Keywords

Hinge, Pre-existing Population, Ligand Binding, Protein Flexibility, Review, Protein Science

Reference

DOI: 10.1110/ps.21302

Abstract

Proteins with specificity can bind ligands of diverse shapes, sizes, and composition. This phenomenon stems from binding as a dynamic process governed by populations in equilibrium, where the binding site’s shape is influenced by the ligand. All proteins, whether specific or not, exist as ensembles of substates. If enough ligands are present in solution, favorable matches with varying shapes and sizes can bind, causing a redistribution of protein populations. Point mutations at distant sites can induce large conformational changes and hinge effects, shifting populations and potentially leading to drug resistance. Similar effects are seen in protein superfamilies with shared topology but different specificities. Thus, binding site shape and size are defined by the ligand and cannot be analyzed independently. Proteins present a dynamic distribution of binding site shapes to incoming ligands, explaining how specific binding proteins can interact with multiple ligands. This concept has implications for drug design, supporting the need for more diverse molecules and flexible docking approaches.

Notes

1. General Principles

  • Proteins are classified as specific or broad-range binders, but both cases reflect conformational distribution and hinge-bending motions around native states.
  • Binding principles apply to both selective and broad ligand recognition.
  • Proteins exist as conformational populations influenced by topology, residue type, and ligand presence.
  • Mutations and ligand binding shift conformational populations.

2. Dynamic Nature of Binding

  • Binding involves side-chain movements and hinge-bending, even if not always visible in structural comparisons.
  • Hinge bending allows a variety of conformations separated by low-energy barriers.
  • Binding of different ligands with various sizes and shapes suggests hinge-type motions facilitate adaptability.
  • Specific proteins also exhibit multiple conformations, with domain movements allowing adaptable binding.
  • Ligands can shift equilibrium toward favorable conformers, enabling binding even if transient in the unbound state.

3. Binding Site Structure and Ligand Dependence

  • Binding site shape and size are ligand-defined, not intrinsic to the protein alone.
  • Proteins present a range of conformers, and the best-matching one binds the ligand.
  • Unbound crystal structures capture only one of many possible conformations.
  • Binding relies on both geometry and polar residue hotspots, facilitating hydrogen bonding and complementarity.

4. Conformational Ensembles and Mutation Effects

  • Conformational space is unevenly populated, driven by protein topology.
  • Mutations shift population distributions, rather than creating new motions.
  • Certain regions (hinges) consistently exhibit motion, regardless of mutation sites.
  • Binding site variability allows diverse ligand binding and is fine-tuned by topology and flexibility.

5. Stability and Function

  • Structural stability is unevenly distributed: some regions are prone to unfolding, creating conformational diversity.
  • Mutations that increase stability (Shoichet et al., T4 lysozyme) can reduce activity, showing flexibility is crucial for function.
  • Binding sites have both low and high stability regions (Freire et al.).
  • Low-stability loops play roles in allostery and functional dynamics.
  • COREX algorithm shows binding sites as dual-nature regions — dynamic yet partially stable.
  • Conformational substates at low-stability regions impact ligand variability, but usually on smaller scales than hinge motions.

6. About Phosphorylation and Conformational Shifts (Kern, 2001)

  • Phosphorylation shifts populations, stabilizing active-like states.
  • Active-like species constitute only 2%-10% of unphosphorylated molecules.
  • Unphosphorylated proteins are more dynamic, while phosphorylation stabilizes specific conformers.
  • Side-chain motions remain rapid even after phosphorylation.
  • Phosphorylation promotes oligomerization, enabling sigmoidal activity curves.
  • Complex formation involves cascading redistribution of conformers.

7. Binding Mechanisms and Multi-Ligand Binding

  • Single-site proteins bind ligands via collision and diffusion, saturating at high ligand concentrations.
  • Multivalent proteins engage in cascading conformational redistribution — binding at one site influences others, enhancing overall binding with increased ligand.
  • Residues like glycine, promoting flexibility, may be targeted to modulate stability and binding efficiency.

8. Take-home Concept

  • Binding sites are defined by ligands, not fixed by the protein.
  • Proteins are dynamic distributions, sampling a range of conformers that ligands can select from.
  • Mutations, environment, and ligands all shift these conformational populations.
  • Hinge-bending motions and dynamic ensembles underpin diverse ligand binding.
  • Protein-ligand interaction is a matter of population redistribution, not simple induced fit or lock-and-key.
  • Understanding these principles is essential for drug design, allostery, and understanding resistance.

Sth else

“All those pieces weaves a logical scheme.” — loved this writing.

  • Single-site proteins reach binding saturation — plateau effect.
  • Multivalent proteins (e.g., in large complexes) redistribute conformations as ligand concentration increases, allowing enhanced and dynamic binding.
  • Flexibility residues (e.g., glycine) are key to adaptable binding and can be targeted for fine-tuning.