Optimization of CD4/gp120 inhibitors by thermodynamic-guided alanine-scanning mutagenesis.
As protein/protein interactions usually trigger signalling processes, inhibitors of those interactions must preclude protein binding without eliciting the signalling process themselves. To accomplish those goals, small molecules need to target those protein residues that contribute the most to binding (binding hotspots) without disturbing those residues that initiate signalling processes (allosteric hotspots). The availability of a blueprint identifying binding and allosteric hotspots will significantly aid inhibitor design and optimization. In this study, we show that in some situations the blueprint can be constructed by combining the standard technique of alanine-scanning mutagenesis with isothermal titration calorimetry (ITC). We demonstrate the approach by developing the combined binding and allosteric hotspots blueprint for CD4/gp120, the initial interaction leading to HIV-1 cell infection. A major finding of these studies is that not all binding hotspots are allosteric hotspots opening the possibility for the rational design of inhibitors and antagonist or agonist modulators.
Department of Biology, The Johns Hopkins University, 3400 North Charles, Baltimore, MD 21218, USA.
Yingyun
Y
Liu
Arne
A
Schön
Ernesto
E
Freire
D000409:Alanine / Q000378:metabolism*; D000494:Allosteric Regulation; D015704:CD4 Antigens / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002151:Calorimetry; D057809:HEK293 Cells; D015699:HIV Envelope Protein gp120 / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D015497:HIV-1 / Q000378:metabolism; D006801:Humans; D016296:Mutagenesis; D060066:Protein Interaction Maps; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D013816:Thermodynamics; D014162:Transfection
D015704:CD4 Antigens; D015699:HIV Envelope Protein gp120; D011994:Recombinant Proteins; D000409:Alanine