NMR Studies Of Angiotensin â…¡ Type1Receptor And The Interaction Between Valsartan And Mimetic Membranes

Valsartan （VST） is one of the Angiotensin II receptor antagonists, which is widelyused in clinical hypertension treatment. However, the tertiary structure of the targetprotein angiotensin â…¡ type1receptor（AT₁） has not been solved, so that the mechanismof the protein-drug interaction is not clear. It is believed that VST incorporates intobiological membranes before it binds to AT₁receptor. Nuclear Magnetic Resonance（NMR） is a powerful tool for investigating the interaction between molecules at atomicand molecular level. Herein the interactions between VST and detergent-mimickingmembrane environment were investigated by using NMR and Molecular Dynamics （MD）simulation. Two AT₁transmembrane peptides were synthesized or expressed, andpreliminarily explored by solution NMR techniques.The interaction between VST and membrane is of importance for furtherunderstanding the antagonism mechanism of the drug. By using NMR techniques and MDsimulation, we studied VST’s penetration in the detergent mimic membrane before itreached the AT₁receptor. We observed that VST has two conformers （trans and cis）exchanging slowly in DPC micelles. The changes of chemical shifts, relaxation rates andself-diffusion coefficients of VST protons indicate that both conformers have stronginteractions with DPC. NOE cross peaks and MD simulation reveal that DPC interactswith VST not only through the hydrophobic lipid chain, but also the hydrophilic headgroup, locating VST at the charged head group and upper part of the micelles. Theconcentration ratio of trans over cis conformers is0.94, showing that two conformers havesame affinities with DPC, which is significantly smaller than our previous results obtainedin SDS micelles. MD simulation suggested that cis conformer has slightly lower bindingfree energy than trans conformer when interacting with DPC. The conformational changeof VST was further investigated in two detergents, CTAB and Tween-20. Ratios ofconformer trans and cis in the presence of detergents are in the order of DPC，CTAB <Tween-20<SDS, which is correlated with the charge characters of their head groups. These results indicate that the electrostatic interaction plays an essential role in the bindingprocess of VST with detergents and the hydrophobic interaction influences the packing ofthe drug in the micelles. These results may be of help in understanding delivery processesof sartan drugs in cell membranes.AT₁receptor belongs to the GPCR family, consisting of seven transmembrane helices.Based on the two-step folding model of the multi-transmembrane protein, the fifthtransmembrane domain of AT₁（AT₁TM5） which directly involved in the binding of VSTwas treated as an independent structural element. We obtained over-expressed and purifiedfusion protein （MBP-AT₁TM5） by the prokaryotic expression. The results showed thatMBP-tag can dramatically improve the solubility of the peptide and the proper length ofthe peptide is of significant importance to the solubility.We chemically synthesized the third transmembrane domain of AT₁（AT₁TM3），whichdirectly participates in the binding of VST. We acquired and assigned the homonucleartwo-dimensional NMR spectra of the peptide in the DPC micelles. The assignments wereonly achieved between Leu¹⁹and Lys²⁹, and the fragment does not form a stableconformation. It indicates that the two-step folding model might not be suitable to our case,and the screening of a proper detergent is still bottle-neck of the structure analysis ofmembrane proteins. Our investigations may provide some useful data to the understandingof the structure of AT₁receptor.