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 (2 nd 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..