Angiotensinogen and the Modulation of Blood Pressure.

The angiotensin peptides that control blood pressure are released from the non-inhibitory plasma serpin, angiotensinogen, on cleavage of its extended N-terminal tail by the specific aspartyl-protease, renin. Angiotensinogen had previously been assumed to be a passive substrate, but we describe here how recent studies reveal an inherent conformational mechanism that is critical to the cleavage and release of the angiotensin peptides and consequently to the control of blood pressure. A series of crystallographic structures of angiotensinogen and its derivative forms, together with its complexes with renin show in molecular detail how the interaction with renin triggers a profound shift of the amino-terminal tail of angiotensinogen with modulation occurring at several levels. The tail of angiotensinogen is restrained by a labile disulfide bond, with changes in its redox status affecting angiotensin release, as demonstrably so in the hypertensive complication of pregnancy, pre-eclampsia. The shift of the tail also enhances the binding of renin through a tail-in-mouth allosteric mechanism. The N-terminus is now seen to insert into a pocket equivalent to the hormone-binding site on other serpins, with helix H of angiotensinogen unwinding to form key interactions with renin. The findings explain the precise species specificity of the interaction with renin and with variant carbohydrate linkages. Overall, the studies provide new insights into the physiological regulation of angiotensin release, with an ability to respond to local tissue and temperature changes, and with the opening of strategies for the development of novel agents for the treatment of hypertension.

Heparin Blocks the Inhibition of Tissue Kallikrein 1 by Kallistatin through Electrostatic Repulsion.

Kallistatin, also known as SERPINA4, has been implicated in the regulation of blood pressure and angiogenesis, due to its specific inhibition of tissue kallikrein 1 (KLK1) and/or by its heparin binding ability. The binding of heparin on kallistatin has been shown to block the inhibition of KLK1 by kallistatin but the detailed molecular mechanism underlying this blockade is unclear. Here we solved the crystal structures of human kallistatin and its complex with heparin at 1.9 and 1.8 Å resolution, respectively. The structures show that kallistatin has a conserved serpin fold and undergoes typical stressed-to-relaxed conformational changes upon reactive loop cleavage. Structural analysis and mutagenesis studies show that the heparin binding site of kallistatin is located on a surface with positive electrostatic potential near a unique protruded 310 helix between helix H and strand 2 of ß-sheet C. Heparin binding on this site would prevent KLK1 from docking onto kallistatin due to the electrostatic repulsion between heparin and the negatively charged surface of KLK1, thus blocking the inhibition of KLK1 by kallistatin. Replacement of the acidic exosite 1 residues of KLK1 with basic amino acids as in thrombin resulted in accelerated inhibition. Taken together, these data indicate that heparin controls the specificity of kallistatin, such that kinin generation by KLK1 within the microcirculation will be locally protected by the binding of kallistatin to the heparin-like glycosaminoglycans of the endothelium.

Measurement of the total angiotensinogen and its reduced and oxidised forms in human plasma using targeted LC-MS/MS.

Angiotensinogen (AGT) is a critical protein in the renin-angiotensin-aldosterone system and may have an important role in the pathogenesis of pre-eclampsia. The disulphide linkage between cysteines 18 and 138 has a key role in the redox switch of AGT which modulates the release of angiotensin I with consequential effects on blood pressure. In this paper, we report a quantitative targeted LC-MS/MS method for the reliable measurement of the total AGT and its reduced and oxidised forms in human plasma. AGT was selectively enriched from human plasma using two-dimensional chromatography employing concanavalin A lectin affinity and reversed phase steps and then deglycosylated using PNGase F. A differential alkylation approach was coupled with targeted LC-MS/MS method to identify the two AGT forms in the plasma chymotryptic digest. An additional AGT proteolytic marker peptide was identified and used to measure total AGT levels. The developed MS workflow enabled the reproducible detection of total AGT and its two distinct forms in human plasma with analytical precision of ≤ 15%. The LC-MS/MS assay for total AGT in plasma showed a linear response (R2 = 0.992) with a limit of quantification in the low nanomolar range. The method gave suitable validation characteristics for biomedical application to the quantification of the oxidation level and the total level of AGT in plasma samples collected from normal and pre-eclamptic patients.

Structural basis for the specificity of renin-mediated angiotensinogen cleavage.

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.

How serpins transport hormones and regulate their release.

The adaptation of the serpin framework and its mechanism to perform diverse functions is epitomised in the hormone carriers of the blood. Thyroxine and the corticosteroids are transported bound in a 1:1 ratio on almost identical sites in the two homologous binding-globulins, TBG and CBG. Recent structural findings show an equilibrated, rather than on-and-off, release of the hormones from the carriers, reflecting small reversible movements of the hinge region of the reactive loop that modify the conformational flexibility of the underlying hormone-binding site. Consequently, contrary to previous concepts, the binding affinities of TBG and CBG are not fixed but can be allosterically modified to allow differential hormone delivery. Notably, the two carriers function like protein thermocouples with a surge in hormone release as body temperatures rise in fevers, and conversely a large diminution in free hormone levels at hibernation temperatures. By comparison angiotensinogen, the source of the angiotensin peptides that control blood pressure, does not appear to utilise the serpin mechanism. It has instead evolved a 63 residue terminal extension containing the buried angiotensin cleavage site, which on interaction moves into the active cleft of the renin. The conformational shift involved is critically linked by a labile disulphide bridge. The observation of changes in the redox status of this S-S bridge, in the hypertensive complication of pregnancy, pre-eclampsia, has opened an unexpected level of regulation at what is the initial stage in the control of blood pressure.

Molecular Mechanism of Z α1-Antitrypsin Deficiency.

The Z mutation (E342K) of α1-antitrypsin (α1-AT), carried by 4% of Northern Europeans, predisposes to early onset of emphysema due to decreased functional α1-AT in the lung and to liver cirrhosis due to accumulation of polymers in hepatocytes. However, it remains unclear why the Z mutation causes intracellular polymerization of nascent Z α1-AT and why 15% of the expressed Z α1-AT is secreted into circulation as functional, but polymerogenic, monomers. Here, we solve the crystal structure of the Z-monomer and have engineered replacements to assess the conformational role of residue Glu-342 in α1-AT. The results reveal that Z α1-AT has a labile strand 5 of the central ß-sheet A (s5A) with a consequent equilibrium between a native inhibitory conformation, as in its crystal structure here, and an aberrant conformation with s5A only partially incorporated into the central ß-sheet. This aberrant conformation, induced by the loss of interactions from the Glu-342 side chain, explains why Z α1-AT is prone to polymerization and readily binds to a 6-mer peptide, and it supports that annealing of s5A into the central ß-sheet is a crucial step in the serpins' metastable conformational formation. The demonstration that the aberrant conformation can be rectified through stabilization of the labile s5A by binding of a small molecule opens a potential therapeutic approach for Z α1-AT deficiency.

Temperature-responsive release of thyroxine and its environmental adaptation in Australians.

The hormone thyroxine that regulates mammalian metabolism is carried and stored in the blood by thyroxine-binding globulin (TBG). We demonstrate here that the release of thyroxine from TBG occurs by a temperature-sensitive mechanism and show how this will provide a homoeostatic adjustment of the concentration of thyroxine to match metabolic needs, as with the hypothermia and torpor of small animals. In humans, a rise in temperature, as in infections, will trigger an accelerated release of thyroxine, resulting in a predictable 23% increase in the concentration of free thyroxine at 39°C. The in vivo relevance of this fever-response is affirmed in an environmental adaptation in aboriginal Australians. We show how two mutations incorporated in their TBG interact in a way that will halve the surge in thyroxine release, and hence the boost in metabolic rate that would otherwise occur as body temperatures exceed 37°C. The overall findings open insights into physiological changes that accompany variations in body temperature, as notably in fevers.

How changes in affinity of corticosteroid-binding globulin modulate free cortisol concentration.

CONTEXT: Recent studies of corticosteroid-binding globulin (CBG) indicate that it does not merely transport cortisol passively but also actively regulates its release in the circulation. We show how CBG binding affinity can vary to give changes in free cortisol concentration in a physiologically relevant range. OBJECTIVE: The objective was to determine how the binding affinity of plasma CBG is affected by glycosylation, changes in body temperature, and the conformational change induced by proteases at sites of inflammation. DESIGN: Binding assays were performed over a range of temperatures with plasma and recombinant CBG to determine the contribution of glycosylation. The role of conformational change was assessed by measuring binding affinities of plasma CBG before and after reactive loop cleavage by neutrophil elastase. MAIN OUTCOME MEASURES: Determination of binding constants allows calculation of clinically relevant changes in CBG saturation and free cortisol concentrations. RESULTS: On reactive loop cleavage at inflammation sites, CBG can continue to act as a buffered source of cortisol, although with a much reduced affinity, to give a potential quadrupling of free cortisol. Predicted increases in systemic free cortisol resulting from elevated body temperatures, previously reported based on affinity measurements using nonglycosylated recombinant CBG, were shown here to be considerably increased using glycosylated plasma CBG, with a doubling for every 2°C rise in body temperature. CONCLUSIONS: The ability of CBG to modulate free cortisol levels in blood must be considered in the understanding and management of disease processes, as illustrated here with predictable changes in inflammation and fever.

Allosteric modulation of hormone release from thyroxine and corticosteroid-binding globulins.

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 ß-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 ß-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 Thr(342) of the reactive loop and Tyr(241) 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 Lys(243), which is, in turn, linked to the s4/5B loop, flanking the thyroxine-binding site, by Arg(378). 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.

A redox switch in angiotensinogen modulates angiotensin release.

Blood pressure is critically controlled by angiotensins, which are vasopressor peptides specifically released by the enzyme renin from the tail of angiotensinogen-a non-inhibitory member of the serpin family of protease inhibitors. Although angiotensinogen has long been regarded as a passive substrate, the crystal structures solved here to 2.1 Å 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 our 4.4 Å 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. Here we show that the reduced unbridged form of angiotensinogen is present in the circulation in a near 40:60 ratio with the oxidized 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.

Crystal structure of protein Z-dependent inhibitor complex shows how protein Z functions as a cofactor in the membrane inhibition of factor X.

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 Ca2+. A 2.3A 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. The 2 molecules bind with fully complementary surfaces spanning over 2400A(2) and involving extensive ionic and hydrophobic interactions. ZPI has an unusual shutter region with a negatively charged residue buried within the hydrophobic core of the molecule. This unique Asp(213) is critical in maintaining the balanced metastability required for optimal protease inhibition, especially when PZ is bound, with its replacement with Asn resulting in increased thermal stability, but decreased efficiency of protease inhibition. The structure of ZPI shows negatively and positively charged surfaces on top of the molecule, in keeping with mutagenesis studies in this work indicating exosite interactions with FXa when it docks on top of ZPI. As modeled in this study, the gamma-carboxy-glutamic acid-containing domains of PZ and FXa enable them to bind to the same phospholipid surfaces on platelet and other membranes, with optimal proximity for the inhibition of FXa by the complexed ZPI.

Serpins show structural basis for oligomer toxicity and amyloid ubiquity.

Many disorders, including Alzheimer's, the prion encephalopathies and other neurodegenerative diseases, result from aberrant protein aggregation. Surprisingly, cellular toxicity is often due not to the highly-ordered aggregates but to the oligomers that precede their formation. Using serpins as a paradigm, we show how the active and infective interface of oligomers is inherently toxic and can promiscuously bind to unrelated peptides, including neurotransmitters. Extension of the oligomer and its eventual sequestration as amyloid can thus be seen as a protective response to block the toxic interface. We illustrate how the preferential self-association that gives this protection has been selectively favoured.

The S-to-R transition of corticosteroid-binding globulin and the mechanism of hormone release.

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.

Dimers initiate and propagate serine protease inhibitor polymerisation.

The serine protease inhibitor (serpin) family can readily form long-chain polymers by a process that underlies a variety of diseases. We show here that monomers of plasma serpins alpha(1)-antitrypsin and antithrombin are stable on incubation with the rate-limiting step in their polymerisation being the formation of the initial dimer. Once formed, the dimers readily interlink to form tetramers and can bind monomers to form trimers and longer oligomers. Cleavage of the only exposed reactive loop, in unit I of the dimers, prevents their interlinkage, but these cleaved dimers can still link to monomers. The rapid binding by the cleaved dimers of a peptide specific to the lower half of beta-sheet A of the molecule indicates the ready opening of this beta-sheet in unit II of the dimers. The failure of the cleaved dimers to bind peptide-complexed monomers, together with the relative inaccessibility of the P14 hinge residue in the oligomers, is evidence that partial insertion of the reactive loop into its own A-sheet is required for polymer formation. We propose that serpin dimers initiate and propagate polymerisation by having one exposed loop with an optimal conformation as a beta-strand donor and a readily opened beta-sheet as an acceptor. The sequential reformation of these activated beta-interfaces as the oligomer extends, molecule by molecule, provides a model for the fibril and amyloid formation of conformational diseases in general as well as for the infectivity of prion encephalopathies.

Structural mechanism for the carriage and release of thyroxine in the blood.

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 A, 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 beta-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.

Cell toxicity and conformational disease.

Numerous disorders, including Alzheimer's, Parkinson's and other late-onset neurodegenerative diseases, arise from the conformationally driven aggregation of individual proteins. Previous focus on just one end-product of such aggregation - extracellular deposits of amyloid - has diverted attention from what is now recognized as being primarily intracellular disease processes. Recent structural findings show how cytotoxicity can result from even minor changes in conformation that do not lead to amyloid formation, as with the accumulation within the endoplasmic reticulum of intact mutant alpha-1-antitrypsin in hepatocytes and of neuroserpin in neurons. Studies in Alzheimer's and other dementias also indicate that the damage occurs at the stage of the initial intermolecular linkages that precede amyloid formation. The challenge now is to determine the detailed mechanisms of this cytotoxicity.

Thrombosis as a conformational disease.

Conformational diseases are a newly recognized group of heterogeneous disorders resulting from the conformational instability of individual proteins. Such instability allows the formation of intermolecular linkages between b-sheets, to give protein aggregation and inclusion body formation. The serpin family of serine protease inhibitors provides the best-studied examples of the structural changes involved. Notably, mutations of a-1-antitrypsin result in its intracellular polymerization and accumulation in the liver leading eventually to cirrhosis. Here we consider how other conformational changes in another serpin, antithrombin, can cause its inactivation with consequent thrombosis. Thirteen different missense mutations in antithrombin are associated with either oligomer formation or with conversion of the active molecule into an inactive latent form. Each of these variant antithrombins is associated with an increased risk of thrombosis that typically occurs in an unexpectedly severe and sudden form. The trigger for this episodic thrombosis is believed to be the sudden conformational transition of the antithrombin with an accompanying loss of inhibitory activity. But what causes the transition? This is still unclear, though a likely contributor is the increased body temperature that occurs with infections hence the frequency of episodes associated with the urinary infections of pregnancy. The search for other causes is important, as the conformational perturbation of normal antithrombin is likely to be a contributory cause to the sporadic and apparently idiopathic occurrence of venous thrombosis.