Interactions Between AqpZ And DAGK With Small Molecules Investigated By Solid-State NMR

Membrane proteins are the bridge for material exchange,energy conversion and signal transmission between intracellular and extracellular.Currently,about 60%of the drug targets on the market are membrane proteins.Studying the interaction between membrane proteins and small molecules is very important for screening and optimizing potential drug molecules.Magic Angle Spinning Solid State Nuclear Magnetic Resonance(MAS SSNMR)is an excellent method to study non-crystalline and insoluble biological macromolecules,which are difficult to be studied by traditional X-ray crystallography and solution-state NMR methods.MAS SSNMR has three advantages in studying the interaction between membrane proteins and small ligands.It can be used to study membrane proteins in(near)native cell membrane environments,to obtain 3D structure and dynamics information with atomic resolution,and to monitor the interaction process in real-time.In this paper,the author studied the interactions between two membrane proteins(E.coli aquaporin,i.e.AqpZ and E.coli diacylglycerol kinase,i.e.DAGK)with other molecules/ions(e.g.water,phospholipid,metal ions and nucleotides)using MAS SSNMR.The research work is divided into four parts as follows.In the first part,we developed a SSNMR method to measure the chemical exchange rate between protein and water.The method suppressed the spin diffusion between 1H by locking it in the direction of the magic angle,and then transferred the water 1H polarization to protein labile 15N whose signal has been dispersed by the REDOR.The chemical exchange process of water and protein residues was indirectly monitored by the 15N?13C? signal.Through this method,we obtained the chemical exchange rates of 11 backbone atoms and 3 side chain atoms of AqpZ,which provided important information for understanding the interaction between AqpZ and the surrounding water.We also established an SSNMR method for characterizing the water cavity in the membrane protein,and found two conserved water cavity in the AqpZ CS-Rosetta structure using the method.These studies demonstrated the ability of SSNMR to characterize water cavity in membrane proteins.In the second part,we characterized the topology structure and proposed a permanent opening mechanism of AqpZ in the native E.coli inner membrane.We prepared high quality AqpZ samples for SSNMR by optimizing the sample preparation strategy.We collected a set of 3D SSNMR spectra and assigned the NMR signals of more than 90%amino acids of AqpZ.We identified the residues that can be accessed by external water molecules using the H/D exchange experiment and the residues close to lipid molecules using the lipid-CH2 edited experiment.Based on the above data,we at the first time revealed the AqpZ topology structure in its native E.coli inner membrane.Besides,we demonstrated that there was only one stable open conformation of Arg189 that was thought to gate the AqpZ water channel using the 1H-15N DIPSHIFT experiments and proposed a permanently open gating mechanism of AqpZ in the native E.coli inner membrane.The high-quality distance constrained spectra provide a solid basis for the calculation of the high-resolution NMR structure of AqpZ in the native E.coli inner membrane.In the third part,we elucidated the molecular mechanism of AqpZ inhibition by mercury.We prepared AqpZ samples of different cysteine(Cys)mutants,and collected a set of 3D SSNMR spectra of those samples with and without mercury ions.We then analyzed the residues that changed with mercury and obtained the coordination of mercury ions in different AqpZ mutants.Combined with molecular dynamics simulation,the different mercury-inhibition mechanism of different cysteine sites in the AqpZ water channel was determined.In the last part,we proposed the molecular mechanism of adenosine triphosphate(ATP)entering DAGK and adenosine diphosphate(ADP)leaving DAGK.Through the SSNMR titration experiment in E.coli membrane environment,we systematically analyzed the binding sites of ATP and ADP to DAGK,the dynamic changes in DAGK cytosolic loop(CL)region during ATP binding and ADP leaving,the affinity of ATP binding sites,and the change in affinity when ADP left We finally proposed the molecular mechanism of ATP entering DAGK and ADP leaving from DAGK in the membrane environment.