO binds to the porphyrin ring of heme exactly where O2 binds

nn

n

KRL

RL 1

][][

][

What if the binding to multiple sites on the same receptor is strictly interdependent (i.e. cooperative)?

nntotn

n

KRLRL

RL 1

][][

][

nn

n

tot

nn LK

L

R

RLB

][

][

][

][

Hill equation, n is Hill coefficient 0 1 2 3 4 50

0.2

0.4

0.6

0.8

1

B1 x( )

B2 x( )

B3 x( )

L

n=2n=4

n=1

rearrange

Probability of binding to one

site ~[L]

Probability of binding

simultaneously to n sites ~[L]n

Myoglobin, n = 1

Hemoglobin, n = 2.8

pO2 (kPa)

0 2 4 6 8 100

0.2

0.4

0.6

0.8

1

B1 x( )

B3 x( )

nn

n

n LK

LB

][

][

pO2 in tissues

Hemoglobin vs Myoglobin

Cooperativity is due to tight intersubunit interactions

xkxB

d

n

d

n

xkxB

n – Hill coefficient

independent binding

cooperative binding

Protein Kinase A spatially organizes ATP and peptide chain to facilitate the phosphorylation reaction

(old book)

Intracellular signaling adapter domains SH2 and SH3

Proline-rich sequenceSegment containing phosphotyrosine

Fig 16-11 Fig 16-23

PDZ domains spatially organize ion channel/receptor complexes in synapses

“Postsynaptic density” complex

(old book)

Fatty acid binding protein (FABP)

Fig. 10-24

Common theme: hormones promote dimerization of receptors

Fig. 16-7

The Growth Hormone sequentially binds to two receptors

first binding event second receptor is then recruited

Fig 15-3

Binding of the Epidermal Growth Factor (EGF) leads to receptor dimerization not by cross-linking but by exposing ‘sticky’ loops

Fig. 16-17

Antibody (IgG)

CDR = complementarity determining region

The lymph system and lymph nodesSee Chapter 24

Clonal selection of B lymphocytes: prolifereation and differentiation of these cells is induced by an encounter with an antigen recognized by the surface receptor

The immunoglobulin fold and the hypervariable regions

Fig. 24-12

Variability of sequence in hypervariable loops

The antigen recognition site

Fig. 24-13

Light chain coding regions:

VLCL

100 5 variants

Heavy chain coding regions:

VHD CH

100 30 4 variants

therefore, total number of combinations is ~ 6,000,000

Combinatorial diversity of antibodies

see Lodish (4th edition)

V – variableC – constant

The recognition site exposes flexible loops typically with many polar residues