Structural basis for the specificity of renin-mediated angiotensinogen cleavage

Abstract

The renin-angiotensin cascade is a hormone system that regulates blood pressure and fluid balance. Renin-mediated cleavage of the angiotensin I peptide from the N terminus of angiotensinogen (AGT) is the rate-limiting step of this cascade; however, the detailed molecular mechanism underlying this step is unclear. Here, we solved the crystal structures of glycosylated human AGT (2.30 Å resolution), its encounter complex with renin (2.55 Å), AGT cleaved in its reactive center loop (RCL; 2.97 Å), and spent AGT from which the N-terminal angiotensin peptide was removed (2.63 Å). These structures revealed that AGT undergoes profound conformational changes and binds renin through a tail-into-mouth allosteric mechanism that inserts the N terminus into a pocket equivalent to a hormone-binding site on other serpins. These changes fully extended the N-terminal tail, with the scissile bond for angiotensin release docked in renin's active site. Insertion of the N terminus into this pocket accompanied a complete unwinding of helix H of AGT, which, in turn, formed key interactions with renin in the complementary binding interface. Mutagenesis and kinetic analyses confirmed that renin-mediated production of angiotensin I is controlled by interactions of amino acid residues and glycan components outside renin's active-site cleft. Our findings indicate that AGT adapts unique serpin features for hormone delivery and binds renin through concerted movements in the N-terminal tail and in its main body to modulate angiotensin release. These insights provide a structural basis for the development of agents that attenuate angiotensin release by targeting AGT's hormone binding pocket.

Keywords: angiotensinogen; aspartic protease; conformational change; crystal structure; hypertension; kinetics; proteolysis; renin angiotensin system; serpin; site-directed mutagenesis.

Figure 1.

The crystal structure of human glycosylated AGT. A, the N-terminal tail sequence of AGT, indicating the renin and ACE cleavage sites, the glycosylation site, and the conserved disulfide bond. B, the structure of AGT is shown as a cartoon. The serpin template is in gray, and helix A (hA) is in marine with the A-sheet (sA) in light blue and the disordered RCL in red dashes. The Ang I segment is in magenta, and the following amino-tail is in green with the scissile bond shown as magenta and green spheres. Cysteine 18 in the amino tail forms a disulfide bond with cysteine 138 of the CD-loop (brown). The glycan attached to Asn¹⁴ is shown as green sticks. The segment from Glu²⁰ to Pro²⁹ (dashed, pale green) is disordered in the structure and modeled for illustration. Helix H (hH) is in red, and helix G (hG) is in wheat color. C, surface representation of the main body of AGT, with the extra N terminus (residues 1–63) shown in a cartoon representation. The Ang I peptide is mainly stabilized by hydrophobic interactions with the main body. Residues Ile⁵, Phe⁸, Leu¹⁰, Val¹¹, and Ile¹² (shown as sticks) form hydrophobic interactions with residues in the CD-loop (Val¹³¹, Pro¹³², and Trp¹³³ as brown surface), helix A (Leu⁶⁸, Met⁷², Leu⁷⁶, and Phe⁷⁹ as marine surface), and helix D (hD; Leu¹⁴² and Val¹⁴⁷ as cyan surface). The scissile bond (Leu¹⁰–Val¹¹) is buried in the hydrophobic cavity.

Figure 2.

The crystal structure of the AGT–renin complex. The color scheme of the AGT moiety in the complex is the same as in Fig. 1, whereas the renin moiety is shown as gray with yellow N-flap and purple C-flap. The renin molecule is in contact with the surface of AGT containing helix H (hH), helix A (hA), helix C (hC), and the CD-loop. The scissile bond (shown as spheres) is located in the renin active cleft. The fragments without clear electron density are shown with dashed lines for illustration.

Figure 3.

Interactions inside the renin active cleft. A, renin residues are shown as gray, and the AGT peptide is in magenta. Asp³⁸ in renin is hydrogen-bonded to the carbonyl oxygen of the scissile bond and a water (W1) that is also within hydrogen-bond range to the other mutated aspartic acid (Ala²²⁶). The conserved hydrogen bond network Trp⁴⁵-Tyr⁸³-water (W2)-Ser⁴¹-Asp³⁸ is present. B, His⁹ in AGT is hydrogen-bonded to Ser²³³. His⁹ also connects to His¹³ through a bridging water (W3). Ser⁸⁴ in the N-flap forms a hydrogen bond with a water (W4), which connects to the carbonyl oxygen of His⁹. C, binding subsites in the renin active cleft. Subsite S3 (Pro¹¹⁸, Phe¹¹⁹, Leu¹²¹, Ala¹²², and Phe¹²⁴ of renin) accommodates Phe⁸; S1 (Phe¹¹⁹, Phe¹²⁴, Val¹²⁷, Val³⁶, and Tyr⁸³) accommodates Leu¹⁰; S1′ (Leu²²⁴ and Ile³⁰⁵) accommodates Val¹¹; and S2′ (Ile¹³⁷, Leu⁸¹, and Tyr⁸³) accommodates Ile¹². The N-flap of renin is shown as yellow, and the C-flap is in purple. D, the N-flap of renin (yellow) in the AGT–renin complex adopts an “open” position with a movement of 4.8 Å compared with its conformation in the unbound renin (cyan, PDB code 1BBS). There is no such conformational change when a decapeptide substrate (corresponding to residues 4–14 of rat AGT) is bound to mouse submaxillary renin (pink; PDB entry 1SMR). The N-terminal residues 1–10 of the AGT peptide are in magenta and the following residues 11–15 and the glycan are shown as green.

Figure 4.

Interactions between renin and angiotensinogen in the complex. A, an opened-up cartoon view of the interface (with the renin moiety rotated 180° around the y axis) illustrating the hydrophobic interactions and hydrogen-bonded residues (sticks). B, an opened-up surface representation highlights hydrophobic interactions at the interface. C, cartoon view of the interface, with hydrogen bonds shown as red dashes between interacting residues (sticks). Renin, on the left, is shown as gray with interacting residues in yellow. AGT, on the right, is shown using the colors defined for Fig. 1. D, residues involved in hydrogen-bonding interactions are listed, along with the identities of corresponding residues in AGT from other species. The plot shows that cleavage efficiency is reduced when Asn³³¹ or Arg⁸³ of human AGT is replaced by an equivalent residue from rat or mouse AGT, respectively. The detailed kinetic constants are listed in Table 2.

Figure 5.

Conformational changes of angiotensinogen upon renin interaction. Side views of AGT alone (A) and from the complex with renin (B) illustrate the substantial movement of the N terminus of AGT into the renin active cleft. The scissile bond (shown as spheres) moves 18.6 Å during the complex formation. The color coding and abbreviations are the same as in Fig. 1. C, superposed structures of native AGT and AGT from the complex with renin show significant structural rearrangement of helices H, A, I, and J (hH, hA, hI, and hJ) of AGT upon renin binding. Native AGT is shown as gray with a cyan helix H, and AGT in the complex is color-coded as AGT in Fig. 1. Helix H is completely unwound with the C^α atom of Asn³³¹ (shown as a cyan stick in native AGT and red stick in AGT complexed with renin) shifting more than 6 Å to interact with Tyr⁶⁰ of renin. Helix A is extended by two turns with the side chain of Trp⁹² (shown as a magenta stick in native AGT and a marine stick in AGT complexed with renin) at the tip of helix A being flipped out. D, superposed structures of native (gray) and spent AGT (same color coding as AGT in Fig. 1) show that spent AGT largely resembles native AGT, but with differences in helix H and the CD-loop (blue in spent AGT). Helix H of spent AGT is completely disordered.

Figure 6.

The insertion of the N-terminal peptide into the hormone binding pocket. A, renin binding results in the N terminus of AGT being inserted into the hormone binding pocket and in unwinding of helix H. The side chain of Arg² is largely buried and forms stabilizing interactions with Gln³⁰⁰. Val³ and Tyr⁴ also form hydrophobic interactions with surrounding residues. B, there is clear electron density for all of the residues in the N terminus (gray mesh, 2mF_o − DF_c map, contoured at 1.0σ, including density within 1.6 Å of atoms in the N terminus). C, a similarly located pocket is used to bind thyroxine (T4; spheres) in TBG.