Transcript Review of “Stability of Macromolecular Complexes”

Review of “Stability of
Macromolecular
Complexes”
Dan Kulp
Brooijmans, 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 stability
Relationship between maximal affinity of
macromolecular ligands and interface size
Subject of Study: Highest affinity complexes
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
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.
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
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
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.
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
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
Binding free energy vs # atoms
Binding free energy per atom
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
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?)
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..