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..
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