Blood pressure is critically controlled by angiotensins1, vasopressor peptides specifically released by the enzyme renin from the tail of angiotensinogen, a non-inhibitory member of the serpin family of protease inhibitors2,3. Although angiotensinogen has long been regarded as a passive substrate, the crystal structures solved here to 2.1Å resolution show that the angiotensin cleavage-site is inaccessibly buried in its amino-terminal tail. The conformational rearrangement that makes this site accessible for proteolysis is revealed in a 4.4Å structure of the complex of human angiotensinogen with renin. The co-ordinated changes involved are seen to be critically linked by a conserved but labile disulphide bridge. We show that the reduced unbridged form of angiotensinogen is present in the circulation in a near 40:60 ratio with the oxidised sulphydryl-bridged form, which preferentially interacts with receptor-bound renin. We propose that this redox-responsive transition of angiotensinogen to a form that will more effectively release angiotensin at a cellular level contributes to the modulation of blood pressure. Specifically, we demonstrate the oxidative switch of angiotensinogen to its more active sulphydryl-bridged form in the maternal circulation in pre-eclampsia - the hypertensive crisis of pregnancy that threatens the health and survival of both mother and child.

The hormones that most directly control tissue activities in health and disease are delivered by two noninhibitory members of the serpin family of protease inhibitors, thyroxine-binding globulin (TBG) and corticosteroid-binding globulin. The structure of TBG bound to tetra-iodo thyroxine, solved here at 2.8 Å, shows how the thyroxine is carried in a surface pocket on the molecule. This unexpected binding site is confirmed by mutations associated with a loss of hormone binding in both TBG and also homologously in corticosteroid-binding globulin. TBG strikingly differs from other serpins in having the upper half of its main ␤-sheet fully opened, so its reactive center peptide loop can readily move in and out of the sheet to give an equilibrated binding and release of thyroxine. The entry of the loop triggers a conformational change, with a linked contraction of the binding pocket and release of the bound thyroxine. The ready reversibility of this change is due to the unique presence in the reactive loop of TBG of a proline that impedes the full and irreversible entry of the loop that occurs in other serpins. Thus, TBG has adapted the serpin inhibitory mechanism to give a reversible flip-flop transition, from a high-affinity to a low-affinity form. The complexity and ready triggering of this conformational mechanism strongly indicates that TBG has evolved to allow a modulated and targeted delivery of thyroxine to the tissues.corticosteroid-binding globulin ͉ serpin ͉ thyroxine-binding globulin ͉ crystal structure T hyroxine is the major hormone controlling cellular development as well as the rate of body metabolism. It is a small molecule formed by the linkage of two tyrosines, which are iodinated to give the alternative tri-or tetra-iodo forms of the hormone. Thyroxine is principally carried in the blood by thyroxine-binding globulin (TBG), which binds it with high affinity (K d ϭ 0.1 nM) in equilibrium with steady-state low levels of free thyroxine (1-3). Although TBG is not a protease inhibitor, it is otherwise a typical member of the serpin family of protease inhibitors. The alignment of its sequence shows that it retains the same framework structure as the archetypal inhibitory members of the family, ␣ 1 -antitrypsin, antithrombin, and antichymotrypsin (4). It has similarly retained a typical reactive site loop, with a putative reactive center at the position denoted P1 and, with 17 residues before it, the hinge of the loop at P17 (see Fig. 1 and Table 1). In particular, TBG undergoes the profound and irreversible conformational change on cleavage of its reactive loop, which is characteristic of the serpins (5, 6). Such cleavage of the reactive loop of TBG by proteases does occur during sepsis to give a 3-fold reduction in its binding affinity (7-9). However, because only a minor proportion, Ͻ20%, of the circulating TBG is bound to thyroxine, even this relatively small decrease in affinity will result in an effective release of thyroxine (10), as demonstrably occurs at sites of inf lammation (11). Normally, ho...

Corticosteroids are transported in the blood by a serpin, corticosteroid-binding globulin (CBG), and their normally equilibrated release can be further triggered by the cleavage of the reactive loop of CBG. We report here the crystal structures of cleaved human CBG (cCBG) at 1.8-A resolution and its complex with cortisol at 2.3-A resolution. As expected, on cleavage, CBG undergoes the irreversible S-to-R serpin transition, with the cleaved reactive loops being fully incorporated into the central beta-sheet. A connecting loop of helix D, which is in a helix-like conformation in native CBG, unwinds and grossly perturbs the hormone binding site following beta-sheet expansion in the cCBG structure but shifts away from the binding site by more than 8 A following the binding of cortisol. Unexpectedly, on cortisol binding, the hormone binding site of cCBG adopts a configuration almost identical with that of the native conformer. We conclude that CBG has adapted an allosteric mechanism of the serpins to allow equilibrated release of the hormones by a flip-flop movement of the intact reactive loop into and out of the beta-sheet. The change in the hormone binding affinity results from a change in the flexibility or plasticity of the connecting loop, which modulates the configuration of the binding site.

The release of hormones from thyroxine-binding globulin (TBG) and corticosteroid-binding globulin (CBG) is regulated by movement of the reactive center loop in and out of the β-sheet A of the molecule. To investigate how these changes are transmitted to the hormone-binding site, we developed a sensitive assay using a synthesized thyroxine fluorophore and solved the crystal structures of reactive loop cleaved TBG together with its complexes with thyroxine, the thyroxine fluorophores, furosemide, and mefenamic acid. Cleavage of the reactive loop results in its complete insertion into the β-sheet A and a substantial but incomplete decrease in binding affinity in both TBG and CBG. We show here that the direct interaction between residue Thr342 of the reactive loop and Tyr241 of the hormone binding site contributes to thyroxine binding and release following reactive loop insertion. However, a much larger effect occurs allosterically due to stretching of the connecting loop to the top of the D helix (hD), as confirmed in TBG with shortening of the loop by three residues, making it insensitive to the S-to-R transition. The transmission of the changes in the hD loop to the binding pocket is seen to involve coherent movements in the s2/3B loop linked to the hD loop by Lys243, which is, in turn, linked to the s4/5B loop, flanking the thyroxine-binding site, by Arg378. Overall, the coordinated movements of the reactive loop, hD, and the hormone binding site allow the allosteric regulation of hormone release, as with the modulation demonstrated here in response to changes in temperature.

Protein Z (PZ) binds to PZ-dependent inhibitor (ZPI) and accelerates the inhibition of the coagulation protease, activated factor X (FXa), in the presence of phospholipids and Ca 2؉ . A 2.3Å resolution crystal structure of PZ complexed with ZPI shows that ZPI is a typical serine protease inhibitor and that PZ has a serine protease fold with distorted oxyanion hole and S1 pocket.