Rob Meijers

EMBL Hamburg

EMBO Global Exchange Lecture Course

Hyderabad 2012

Biophysical characterization of protein-

protein interactions

Bottom up look at protein-protein interactions

• Role of hydrogen bonds

• Role of hydrophobic interactions

• Techniques to measure kinetics:

• Surface plasmon resonance

• Isothermal titration calorimetry

• Thermophoresis

• Cross-linking

• Native mass spectrometry

A hydrogen-bonds only interface: Dscam

• Each bristle is innervated by a single neuron

• Each mechanosensory axon pathfinds into the CNS

A simple mechanosensory neuron

R265/R265

R87/R87

R265/R265 50μm

Exon 4.1 .2 .5 .7 .10

R265: Exon 4.2-4.6 deleted

R265/R265 R265/R265

Reducing Dscam diversity impairs

precise axon targeting

Chen et al 2006 Cell 125,607

Exclusive homophilic adhesion

From: Woj et al. Cell (2007) 130 1134

A horse shoe shape

• D1-D2 fold onto D3-D4

• Confirmed by EM

Meijers et al, Nature 449 (2007) 487

Dimerization mode for two isoforms

Homophilic interactions

• D2-D2 & D3-D3

• Most variable parts

• Anti-parallel

• Little overlap between D2 and D3

D3-D3

D2-D2

Antiparallel, symmetric interactions

D2-D2 D3-D3

Epitope swap

Binding swap

Hydrogen bonds contribution

• Highly specific

• 1-2 kcal/mol per hydrogen bond

• Not very strong

Quaternary complex

T cell

Antigen Presenting Cell

TCR

pMHC

CD4

Wang, Meijers et al (2001) PNAS 98, 10799

TCR recognition unit crystallized

• Murine MHC+Human

CD4

• Mouse model produced

• 10 years later…

Yin et al. (2012) PNAS 108, 15960

Immunodeficiency on a molecular level

T cell

Antigen Presenting Cell HIV

T cell

TCR

pMHC

CD4 CD4

gp120

Kwong et al 1998 Nature 393, 648

CD4 structure

• 4 immunoglobulin

domains

• N-terminal domain:

Phenylalanine sticks

out into the solvent

Wu et al 1997 Nature 387, 527

A buried hydrophobic residue

• Cd4/MHC class II

structure:

• Phenylalanine 49 is

buried

• Surrounded by

hydrogen bonds

Hotspot theory

• Hydrophobic residues lie

at the center of the

binding interface

• Contribute most free

energy

• Water displacement =

entropy gain

Clackson & Wells, Science (1995) 267, 383

Alanine scanning on TCR/pMHC binding

Wu et al. Nature (2002) 418, 552

Resulting model; used SPR

Surface plasmon resonance

• Allows you to measure:

• Specificity

• Affinity

• Kinetics

• One interacting partner (“ligand”) is attached to the

surface of a chip

• The second interaction partner (“analyte”) is flown over

the surface of the chip.

• Binding of molecules to the sensor surface generates a

response that is proportional to the bound mass.

Applications

• Specificity (yes/no binding response)

– Search for binding partners

– Screen for inhibitors

– Look for activity after protein purification

– Test cell culture lines for expression of a given protein

• Kinetics (rates of reactions)

– Complex formation

– Complex dissociation

• Affinity (strength of binding)

• Ligand Fishing • Recover analyte from a complex mixture with mass spec

The chip: 200 euros per

pH scouting

Immobilization

Basic steps during an SPR experiment

1) Decide on chip to use

2) Decide on which molecule will be the ligand and which will be the analyte

3) Prepare the ligand and analyte

4) Choose immobilization strategy

5) Pre-concentrate ligand on chip prior to activation (“pH scouting”)

6) Immobilize ligand onto sensor chip

7) Inject analyte and record response

8) Regenerate surface

9) Analyze data

The sensogram

Overall structure and SDS/PAGE

Song G et al. PNAS 2005;102:3366-3371

©2005 by National Academy of Sciences

Binding of the L I domains to ICAM-3 measured with SPR

Song G et al. PNAS 2005;102:3366-3371

©2005 by National Academy of Sciences

ICAM

ICAM

ICAM

I

I

Surface acoustic wave resonance

• Sound wave instead of

light

• Better penetration, higher

sensitivity

• Can be used to

immobilize cells and check ligand binding

Thermodynamics of protein-protein

interactions

• G = H-T S = -RTlnKa

In a single ITC experiment you get…

• Affinity (KD): strength of binding

• Heat of binding ( H) and entropy ( S):

mechanism

• Stoichiometry (n): number of binding sites

• Enzyme kinetics

ITC experimental principle

X + M XM

Reference Sample

Syringe

Fit to model (1)

One Set of Sites

• Symmetrical S-shaped curve

• One or more binding sites with the same affinity

• The number of sites is determined by the stoichiometry N

• Same result by loading the ligand in the syringe or in the cell

Two Sets of Sites

• Non symmetrical curve

• Two set of Indipendent binding sites with different affinity

• The number of sites is determined by the stoichiometry N1 and N2.

• Different result by loading the ligand in the syringe or in the cell.

Sequential Binding Sites

• Non symmetrical curve

• Non-indepoendent binding sites

• The number of binding sites is defined by the user

• Different result by loading the ligand in the syringe or in the cell

N < 1 the protein concentration is lower than you think

the protein is impure the protein (polypeptide) is pure but not all correctly folded

the ligand concentration is higher than you think the simple single site binding model is inappropriate

N > 1 your protein has multiple binding sites the ligand concentration is lower than you think

the simple single site binding model is inappropriate,

N = 1 You’re lucky

Binding stoichiometry: common problems

SPR vs ITC

• Advantages:

• You can work with ‘dirty’ sample to fish out specific interactions

• You need less material (~20 ug of immobilized ligand)

• -You can compare different ligands at the same time

• Disadvantages:

• ITC gives you thermodynamic data (dG)

• ITC kinetics is more accurate

• Topology of binding is limited in SPR

• Small molecules <100 Da don’t work

• But now, there is….

Thermophoresis

• Heat sample by 2K

• Molecules dissipate

• When fluorescently

labeled

• Equilibrate depending

on size of hydration

shell, charge

distribution

Duhr and Braun (2006) PNAS 103, pp19678

Current technology

Jerabek-Willemsen et al. 2011

??

Mass spectrometry

• MS/MS + Ion mobility :

• Native mass spec

• Detailed folding state

• Protein-protein

interactions

Synapt G2-S HDMS – Waters, UK

Instrumentation

• Synapt G2-S HDMS – Waters, UK

S.D. Pringle et al., Int J Mass Spectrom 2007

Ion mobility derived particle size

12/12/12 49

Ruotolo et al Nature Protocols (2008) 3, 1139

in combination with SAXS…

Collision cell gives activation energy

• Oligomers and/or

complexes fly

• Can be perturbed by

increased gas

pressure

• Gives indication of

complex integrity

Barrera & Robinson, Ann Rev Biochem (2011) 80, 247