Identification Of Allosteric Site In Proteins:Methodology Development And Mechanism Study

Allostery is the most direct,rapid and efficient way of regulating protein function induced by the binding of a ligand at a site topographically distinct from the orthosteric site,also termed as the allosteric site.Targeting allosteric sites can provide unprecedented advantages in terms of higher specificity and lower toxicity.This is due to the allosteric sites evolved under lower sequence-conservation pressure compared with the evolutionarily conserved active sites.Thus,the discovery and development of small-molecule allosteric drugs has drawn increasing attention as potential therapeutic agents in the international pharmaceutical industry.Currently,because of the devoid of feasible methods to identify the allosteric sites,most of the known allosteric sites are serendipitously found by experiments.This severely hinders the exploitation of allosteric regulation and the development of small-molecule allosteric drugs.In the second chapter of the thesis,for the first time,we present a web service,Allosite,which uses algorithms such as pocket-based analysis and support vector machine(SVM)classifier to predict the location of allosteric sites in proteins.Using a rigorous selection of high-quality dateasets for training,allosite's model exhibits a high accuracy of 95%.More importantly,the prediction of novel allosteric sites for several proteins using Allosite was experimentally supported by mutagenesis.The third chapter reports the benchmarking data for experimentally determined allosteric sites through a complex process,including a ‘Core set' with 235 unique allosteric sites and a ‘Core-Diversity set' with 147 structurally diverse allosteric sites.Allosite v2.0 discussed in the fourth chapter predicts allosteric sites of proteins by incorporating pocket features with perturbation analysis.When tested on the allosteric benchmark dataset ASBench and recently released allosteric sites,the prediction of Allosite v2.0 is substantially superior to those of the other currently available methods.In addition,we have investigated the allosteric mechanism of allosteric modulator.Adenosine-5'-triphosphate(ATP)is generally regarded as a substrate for energy currency and protein modification.Recent findings uncovered the allosteric function of ATP in cellular signal transduction but little is understood about this critical behavior of ATP.Through extensive analysis of ATP in solution and proteins,we found that the free ATP can exist in the compact and extended conformations in solution,and the two different conformational characteristics may be responsible for ATP to exert distinct biological functions: ATP molecules adopt both compact and extended conformations in the allosteric binding sites but conserve extended conformations in the substrate binding sites.Nudged elastic band simulations unveiled the distinct dynamic processes of ATP binding to the corresponding allosteric and substrate binding sites of uridine monophosphate kinase,and suggested that in solution ATP preferentially binds to the substrate binding sites of proteins.When the ATP molecules occupy the allosteric binding sites,the allosteric trigger from ATP to fuel allosteric communication between allosteric and functional sites is stemmed mainly from the triphosphate part of ATP,with a small number from the adenine part of ATP.Taken together,our results provide overall understanding of ATP allosteric functions responsible for regulation in biological systems.