Conformational dynamics in GPCR signaling by NMR

G-protein-coupled receptors(GPCRs)mediate a wide range of cellular responses to various ligands or stimuli,and are the most important drug targets associated with human diseases.While major advances in GPCR structural biology have greatly deepened our understanding of its activation mechanism,the highly complex changes in the structural dynamics of GPCRs during activation remain underdetermined and their links to physiological functions largely unknown.Solution nuclear magnetic resonance(NMR)spectroscopy is an essential technique that allows the characterization of protein structural dynamics at atomic level,and has been applied in the studies of GPCR structural-function relationship in the past decade.Herein,we summarize a few specific studies in which solution NMR methods were employed and provided novel insights into questions difficult to be addressed by other methods.

Three-dimensional domain swapping as a mechanism to lock the active conformation in a super-active octamer of SARS-CoV main protease

Proteolytic processing of viral polyproteins is indispensible for the lifecycle of coronaviruses.The main protease(M^(pro))of SARS-CoV is an attractive target for anti-SARS drug development as it is essential for the polyprotein processing.M^(pro) is initially produced as part of viral polyproteins and it is matured by autocleavage.Here,we report that,with the addition of an N-terminal extension peptide,M^(pro) can form a domain-swapped dimer.After complete removal of the extension peptide from the dimer,the mature M^(pro) self-assembles into a novel super-active octamer(AO-M^(pro)).The crystal structure of AO-M^(pro) adopts a novel fold with four domainswapped dimers packing into four active units with nearly identical conformation to that of the previously reported M^(pro) active dimer,and 3D domain swapping serves as a mechanism to lock the active conformation due to entanglement of polypeptide chains.Compared with the previously well characterized form of M^(pro),in equilibrium between inactive monomer and active dimer,the stable AO-M^(pro) exhibits much higher proteolytic activity at low concentration.As all eight active sites are bound with inhibitors,the polyvalent nature of the interaction between AO-M^(pro) and its polyprotein substrates with multiple cleavage sites,would make AO-M^(pro) functionally much more superior than the M^(pro) active dimer for polyprotein processing.Thus,during the initial period of SARS-CoV infection,this novel active form AOM^(pro) should play a major role in cleaving polyproteins as the protein level is extremely low.The discovery of AOM^(pro) provides new insights about the functional mechanism of M^(pro) and its maturation process.

Mechanisms of Chaperones as Active Assistant/Protector for Proteins:Insights from NMR Studies

Molecular chaperones are diverse families of proteins that play key roles in protein homeostasis.They assist the folding of client proteins or prevent them from rreversible aggregation under stress conditions.Diverse chaper-one families contribute to different aspects of protein homeostasis by inter-acting with a wide range of client proteins.Despite the vital roles of chaper-ones in cell survival,the molecular mechanisms underlying chaperone func-tions remain elusive,due to the non-specificity of chaperone-client interac-tions and the intrinsic flexibility of the clients.