Figure 1.
The renin-angiotensin system shown in protein structures based on available or modeled structures.
Angiotensinogen (AGT, red) is cleaved by Renin (cyan) producing the ten amino acid Ang I peptide. Ang I is then cleaved by ACE to produce Ang II that is subsequently cleaved by ACE 2 to produce Ang-(1–7). These peptides then bind to AT1, AT2, or MAS (gray).
Table 1.
Known structures of the renin-angiotensin system.
Figure 2.
A) Methodology for creating models of AT1, AT2 and MAS beginning with I-TASSER models, adding cysteine bridges, inserting into a lipid membrane and then running molecular dynamics simulations. B) The averaged models from A for AT1, AT2, and MAS.
Figure 3.
Molecular dynamics of AT1, AT2, and MAS.
Simulations of each receptor (AT1, AT2, or MAS) were done in in a lipid membrane for 2 nanoseconds showing either the potential energy of the receptor (A) or the averaged carbon alpha root-mean squared deviation (RMSD = average movement of the protein backbone at each amino acid from the initial structure (B)).
Table 2.
Sequence and structural alignment values of AT1, AT2, MAS, and Rhodopsin.
Figure 4.
Sequence alignments of AT1, AT2, MAS, and Rhodopsin from human or the consensus sequence.
Consensus sequence alignments show those amino acids conserved as a hydrophobic as α (A, V, L, I, F, W, M, P), polar acidic as β (D, E), polar basic as µ (K, R, H), aromatic as π (F, W, H, Y), ∞ for S and T conservation, and. for no conservation. Cysteines highlighted in yellow are those identified to form cysteine bridges, amino acids highlighted in red commonly conserved in GPCR, cyan conserved in all sequences including Rhodopsin, green those conserved only in AT1, AT2, and MAS, and gray/magenta those conserved in only AT1 and AT2 that were identified in other experiments to be critical to Ang peptide binding or activation.
Figure 5.
Conservation of amino acids shown on the structure of AT1.
View is from looking down the receptor from the extracellular surface. Red indicates amino acids commonly conserved in GPCRs, cyan those conserved with Rhodopsin, and green those conserved only in AT1, AT2 and MAS corresponding to Figure 4. Amino acids shown are those identified in Table S1 to have functional roles in Ang peptides binding and activation of receptors, including the consensus GPCR number used.
Figure 6.
Amino acids involved in activation of AT1 and AT2 but not MAS.
Amino acids 512 and 621 (blue) interact with amino acid 8 (Phe) of Ang II, while 325 (magenta) interacts with amino acid 4 (Tyr) of Ang II displacing 723 (Tyr) in both AT1 (A) and AT2 (B). Aromatic amino acids (red) likely serve to transition Phe 8 from 512 and 621 to the known photolabled interaction sites at 725 for AT1 (A) or 336 for AT2 (B). The basic seven transmembrane domain schematic representation is added below each figure to show the amino acid positions in both AT1 (A) and AT2 (B) with the numbers listed at each site the location in the respective protein, and the number in brackets that used as the common numbering scheme. These mechanisms seen in AT1/AT2 are not conserved in MAS (C).
Figure 7.
Molecular dynamics simulations of the multiple states of AT1 in activation.
The Carbon alpha RMSD of Ang II bound at the multiple points of activation to AT1 (A). The graph shows that the buried position (cyan) yields an increase in overall dynamics of the AT1 receptor. The initial binding (purple) led to a transition in the simulation to yield a similar binding as the buried as the simulation neared 8 ns. B) The distance between two of the amino acids (326 and 618) found in the site of AT1 where the eighth amino acid (Phe) comes to the final photolabled interaction for each of the stages of AT1 activation. This shows that the binding of Ang II in the buried position causes a stretching (around 3 Å), leading to opening of new interaction sites for protein interactions. The initial (purple) binding led to propagation and stretching of the receptor around 2 ns yielding similar values as that of the buried binding.
Figure 8.
Activated vs. non-activated AT1.
The average structure over the 10 ns simulations shown for either AT1 with Ang II free (gray) or in the final buried position (cyan). This shows that Ang II activation likely leads to shifting in helix 3, 5 and 6. This suggests regions of helix 5 (containing the largest movement of the helix) to likely recruit other proteins when Ang II is bound. Additionally some modification made in the intracellular region (due to the shifting of helix 3) could potentially modify intracellular activation.
Figure 9.
Pathway of activation of AT1 by Ang II.
Figure shows the multiple binding states of Ang II activation of AT1 receptor. Initial binding results in movement of helix 7 by Tyr4 of ANG II leading to p42/44 MAPK activation; buried binding results in movement of helix 5 by Phe8 of Ang II leading to Inositol Phosphate response.