Protein–Protein Interactions

Domains – Definition

• A domain is a distinct part of a protein that can:

  • Fold and function independently of the rest of the protein.

  • Form a stable, compact tertiary structure.

• Many proteins evolved through combinations and variations of domains.

• Domains often correspond to exons, reflecting gene structure.

Example: CD4 (Cell Surface Protein)

• Composed of four similar domains from one polypeptide chain.

• Domains are connected by rigid loops.

Exons as Domains

• Protein-coding exons often match domains structurally.

• Evolution may shuffle or recombine exons to generate new proteins.

Motifs vs. Domains

• Motifs: Formed by secondary structures, not independently stable.

• Domains: Structurally and functionally independent, often conserved.

Fibronectin (Fn)

Why Fibronectin?

• A multidomain protein with many key roles:

  • Cell adhesion

  • Migration

  • Wound healing

  • Development

• Interacts with collagen and integrins, contributing to ECM (extracellular matrix) structure.

Fibronectin in the ECM

• Secreted by fibroblasts.

• Supports mechanical strength and elasticity in tissues.

• ECM adhesion relies on fibrillar assemblies of fibronectin.

Structure of Fibronectin

• 500 kDa disulfide-bonded dimer.

• Repeats three domain types: FnI, FnII, FnIII.

• Key motif: RGD (Arg-Gly-Asp) — binds integrins.

Domains in Detail

FnI Domains

• Involved in fibronectin-fibronectin interactions.

• Rich in disulfide bonds.

• Exposed edge strands are common binding sites.

FnII Domain

• Collagen binding domain.

• Recognizes gelatin (denatured collagen).

• NMR reveals flexibility in this region.

FnIII Domains

• Bind integrins at the cell surface.

• Can unfold under force (e.g. stretching).

• Exposes cryptic binding sites that aid crosslinking and tensile strength.

• Reversible — refolds in minutes.

Unfolding & Rebinding

• Stretching exposes hidden regions in FnIII.

• Allows new interactions during mechanical stress.

• Demonstrates functional elasticity.

Protein–Protein Interactions: Fibronectin and Bacteria

FnBPA – Fibronectin Binding Protein A (from Staphylococcus aureus)

• Binds FnI domains using a tandem β-zipper mechanism.

• Contains 11 repeats, each binding one Fn molecule.

• Forms a huge 3 MDa complex.

Staphylococcus Infections

• Causes infections like:

  • Bacteremia

  • Endocarditis

  • Toxic Shock Syndrome

  • MRSA (antibiotic resistance)

• Uses virulence factors including:

  • Coagulase

  • Exotoxins

  • Fibronectin-binding proteins (FnBPA, FnBPB)

Mechanism of FnBPA Binding

• Intrinsically disordered repeats form β-strands upon binding.

• Creates a strong, zipper-like interaction with fibronectin.

• High affinity (low Kd) ensures bacterial attachment to tissues.

Intrinsic Disorder

• Many proteins (including FnBPA) have unstructured regions:

  • Also called IDPs (intrinsically disordered proteins).

  • Still functional and often central in binding.

• Advantages:

  • Large interfaces with fewer residues.

  • Compact size.

  • Flexible binding to multiple partners.

Affinity and Kd (Dissociation Constant)

What is Kd?

• Measures binding strength:

  • Lower Kd = tighter binding.

  • At Kd, half of proteins are bound to ligand.

• Analogous to Km in enzyme kinetics.

How to Measure Kd

• One method: Isothermal Titration Calorimetry (ITC):

  • Measures heat changes during binding.

  • Provides:

    • Kd

    • Binding stoichiometry

    • Enthalpy (ΔH)

    • Free energy (ΔG)

Example: Factor H and Bacterial Mimicry

Factor H

• 155 kDa plasma protein that regulates complement immune system.

• Prevents immune attack on self-cells.

Pathogen Evasion

• Neisseria meningitidis protein fHbp mimics host targets to bind Factor H.

• Achieves tight binding (Kd = 5 nM) via:

  • Shape complementarity

  • Hydrogen bonding

  • Hydrophobic and ionic interactions

Summary

• Protein–protein interactions are central to biological function and pathogenesis.

• Domains and disorder both play critical roles.

• Bacteria exploit host proteins like fibronectin via evolved, high-affinity mechanisms.

• Structural understanding (NMR, X-ray, ITC) is key to characterizing these interactions.