Review of “Stability of Macromolecular Complexes”

1. Review of “Stability of Macromolecular Complexes” Dan Kulp Brooijmans, Sharp, Kuntz

2. Purpose • Search for general principles governing macromolecular interactions • Protein-Protein (Dimers) • Nucleic Acid-Ligand (Aptamers) • Nucleic Acid–Nucleic Acid (Duplexes) • Interactions/Contributions of specific forces to overall stability • Relationship between maximal affinity of macromolecular ligands and interface size • Subject of Study: Highest affinity complexes

3. Background Research • Protein – Ligand interaction study • Look at strongest binding ligands • Two modes of free energy: • Linear increase w/ increasing molecular size • Plateau, no increase w/increasing mol. Size • Free Energy calculations of binding

4. Differences in Interfaces… • Large macromolecular interfaces are flat • Small ligand binding sites are rough Pettit FK, Bowie JU. Protein surface roughness and small molecular binding sites. J Mol Biol 1999;285: 1377–1382.

5. Other differences.. • Atomic composition • Small ligands • Diverse set, topology • Amino Acid side chains / Nucleic Acids • Evolutionary pressures • Small ligands = shorting binding period • Regulation • Protein-Protein binding = longer binding

6. Selection of complexes • Protein – Protein Complexes • Homodimeric • 3 state denaturation (dissociation to monomers) • Resolution 3.1 Angstroms or better • Heterodimeric • Alanine mutants G > 5 kcal/mol • Nucleic Acid Complexes • DNA Duplex • Two state thermodynamics • Nucleic Acid aptamers • Bind small molecules/peptide ligands w/ high selectivity

8. Calculations • Total binding energy • Attributed to ligand atoms only • Simplify calculation • Interface areas (IA) – dms/MidasPlus • Accessible Surface Area (ASA) • IA = ASA receptor + ASA ligand – ASA complex • Interface atoms • Non-hydrogen, “heavy” atoms • atoms that lose ASA during complex formation • DNA Duplex – non sugar/phosphate atoms Connolly ML. Analytical molecular surface calculation. J Appl Crystallogr 1983;16:548–558.

9. Findings • Some Linear increase free energy w/ size • Maximal affinity plateau > 20 residues • 1.5 kcal/mol per interface atom • 120 cal/mol Angstrom^2 • Apparent differences in maximal affinity based on biological function • Protein-inhibitor complexes higher free energy compared to other interfaces of the same size

10. Findings… • Homodimers vs Heterdimers • Expect Homodimers have higher max. affinity • NO! • Dissociation constants are more permanent and more difficult to measure correctly • Comparison inside biological classes • Max contribution per interface atom is less for larger complexes = plateau behavior

11. Binding free energy vs # atoms

12. Binding free energy per atom

13. Exceptions • DNA Duplexes • Additive(Linear) Free Energy • Less per atom energy • Simple accounting scheme (2nd Structures) • Open Structure w/ size • NA aptamer • NA unstructured w/o ligand. • Ligand binding causes refolding • Hot spots • Contribute more per atom • K15A mutation in BPTI-trypsin complex • > 3 Kcal/mol

14. Previous Study • Chothia et al. Nature, 1975 • Positive correlation between interaction surface size and stability. • More data available • Maximal useful affinity makes sense • Long dissociation times (years?)

15. Better Interactions? • Atoms of low-molecular-weight ligands contribute more to energy than atoms of larger ligands. • More stable protein-protein complexes. Supported by finding that better than wild-type affinity achieved using phage display in vitro evolution. • Drug design – small molecule inhbitiors Dalby PA, Hoess RH, DeGrado WF. Evolution of binding affinity in a WWdomain probed by phage display. Protein Sci 2000;9:2366–2376.

16. Free Energy per class..