Structural basis for the specificity of renin-mediated angiotensinogen cleavage

Yahui Yan,Aiwu Zhou,Robin W Carrell,Randy J Read

doi:10.1074/jbc.ra118.006608

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.

Highlights

The renin–angiotensin cascade is a hormone system that regulates blood pressure and fluid balance
Human blood pressure is mainly controlled by the renin– angiotensin system, which consists of several key components, including renin, angiotensinogen (AGT),3 angiotensin-converting enzyme (ACE), and angiotensin receptors [1]
The serpin superfamily is classically characterized by proteins that fold into a conserved metastable tertiary structure that has the ability to undergo profound conformational changes for protease inhibition

Summary

Results

Crystal structures of human glycosylated AGT, its complex with renin, loop-cleaved AGT, and spent AGT. This indicates that residues involved in the body-tobody interface between AGT and renin play a key role in determining the catalytic efficiency and binding affinity of renin This explains the species specificity of human renin toward AGT from other mammals and explains why intact AGT has a 10-fold lower Km value compared with the tetradecapeptide derived from its N terminus [6, 7]. Comparison of structures of native AGT and its complex with renin shows that the N-terminal tail of AGT undergoes significant conformational changes to dock into the substrate binding site of renin with the scissile bond presented appropriately for hydrolysis by the two active aspartate residues (Fig. 5, A and B). In native AGT, the region of the N-terminal tail near the scissile bond Leu10–Val forms hydrophobic interactions with helix A residues, such as Leu, Met, Leu, and Phe (Fig. 1C).

A RCL sA hA1

Discussion

Experimental procedures