Review of “Stability of Macromolecular

Complexes”

Dan KulpBrooijmans, Sharp, Kuntz

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 stabilityRelationship between maximal affinity of macromolecular ligands and interface sizeSubject of Study: Highest affinity complexes

Background ResearchProtein – Ligand interaction studyLook at strongest binding ligandsTwo modes of free energy:

Linear increase w/ increasing molecular sizePlateau, no increase w/increasing mol. Size

Free Energy calculations of binding

Differences in Interfaces…

Large macromolecular interfaces are flatSmall ligand binding sites are rough

Pettit FK, Bowie JU. Protein surface roughness and small molecular binding sites. J Mol Biol 1999;285: 1377–1382.

Other differences..Atomic composition

Small ligands • Diverse set, topology

Amino Acid side chains / Nucleic Acids

Evolutionary pressuresSmall ligands = shorting binding period

• Regulation

Protein-Protein binding = longer binding

Selection of complexesProtein – Protein Complexes

Homodimeric• 3 state denaturation (dissociation to monomers)• Resolution 3.1 Angstroms or better

Heterodimeric• Alanine mutants G > 5 kcal/mol

Nucleic Acid ComplexesDNA Duplex

• Two state thermodynamics Nucleic Acid aptamers

• Bind small molecules/peptide ligands w/ high selectivity

CalculationsTotal binding energy

Attributed to ligand atoms onlySimplify calculation

Interface areas (IA) – dms/MidasPlusAccessible Surface Area (ASA)IA = ASA receptor + ASA ligand – ASA complex

Interface atoms Non-hydrogen, “heavy” atomsatoms that lose ASA during complex formation

DNA Duplex – non sugar/phosphate atomsConnolly ML. Analytical molecular surface calculation. J Appl Crystallogr 1983;16:548–

558.

Findings

Some Linear increase free energy w/ sizeMaximal affinity plateau > 20 residues1.5 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

Findings…Homodimers vs Heterdimers

Expect Homodimers have higher max. affinityNO!Dissociation constants are more permanent and more difficult to measure correctly

Comparison inside biological classesMax contribution per interface atom is less for larger complexes = plateau behavior

Binding free energy vs # atoms

Binding free energy per atom

ExceptionsDNA Duplexes

Additive(Linear) Free EnergyLess per atom energy

• Simple accounting scheme (2nd Structures)Open Structure w/ size

NA aptamerNA unstructured w/o ligand. Ligand binding causes refolding

Hot spotsContribute more per atomK15A mutation in BPTI-trypsin complex

• > 3 Kcal/mol

Previous Study Chothia et al. Nature, 1975Positive correlation between interaction surface size and stability.More data availableMaximal useful affinity makes sense

Long dissociation times (years?)

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.

Free Energy per class..