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.

Angiotensinogen and the Modulation of Blood Pressure

“…The role of angiotensinogen in the RAS was for long considered as merely that of an inert substrate. The previous questioning of this passive role ( 6 , 8 ), suggesting an inherently active role of angiotensinogen in the overall control of the release of angiotensin and, hence, in the modulation of blood pressure, has now been definitively confirmed by our recent structural studies ( 9 , 10 ). The solving of a series of crystallographic structures at high resolution of human angiotensinogen, together with its physiologically truncated forms and complexes with renin, now provides a video view of the conformational shifts that take place on the interaction of angiotensinogen with renin.…”

“…This is even more evident in the interspecies selectivity of the interaction with renin, thus human renin will only cleave human angiotensinogen and not that of the mouse or rat ( 13 ). The critical factor in this selectivity has now been shown ( 10 ) to be due to changes not as expected from other protease studies in the residues surrounding the scissile site, but rather in more peripheral residues involved in the body-to-body interface between the two molecules.…”

“…This is accompanied by a 10-Å displacement of the CD loop of angiotensinogen, which would otherwise sterically block the binding of renin, the two concerted movements being linked by a conserved disulfide bond. The widespread nature of other changes that take place on the binding of the two molecules has been further revealed in the most recent high-resolution structures ( 10 ), which show an accompanying rearrangement of helix H of angiotensinogen to allow more extensive bonding between the two molecules.…”

“…The less active unbridged form of angiotensinogen, with reduced sulfydryls, is also demonstrably stabilized by nitrosylation ( 9 ) in keeping with the known hypotensive action of nitric oxide. Other adjustments of the efficiency of the renin-release of angiotensin were shown ( 10 ) to arise from variations of glycan composition, notably so at Asn14 in close proximity to the renin-cleavage site at Leu10. The overall clinical and physiological message is that angiotensinogen is not an inert substrate.…”

A comprehensive phylogenetic analysis of the serpin superfamily

“…As the standard for chordate serpin nomenclature is derived from cladistic grouping, the divergence of angiotensinogen (AGT) from the last common ancestor of Groups A (excluding AGT) and D (supported by BS > 99) means that current classification standards should be amended to either recognise AGT as evolutionarily distinct from Group A, or redefine Group D as redundant within Group A. AGT is not a canonical protease inhibitor and instead functions in blood plasma redox sensing and blood pressure regulation, highlighting its divergence from a canonical Group A serpin (Zhou et al 2010;Yan et al 2019).…”