What determines
the binding affinity?

M1 ZHAI 20210430
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Contents• Binding affinity• Assays for binding affinity• Overall determinants for binding affinity• Different binding affinity between in vivo and in vitro• Structure prediction of macromolecular complexes2Binding affinity• The binding affinity is the strength of the interaction between two (or more thantwo) molecules that bind reversibly (interact).
• It is translated into physicochemical terms in the dissociation constant (Kd), thelatter being the concentration of the free protein that occupies half of the overallsites of the second protein at equilibrium.

• The binding affinity can also be translated in physical terms into the Gibbs freeenergy of dissociation.
• The binding affinity is related to the Gibbs free energy of association (▵Ga) as3

Binding affinity• Correct and precise estimation of the binding affinity is
crucial throughout these essential drug design stages.
• This high demand has facilitated the development of anumber of different techniques to assess or predict ligandbinding.

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CETSA• Cellular thermal shiftassay• The method allows
studies of target
engagement of drugcandidates in acellular context.
• Melting temperature(Tm) shift assays

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BLI• Biolayer interferometry• Label-free technology for measuring biomolecularinteractions

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Overall determinants for binding affinity• Buried surface area
• Hot spots and anchor residues• Allosteric regulators and non-interface affinity modifiers

7Overall determinants for binding affinity• The buried surface area (BSA) is defined as the surface buried away from thesolvent when two or more proteins or subunits associate to form a complex.
• The BSA has been the primary descriptor to be related to binding affinity, andmore specifically, to the intrinsic bond energy.
• BSA is a macroscopic descriptor for the hydrophobic interactions.

Panagiotis L., J. R. Soc. Interface, 20128

Overall determinants for binding affinityHot spots and anchor residues• In the context of protein-protein interactions, the term "hotspot" refers to a residue or cluster of residues that makes amajor contribution to the binding free energy.
• They are most often found in central regions of theinterface .
• Their amino acid composition differs from that of nonhotspot residues.

9Overall determinants for binding affinityHot spots and anchor residues• Anchor chains act as ready-made recognition motifs byacquiring native-like conformations before any physicalinteraction with the receptor.
• Anchors are proposed to reduce the number of possible
binding pathways and therefore avoid structuralrearrangements.
• Anchor residues must provide most of the specificity
necessary for protein–protein recognition whereas otherimportant residues on the interface contribute to thestabilization.
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Overall determinants for binding affinityAllosteric regulators and non-interface affinity modifiers• Definiton: account for regulation of a protein by a change in its
tertiary structure induced by a small molecule.
• Changes in the dynamics or structure of a protein by a modulator.
• Such changes shift the population of the inactive protein to its
active form, thereby significantly altering its binding affinity.
• The binding of oxygen to haemoglobin.

Allison AC. Br Med J., 1954, 1, 290-4

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Diﬀerent binding aﬃnity between in vivo and in vitro• Experimental aspect• Cell environment

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Different binding affinity between in vivo and in vitroExperimental aspect• Lack of contextual data (target physiology, pathology andmicro-environment) in samples and an assay platform
capable of probing the interactome.
• Binding differences between soluble and membrane-boundforms of target.
• Performed in non-native environments, are restricted torelatively simple matrices such as buffer.

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Different binding affinity between in vivo and in vitro-Macromolecular crowding• Refers to a phenomenon that alters the properties ofmolecules in a solution when high concentrations ofmacromolecules such as proteins are present.

David G., Biol.Chem., 2016, 397, 37

14Macromolecular crowding-excluded volume effect

Erica Fiorini, CHIMIA International Journal for Chemistry, 2015, 207–212

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Macromolecular crowding-excluded volume effectproteins are more stable in a crowded
solution of macromolecules compared todilute aqueous solution

David G., Biol.Chem., 2016, 397, 37

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Macromolecular crowding-excluded volume effectGermán Rivas., Trends in Biochemical Sciences, 2016, 41

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Analysing macromolecular crowding effects inthe living cellDavid G., Biol.Chem., 2016, 397, 3718

Macromolecular crowding-excluded volume effectand nonspecific interactions

David G., Biol.Chem., 2016, 397, 37

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Macromolecular crowding affects diffusion andthe rates of enzyme-catalyzed reactions.

Ma ́rcio A. Moura ̃o, Biophysical Journal, 2014, 107, 2761

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Modulation of cellular volume to control physiologicalprocesses via macromolecular crowding

Ma ́rcio A. Moura ̃o, Biophysical Journal, 2014, 107, 2761

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A specific example- the effects of macromolecular
crowding on antibody function：HSA&mAbsIonic strength dependence of mAb1/HSA andmAb2/HSA cross interactions measured by CG-MALS
Dorothy M Kim, mAbs, 2019, 1319

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A speciﬁc example- the eﬀects of macromolecular
crowding on anEbody funcEon
mAb1

mAb2

Binding of 40 nM mAb1 andmAb2 to biotinylated antigen in
the absence and presence ofHSA was observed by biolayerinterferometry at 10 (panel a)and 137 mM NaCl (panel b) inphosphate buffer
Dorothy M Kim, mAbs, 2019, 1319 23

A specific example- the effects of macromolecular crowdingon antibody function
Low salt condition

physiological salt condition

Dorothy M Kim, mAbs, 2019, 1319

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A speciﬁc example- the eﬀects ofmacromolecular crowding on anEbody funcEon• The crowding agent ficoll 70 does not produce the same
effect on mAb binding to antigen
Low salt condition

physiological salt condition

Dorothy M Kim, mAbs, 2019, 131925

Binding affinity• The physicochemical complexity of the solvent-accessible
surface areas presented by different proteins plays afundamental role in the diversity of non-specificmacromolecular interactions.
Low salt condition

physiological salt condition

Dorothy M Kim, mAbs, 2019, 1319 26

A speciﬁc example- the eﬀects ofmacromolecular crowding on anEbody funcEon• The crowding agent ficoll 70 does not produce the same
effect on mAb binding to antigen
Low salt condition

physiological salt condition

Dorothy M Kim, mAbs, 2019, 131927

Different macromolecular crowding agent cause
different results
Differential effect of HSA and RNase A as crowder proteins

Silvia Z., Protein Sci., 2004, 13, 296028Macromolecular crowding• Contrary to the typical in vitro media, the intracellularenvironment is densely packed with macromolecules.
• Excluded volume effect + nonspecific interaction• Polymer crowders do not consistently produce an effecton ligand binding, and may even have totally different
effect on different proteins

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Structure prediction of macromolecular complexes• Scheme of a typical drug discovery process.

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Structure predicEon of macromolecular complexes• Docking: developed but still influenced by several factors- Proteins are not static structures.
- The binding site is not always conserved or cannot always
be identified.
- Current docking methods cannot distinguish whether two
proteins will bind or not, (predict the binding affinity).

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Structure prediction of macromolecular complexes
The binding site is not always conserved or cannot always
be identified.
The most conserved surface patch
on a protein was rarely found toshare >50% residue overlap with thereal interface.
The data set consists of 42 chains that form
homodimers, 12 chains that form heterodimers,
and 10 chains that form transient complexes as
described
Overall, the results suggest that one will havea small chance (17/64) of correctly predicting
50% of the interface residues
Caffrey DR., Protein Sci., 2004, 190

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Structure prediction of macromolecular complexes
Current docking methods cannot distinguish whether two
proteins will bind or not, (predict the binding affinity).
Barstar-barnase complex.Sacquin-Mora S. et al., J. Mol. Biol., 2008, 382, 1276

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Summary• Correct and precise estimation of the binding affinity is
crucial throughout these essential drug design stages.
• CETSA and BLI would be effective method to estimate
binding affinity.
• Buried surface area, hot spots and anchor residues andallosteric regulators and non-interface affinity modifiers
would be the determinant of binding affinity.
• Macromolecular crowding may account for thedifference of binding affinity between in vivo and in vitro.
• Some problems still need to be resolved for the predictionof binding affinity.
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