The central role of angiotensinogen in the control of blood pressure is revealed by a series of crystallographic structures, including complexes with renin. Specifically, the structures provide an understanding of the sequential molecular events that lead to the pre-eclamptic hypertensive crises of pregnancy. The release of the precursor vasopressor peptide from the amino-terminal tail of angiotensinogen appears to be modulated by a redox-sensitive disulphide bridge. Our findings indicate that the activation of the thiol-switch in the circulating maternal angiotensinogen occurs at the placental level in response to oxidative stress, exacerbated by placental insufficiency. We propose here that a contributory factor is the inherent redox stress accompanying the placental exchange of oxygenation between the haemoglobin of the mother (oxy-HbA) and the deoxygenated haemoglobin of the foetus (deoxy-HbF).

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.

The National Women's Hospital in Auckland was from the 1960s New Zealand's only great teaching hospital. Internationally it became a leader in the care of mother and newborn, and nationally it was a forerunner in the wider transition that was taking place to science-led and evidence-based medicine. Its success arose from an outstanding faculty of university consultant obstetricians and gynecologists, medical scientists as well as clinicians. William Liley developed intrauterine detection and then restorative transfusion for Rh incompatibility, and the demonstration by Graham Liggins that corticosteroids promoted the maturation of the lungs has subsequently been lifesaving for countless premature newborns. Their achievements were recognized for each with knighthoods, and Liggins was elected to the Fellowship of the Royal Society of London, FRS. The equal excellence of teaching and clinical care at the National Women's was evident from its annual output of postgraduates who materially lifted the standards in obstetric care throughout the country. At the core of this programme was the teaching and training provided by Dr Herbert Green, an academic gynecologist and an exponent of evidence-based practice. It was however his pursuit of the latter, as outlined in this journal by Raffle and Muir Gray [[1]Raffle A.E. Muir Gray J.A. Review. The 1960s cervical screening incident at National Women's Hospital, Auckland, New Zealand: insights for screening research, policy making, and practice.J Clin Epidemiol. 2020; 122: A7-A12Abstract Full Text Full Text PDF Scopus (11) Google Scholar] and Chalmers [[2]Chalmers I. Commentary. The “unfortunate experiment” that was not, and the indebtedness of women and children to Herbert (“Herb”) Green (1916-2001).J Clin Epidemiol. 2020; 122: A13-A19Abstract Full Text Full Text PDF Scopus (11) Google Scholar], that provided a trigger for the events leading to the dishonoring of the Faculty of the National Women's and to its calamitous loss of repute as a teaching hospital. The question Green was asking in the 1960s was one that needed to be addressed in clinical medicine as a whole. It was becoming evident then that early neoplastic changes could be variable in progression and may regress, or progress so slowly as not to threaten well-being or life expectancy. These were alternative outcomes that Green investigated with respect to changes in cervical cytology, in what was a thoughtful clinical study for its time: carefully planned and documented, with meticulous follow-up, and regular peer-reviewed publications. It was an open study, with the full support of colleagues at the time as evident in this excerpt from Liley's laudatory letter [[3]Correspondence. W Liley to H Green, 6th May 1975, Valerie Smith archive, University of Auckland Library Special Collections. As supplementary data to this article to be found online at https://doi.org/10.1016/j.jclinepi.2020.09.001Google Scholar] in 1975 quoting the comment of a distinguished US expert in the field ‘I think Green's work on the natural history of carcinoma in situ is just as important as your [Liley's] work on haemolytic disease or Liggin's work on fetal endocrinology. He has saved a lot of young women from mutilating surgery.' The precepts set at the National Women's, in challenging established dogmas of practice as well as in integrating science and medicine, were an inspiration to me as I moved in 1986 from a Professorship in Pathology in New Zealand to take up the foundation Chair of Haematology in the University of Cambridge. It was with bewilderment then that I subsequently learned of the turn of events in Auckland leading to the Cartwright Inquiry [[4]Committee of Inquiry into Allegations Concerning the Treatment of Cervical Cancer at National Women’s Hospital and into Other Related Matters. The report of the cervical cancer inquiry. Government Printing Office, Auckland, NZ1988Google Scholar] and its disastrous aftermaths. An immediate questioning of this outcome came from common sense. In any academic institution, one or even two people may go off the rails, but for this to involve a whole faculty of proven distinction and integrity—Liley, Green, Liggins, Bonham, and Seddon—defies rationality. Something had gone wrong in Auckland? How did all this come about? These were questions I whispered in 2011, in the choir stalls of King's College Chapel in Cambridge, to the young Professor of Surgery from Auckland sitting next to me. “I will send you a book”, he quietly replied. The book, Women's Bodies and Medical Science [[5]Bryder L. A Women's Bodies and Medical Science. An Inquiry into Cervical Cancer. Palgrave Macmillan London; 2010. Previous Auckland edition: Bryder L. A history of the ‘Unfortunate Experiment’ at National Women's Hospital. Auckland University Press, 2009Google Scholar], did indeed answer the questions. Its author, Linda Bryder, a medical historian, gives a tightly referenced account, step by step, of the course of events in Auckland both before and after the Cartwright Inquiry. The book is a landmark, in providing a scholarly account of how an ordered society can implode on itself, with lessons of wider relevance to any in positions of responsibility in science. In reviewing the book for a journal of the Royal Society of London [[6]Carrell R.W. Trial by media.Notes Rec R Soc Lond. 2012; 66: 301-306Crossref Google Scholar], I first spent over 8 months in checking and counterchecking its sources. Not just reference by reference but also by speaking to those who had worked with Green et al., including an independent cervical cytologist. All the inquiries firmly verified Bryder's account, as indeed is true of all subsequent checks made to this day. Bryder's account is now further supported by the independent and authoritative assessment of Raffle and Gray [[1]Raffle A.E. Muir Gray J.A. Review. The 1960s cervical screening incident at National Women's Hospital, Auckland, New Zealand: insights for screening research, policy making, and practice.J Clin Epidemiol. 2020; 122: A7-A12Abstract Full Text Full Text PDF Scopus (11) Google Scholar] of the cervical screening programme at the National Women's Hospital in the 1960s, and of the contentions and Inquiry that arose from it. A detailed and referenced case study in their recent text book [[7]Raffle A.E. Mackie A. Gray J.A.M. Screening, evidence and practice.2nd ed. Oxford University Press, Oxford2019Crossref Google Scholar] points out the fundamental error of the Inquiry in using the term cancer to describe symptomless changes in cervical cell and tissue samples in healthy women. The resulting impression in the public mind was that women with cancer were deliberately left untreated. Raffle and Gray [[1]Raffle A.E. Muir Gray J.A. Review. The 1960s cervical screening incident at National Women's Hospital, Auckland, New Zealand: insights for screening research, policy making, and practice.J Clin Epidemiol. 2020; 122: A7-A12Abstract Full Text Full Text PDF Scopus (11) Google Scholar] make clear that this was not so. Crucially they document the inaccuracies and exaggerations of subsequent retrospective and anecdotal reports of patients claimed to have had treatment withheld in Green's practice. The way the denigration of Green came about has sobering lessons for other medical and scientific investigators. The incident that sparked what was to become a conflagration of public indignation was the labeling of Green's study, as an ‘unfortunate experiment’ [[8]Skegg D.C.G. Cervical screening - correspondence.N Z Med J. 1986; 99: 27PubMed Google Scholar]. This label was soon extrapolated by activists Coney and Bunkle [[9]Coney S. Bunkle P. The unfortunate experiment. Metro. Auckland. Metro Magazine.https://www.metromag.co.nz/Date: 1987Google Scholar] to ‘men carrying out experiments on women's bodies', along with analogies by others of Green's 'experiments' with those at Auschwitz. The resulting furor rapidly culminated in the establishment of the Cartwright Inquiry, the outcome of which was widely acclaimed as confirmation of medical men experimenting on women's bodies. The consequent response to the publication of Bryder's book criticizing the Inquiry and its findings was furious and at times vicious. Not in effectively challenging the narrative of her book, but in continuing attempts to dismiss it: by attacking the credibility of the author, by belittling independent scholars who endorsed it, and by the labeling of supportive obstetricians as partisan, together with efforts to suppress favorable reviews including direct attempts to prevent the publication of its London edition. Can the reputation of the National Women's Hospital be restored from these concerted attacks? Not as such, but the documentation of Bryder along with the independent endorsement of Raffle and Gray clearly vindicates the work and integrity of Green. The question he asked, and the study he carried out, can now be seen as forerunners of what has become a universal approach to screening programmes in general. There is now recognition of the need to balance the benefits of reflex radical treatments with the morbidities from overdetection and overtreatment [[7]Raffle A.E. Mackie A. Gray J.A.M. Screening, evidence and practice.2nd ed. Oxford University Press, Oxford2019Crossref Google Scholar]. The pioneering contribution of Herbert Green in the shift toward active surveillance (‘watchful waiting’) has benefited many who would otherwise have undergone disfiguring disruptions to their lives. He deserves acknowledgment and honor rather than the calumny he received in Auckland. I strongly support Iain Chalmers' proposal [[2]Chalmers I. Commentary. The “unfortunate experiment” that was not, and the indebtedness of women and children to Herbert (“Herb”) Green (1916-2001).J Clin Epidemiol. 2020; 122: A13-A19Abstract Full Text Full Text PDF Scopus (11) Google Scholar] for an award in Green's name that will recognize his contributions and repute. The charges against Herbert Green were central both to the Inquiry and to the tribunals in its aftermath. Once the restoration of the reputation of Green is acknowledged, the dishonoring of his colleagues will fall away. The sudden downfall of the National Women's Hospital, in a matter of months, has lessons for all. Creativity and innovation bring with them changes, and with change comes resentment and tensions—notably so in science and medicine. Such tensions are inherently present in teaching hospitals where the mandate of the academics is to question and innovate, whereas the health service doctors for the most part just get on with it and practice the procedures they were taught in their first graduate years. When the tensions explode, as they did in Auckland in 1987, there needs to be cautious and sensitive adjudication. Justice is not served amidst a storm of media and public indignation [[4]Committee of Inquiry into Allegations Concerning the Treatment of Cervical Cancer at National Women’s Hospital and into Other Related Matters. The report of the cervical cancer inquiry. Government Printing Office, Auckland, NZ1988Google Scholar]. The issues are almost always complex and best resolved using the inbuilt safeguards of science: of careful documentation and scholarship [[5]Bryder L. A Women's Bodies and Medical Science. An Inquiry into Cervical Cancer. Palgrave Macmillan London; 2010. Previous Auckland edition: Bryder L. A history of the ‘Unfortunate Experiment’ at National Women's Hospital. Auckland University Press, 2009Google Scholar], as endorsed here by the authoritative assessments of Raffle and Gray [[1]Raffle A.E. Muir Gray J.A. Review. The 1960s cervical screening incident at National Women's Hospital, Auckland, New Zealand: insights for screening research, policy making, and practice.J Clin Epidemiol. 2020; 122: A7-A12Abstract Full Text Full Text PDF Scopus (11) Google Scholar] and by Chalmers [[2]Chalmers I. Commentary. The “unfortunate experiment” that was not, and the indebtedness of women and children to Herbert (“Herb”) Green (1916-2001).J Clin Epidemiol. 2020; 122: A13-A19Abstract Full Text Full Text PDF Scopus (11) Google Scholar]. In research, the defining criterion is the quality of the question. Green was asking the right questions in the 1960s at a time when his critics were just doing repetitively what everyone else had done before. The ultimate vindication of Green is the benefits, now evident across medicine, of the field of research in which he was an initiator. Download .pdf (.53 MB) Help with pdf files Appendix

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 β-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.

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 (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.

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.

The plasma protein α-1-antitrypsin holds a special place in medicine and biology. In medicine, its common deficiency is a model of the way genetic disease can result in both loss-of-function and gain-of-function disabilities. In biology, α-1-antitrypsin is the archetype of an extensive superfamily of serine proteinase inhibitors, the serpins, which control essential intra- and extracellular functions. A central feature of the serpins is their extraordinary conformational mechanism evolved to irreversibly trap target proteases. A downside of this conformational mobility is the susceptibility to intracellular misfolding and polymerisation, exemplified in Z α-1-antitrypsin but now recognised to result in a range of diseases, from thrombosis to dementia, due to homologous mutations in other serpins. Although the principles of the central mechanism came from original findings with α-1-antitrypsin, subsequent studies of other serpins have revealed subtler interactions, with ligand and receptors and in response to changes in body temperature, that modulate the activity of individual serpins. These later findings provide a challenge in α-1-antitrypsin research. There is much to learn: α-1-antitrypsin deficiency is still a syndrome and not a fully explained disease.

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 β-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 β-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 β-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.

Cortisol is transported in the blood by corticosteroid-binding globulin (CBG), a non-inhibitory member of the serpin family of serine protease inhibitors. Recent structural advances reveal how CBG acts as a releasing-agent as well as a carrier of cortisol. Taken together, the structures of the various forms of CBG and of the closely related thyroxine binding-globulin, show how the inherent conformational mechanism of the serpins has been adapted to modulate hormone release to the tissues by changes in binding affinities. A deduction from this, of the temperature dependence of hormone binding, is remarkably borne out with CBG, with a doubling in plasma free cortisol as the body temperature rises to 39 °C. Another insight, against a dogma in the corticosteroid field, is that the proteolytic cleavage of CBG in inflammation results in a partial and not a complete loss of cortisol binding. This becomes of medical importance in conjunction with recent evidence of a pool of the circulating cleaved-form of CBG. It is now evident that tissue levels of free cortisol are buffered by two responsive plasma pools, intact CBG with a high binding-affinity and, particularly in inflammation and sepsis, a further pool of cleaved-CBG with a ten-fold lower affinity. The new molecular understandings, as well as providing insights into the differential release of circulating hormones, also open prospects for therapeutic interventions and draw attention to the potential of CBG and TBG as vehicles for the targeted delivery of drugs.

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.

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.

Linda Bryder, Women's bodies and medical science: an inquiry into cervical cancer. Palgrave Macmillan, Basingstoke, 2010. Pp. vi + 250. £60 (hardback). ISBN 978-0-230-2603-5. If a professor … neglects research, lets controversy rest, He's but a petty tradesman at best.Kalidasa (Sanskrit poet,

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 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.

The hormone-carrying serpins, thyroxine- and corticosteroid-binding globulins, TBG and CBG, provide a clear example of the way the serpin conformational mechanism can be adapted not only to give an irreversible switching-off of function but also more significantly to allow a constant dynamic modulation of activity. This is illustrated here with the demonstration that hormone release from both TBG and CBG is responsive to changes in ambient temperature and specifically to changes in body temperature. An exception to this adaptation of the serpin mechanism is seen with another family member, angiotensinogen, in which hormone release is modulated by a redox switch and is apparently independent of changes in the serpin framework.

Background: Only 5% of circulating cortisol is active and unbound to carrier proteins. Because cortisol levels vary rapidly due to the pulsatile nature of cortisol secretion, the dynamics of cortisol binding are critical determinants of tissue levels of free cortisol and consequent hormonal signaling. The major glucocorticoid carrier protein is corticosteroid binding globulin (CBG), a member of the serpin family that undergoes conformational changes to bind and release hormones. This mechanism has been noted to be temperature responsive, and we have now investigated the effects of temperature on the binding of human CBG to both cortisol and progesterone.Methods: Recombinant human CBG was synthesized and used for binding studies with cortisol and progesterone between 34 and 43 C. Binding was monitored by recording the change in intrinsic protein fluorescence. Binding of the steroids to the other major carrier, serum albumin, was measured in a similar manner.Results: There was no effect of temperature on the interaction between human serum albumin and either cortisol or progesterone. The association of both cortisol and progesterone with CBG is more than three orders of magnitude greater than that with HSA, and this interaction was extremely responsive to changes in temperature. The affinity of both cortisol and progesterone for CBG drops approximately 16-fold as temperature increases from 35 to 42 C.Conclusions: This study clearly shows that even within the clinically relevant range of temperatures found in humans, CBG acts as a protein thermocouple that is exquisitely sensitive to temperature change and will release cortisol in response to fever or external sources of heat. This has major implications for our understanding of cortisol regulation in febrile patients. (J Clin Endocrinol Metab 95: 4689-4695, 2010)

A 20-year effort has succeeded in identifying the initiating step in the molecular interactions that release the vasopressor peptide angiotensin, and hence control blood pressure. Angiotensins are generated by cleavage of a larger protein, angiotensinogen, by the enzyme renin. Crystal structures of angiotensinogen alone and bound to renin now show that a large conformational change is required to expose the renin cleavage site. This transition is regulated by oxidation, and women with pre-eclampsia — which is associated with high blood pressure — have higher levels of the more active oxidized form. Angiotensins have a crucial role in blood pressure regulation and are generated by cleavage of a larger protein, angiotensinogen, by the enzyme renin. Structures of angiotensinogen alone and in complex with renin show that a large conformational change is required to expose the renin-cleavage site. The authors also show that this transition is regulated by oxidation and that women with pre-eclampsia have higher levels of the more active, oxidized, form. Blood pressure is critically controlled by angiotensins1, 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 inhibitors2,3. 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.

The LKB1 tumor suppressor kinase regulates activity of the AMPK family of kinases.LKB1 is activaty is regulated by the pseudokinase STRADa and the scaffolding protein MO25a through an unknown, phosphorylation-independent, mechanism.Here we describe the 2.65 Å structure of the heterotrimeric LKB1/STRADa/MO25a complex, revealing an unusual allosteric mechanism of LKB1 activation.STRADa adopts a closed conformation typical of active protein kinases, and binds LKB1 as a pseudosubstrate.STRADa binding, promotes the active conformation of LKB1, which is further stabilized by MO25a interacting with the LKB1 activation loop.This represents a previously undescribed mechanism of kinase activation that may be relevant to understanding the evolution of other pseudokinases.The structure also reveals how mutations found in Peutz-Jeghers syndrome and other cancers impair LKB1 function.The body text font is Times New Roman (size 9) with single spacing and full justification.The total allowed area is 8 x 24 cm.

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.