Review of Stability of Macromolecular Complexes

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Review of Stability of Macromolecular Complexes

• Dan Kulp
• Brooijmans, Sharp, Kuntz

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Purpose

• Search for general principles governing
  macromolecular interactions
• Protein-Protein (Dimers)
• Nucleic Acid-Ligand (Aptamers)
• Nucleic AcidNucleic 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

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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

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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
1999285 13771382.
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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

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Selection of complexes

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

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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 198316548558.
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Findings

• Some Linear increase free energy w/ size
• Maximal affinity plateau gt 20 residues
• 1.5 kcal/mol per interface atom
• 120 cal/mol Angstrom2
• Apparent differences in maximal affinity based on
  biological function
• Protein-inhibitor complexes higher free energy
  compared to other interfaces of the same size

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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

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Binding free energy vs atoms
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Binding free energy per atom
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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
• gt 3 Kcal/mol

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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?)

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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 2000923662376.
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Free Energy per class..
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