The 1.5 A crystal structure of a prokaryote serpin: controlling conformational change in a heated environment

Serpins utilize conformational change to inhibit target proteinases; the price paid for this conformational flexibility is that many undergo temperature-induced polymerization. Despite this thermolability, serpins are present in the genomes of thermophilic prokaryotes, and here we characterize the first such serpin, thermopin. Thermopin is a proteinase inhibitor and, in comparison with human alpha(1)-antitrypsin, possesses enhanced stability at 60 degrees C. The 1.5 A crystal structure reveals novel structural features in regions implicated in serpin folding and stability. Thermopin possesses a C-terminal "tail" that interacts with the top of the A beta sheet and plays an important role in the folding/unfolding of the molecule. These data provide evidence as to how this unusual serpin has adapted to fold and function in a heated environment.     

Ovalbumin and angiotensinogen lack serpin S-R conformational change.

Cleavage of ovalbumin and angiotensinogen at sites homologous to the reactive centre loop of alpha 1-antitrypsin is not accompanied by the increase in heat-stability associated with the transition from the native stressed (S) structure to a cleaved relaxed (R) form that is typical of other serpins. Failure to undergo the S-R change in ovalbumin is not due to phosphorylation of Ser-344 near the sites of cleavage on the loop. The suggested explanation is the unique presence of bulky side chains at the P10-P12 site in ovalbumin and angiotensinogen.     

Crystal structure of neuroserpin: a neuronal serpin involved in a conformational disease

The protease inhibitor neuroserpin regulates the development of the nervous system and its plasticity in the adult. Neuroserpins carrying the Ser53Pro or Ser56Arg mutation form polymers in neuronal cells. We describe here the structure of wild-type neuroserpin in a cleaved form. The structure provides a basis to understand the role of the mutations in the polymerization process. We propose that these mutations could delay the insertion of the reactive center loop into the central beta-sheet A, an essential step in the inhibition and possibly in the polymerization of neuroserpin.     

Absence of large-scale conformational change upon limited proteolysis of ovalbumin, the prototypic serpin

1H and 31P NMR spectroscopies have been used to examine the effects of limited proteolysis with subtilisin Carlsberg on the global conformation of ovalbumin and on the local environment of phosphoserine 344, a residue two positions removed from the site of proteolysis. Such limited proteolysis has been shown to result in excision of a hexapeptide from the region of the protein that, in other serine protease inhibitors (serpins), contains the reactive center. Based on the structure of the related serpin alpha 1-antitrypsin, it has been predicted that phosphoserine 344 should undergo a large change in environment upon proteolysis of ovalbumin (L?bermann, H., Tokuoka, R., Deisenhofer, J., and Huber, R. (1984) J. Mol. Biol. 177, 531-550). Proteolysis of ovalbumin produces a small upfield shift (0.15 ppm) of the 31P resonance of phosphoserine 344. In addition, the pKa of phosphoserine 344 is raised by 0.1 pH unit. At pH 8.5, phosphoserine 344 in cleaved ovalbumin (plakalbumin) is as accessible to hydrolysis by Escherichia coli alkaline phosphatase as it is in native ovalbumin. 1H NMR shows that dephosphorylation of serine 344 has an imperceptible effect on the protein's conformation. Similarly, little effect on conformation is seen by 1H NMR upon proteolysis of ovalbumin. These findings suggest that ovalbumin does not undergo a marked conformational change analogous to that inferred for the related members of the serpin superfamily, alpha 1-antitrypsin and antithrombin III, nor do the residues close to the site of proteolysis appear to change environment from that of an exposed loop to a buried strand of beta-sheet. These findings are not consistent with the hypothesis of Carrell and Owen ((1985) Nature 317, 730-732) for the role of the exposed loop in serpins of directly facilitating conformational change upon cleavage of the loop. Instead, it is proposed that cleavage of the exposed loop alters the solvent accessibility of residues formerly covered by the loop and that this provides the thermodynamic impetus for conformational change, perhaps by disruption of a salt bridge crucial to the integrity of the native structure.     

A 2.6 A structure of a serpin polymer and implications for conformational disease.

The function of the serpins as proteinase inhibitors depends on their ability to insert the cleaved reactive centre loop as the fourth strand in the main A beta-sheet of the molecule upon proteolytic attack at the reactive centre, P1-P1'. This mechanism is vulnerable to mutations which result in inappropriate intra- or intermolecular loop insertion in the absence of cleavage. Intermolecular loop insertion is known as serpin polymerisation and results in a variety of diseases, most notably liver cirrhosis resulting from mutations of the prototypical serpin alpha1-antitrypsin. We present here the 2.6 A structure of a polymer of alpha1-antitrypsin cleaved six residues N-terminal to the reactive centre, P7-P6 (Phe352-Leu353). After self insertion of P14 to P7, intermolecular linkage is affected by insertion of the P6-P3 residues of one molecule into the partially occupied beta-sheet A of another. This results in an infinite, linear polymer which propagates in the crystal along a 2-fold screw axis. These findings provide a framework for understanding the uncleaved alpha1-antitrypsin polymer and fibrillar and amyloid deposition of proteins seen in other conformational diseases, with the ordered array of polymers in the crystal resulting from slow accretion of the cleaved serpin over the period of a year.     

C1 inhibitor serpin domain structure reveals the likely mechanism of heparin potentiation and conformational disease

C1 inhibitor, a member of the serpin family, is a major down-regulator of inflammatory processes in blood. Genetic deficiency of C1 inhibitor results in hereditary angioedema, a dominantly inheritable, potentially lethal disease. Here we report the first crystal structure of the serpin domain of human C1 inhibitor, representing a previously unreported latent form, which explains functional consequences of several naturally occurring mutations, two of which are discussed in detail. The presented structure displays a novel conformation with a seven-stranded beta-sheet A. The unique conformation of the C-terminal six residues suggests its potential role as a barrier in the active-latent transition. On the basis of surface charge pattern, heparin affinity measurements, and docking of a heparin disaccharide, a heparin binding site is proposed in the contact area of the serpin-proteinase encounter complex. We show how polyanions change the activity of the C1 inhibitor by a novel "sandwich" mechanism, explaining earlier reaction kinetic and mutagenesis studies. These results may help to improve therapeutic C1 inhibitor preparations used in the treatment of hereditary angioedema, organ transplant rejection, and heart attack.     

Serpin crystal structure and serpin polymer structure

Serpins are a family of structurally homologous proteins having metastable native structures. As a result, a serpin variant destabilized by mutation(s) has a tendency to undergo conformational changes leading to inactive forms, e.g., the latent form and polymer. Serpin polymers are involved in a number of conformational diseases. Although several models for polymer structure have been proposed, the actual structure remains unknown. Here, we provide a comprehensive list of serpins, both free and in complexes, deposited in the Protein Data Bank. Our discussion focuses on structures that potentially can contribute to a better understanding of polymer structure.     

The role of protein conformational fluctuations in allostery, function, and evolution

It is now well-known that proteins exist at equilibrium as ensembles of conformational states rather than as unique static structures. Here we review from an ensemble perspective important biological effects of such spontaneous fluctuations on protein allostery, function, and evolution. However, rather than present a thorough literature review on each subject, we focus instead on connecting these phenomena through the ensemble-based experimental, theoretical, and computational investigations from our laboratory over the past decade. Special emphasis is given to insights that run counter to some of the prevailing ideas that have emerged over the past 40 years of structural biology research. For instance, when proteins are viewed as conformational ensembles rather than as single structures, the commonly held notion of an allosteric pathway as an obligate series of individual structural distortions loses its meaning. Instead, allostery can result from energetic linkage between distal sites as one Boltzmann distribution of states transitions to another. Additionally, the emerging principles from this ensemble view of proteins have proven surprisingly useful in describing the role of intrinsic disorder in inter-domain communication, functional adaptation mediated by mutational control of fluctuations, and evolutionary conservation of the energetics of protein stability.     

The conformational dynamics of a metastable serpin studied by hydrogen exchange and mass spectrometry

Serpins are a class of protease inhibitors that initially fold to a metastable structure and subsequently undergo a large conformational change to a stable structure when they inhibit their target proteases. How serpins are able to achieve this remarkable conformational rearrangement is still not understood. To address the question of how the dynamic properties of the metastable form may facilitate the conformational change, hydrogen/deuterium exchange and mass spectrometry were employed to probe the conformational dynamics of the serpin human alpha(1)-antitrypsin (alpha(1)AT). It was found that the F helix, which in the crystal structure appears to physically block the conformational change, is highly dynamic in the metastable form. In particular, the C-terminal half of the F helix appears to spend a substantial fraction of time in a partially unfolded state. In contrast, beta-strands 3A and 5A, which must separate to accommodate insertion of the reactive center loop (RCL), are not conformationally flexible in the metastable state but are rigid and stable. The conformational lability required for loop insertion must therefore be triggered during the inhibition reaction. Beta-strand 1C, which anchors the distal end of the RCL and thus prevents transition to the so-called latent form, is also stable, consistent with the observation that alpha(1)AT does not spontaneously adopt the latent form. A surprising degree of flexibility is seen in beta-strand 6A, and it is speculated that this flexibility may deter the formation of edge-edge polymers.     

SERPINB11 is a new noninhibitory intracellular serpin. Common single nucleotide polymorphisms in the scaffold impair conformational change

SERPINB11, the last of 13 human clade B serpins to be described, gave rise to seven different isoforms. One cDNA contained a premature termination codon, two contained splice variants, and four contained full-length open reading frames punctuated by eight single nucleotide polymorphisms (SNPs). The SNPs encoded amino acid variants located within the serpin scaffold but not the reactive site loop (RSL). Although the mouse orthologue, Serpinb11, could inhibit trypsin-like peptidases, SERPINB11 showed no inhibitory activity. To determine whether the human RSL targeted a different class of peptidases or the serpin scaffold was unable to support inhibitory activity, we synthesized chimeric human and mouse proteins, in which the RSLs had been swapped. The human RSL served as a trypsin inhibitor when supported by mouse scaffold sequences. Conversely, the mouse RSL on the human scaffold showed no inhibitory activity. These findings suggested that variant residues in the SERPINB11 scaffold impaired serpin function. SDS-PAGE analysis supported this notion as RSL-cleaved SERPINB11 was unable to undergo the stressed-to-relaxed transition typical of inhibitory type serpins. Mutagenesis studies supported this hypothesis, since the reversion of amino acid sequences in helices D and I to those conserved in other clade B serpins partially restored the ability of SERPINB11 to form covalent complexes with trypsin. Taken together, these findings suggested that SERPINB11 SNPs encoded amino acids in the scaffold that impaired RSL mobility, and HapMap data showed that the majority of genomes in different human populations harbored these noninhibitory SERPINB11 alleles. Like several other serpin superfamily members, SERPINB11 has lost inhibitory activity and may have evolved a noninhibitory function.     

Regulation of the function of mammalian myosin and its conformational change

It has been known that the phosphorylation of the regulatory light chain, residing at the head/rod junction of the molecule activates the motor activity of smooth muscle and non-muscle conventional myosin (myosin II), and triggers a large conformational change of the molecule from the inhibited folded conformation to the active extended conformation. Recent structural analysis has revealed the structural basis of the inhibition of the motor function of the two heads in the inhibited conformation. On the other hand, recent studies have revealed that a processive unconventional myosin, myosin V, also shows a large change in the conformation from the folded to an extended form and this explains the activation mechanism of myosin V motor activity. These findings suggest the presence of a common scenario for the regulation of motor protein functions.     

Structural bases of the redox-dependent conformational switch in the serpin PAI-2

Depending on the redox-status, the serpin plasminogen activator inhibitor type 2 (PAI-2) can exist in either a stable monomeric or polymerogenic form. The latter form, which spontaneously forms loop-sheet polymers, has an open beta-sheet A and is stabilized by a disulfide bond between C79 (in the CD-loop) and C161 (at the bottom of PAI-2). Reduction of this bond results in a closing of the beta-sheet A and converts PAI-2 to a stable monomeric form. Here we show that the stable monomeric and polymerogenic forms of PAI-2 are fully interconvertible, depending on redox-status of the environment. Our intramolecular distance measurements indicate that the CD-loop folds mainly on one side of the stable monomeric form of the inhibitor. However, the loop can translocate about 54A to the bottom of PAI-2 so that the C79-C161 disulfide bond can form under oxidizing conditions. We show also that the redox-active C79 can form a disulfide-link to the matrix protein vitronectin, suggesting that vitronectin can stabilize active PAI-2 in extracellular compartments. PAI-2 is therefore a rare example of a redox-sensitive protein for which the activity and polymerization ability are regulated by reversible disulfide bond formation leading to major translocation of a loop and significant conformational changes in the molecule.     

Mapping of a conformational epitope on plasminogen activator inhibitor-1 by random mutagenesis. Implications for serpin function

The mechanism for the conversion of plasminogen activator inhibitor-1 (PAI-1) from the active to the latent conformation is not well understood. Recently, a monoclonal antibody, 33B8, was described that rapidly converts PAI-1 to the latent conformation (Verhamme, I., Kvassman, J. O., Day, D., Debrock, S., Vleugels, N., Declerck, P. J., and Shore, J. D. (1999) J. Biol. Chem. 274, 17511-17517). In an attempt to understand this interaction, and more broadly to understand the mechanism of the natural transition of PAI-1 to the latent conformation, we have used random mutagenesis to identify the 33B8 epitope in PAI-1. This site involves at least 8 amino acids scattered over more than two-thirds of the linear sequence that form a compact epitope on the PAI-1 three-dimensional structure. Surface plasmon resonance studies indicate a high affinity interaction between latent PAI-1 and 33B8 that is approximately 100-fold higher than comparable binding to active PAI-1. Structural modeling results together with surface plasmon resonance analysis of parental and site-directed PAI-1 mutants with disrupted 33B8 binding suggest the existence of a specific PAI-1 intermediate structure that is stabilized by 33B8 binding. These analyses strongly suggest that this intermediate form of PAI-1 has a partial insertion of the reactive center loop into beta-sheet A, and together, these data have significant implications for the general serpin mechanism of proteinase inhibition.     

Sulfmyoglobin conformational change: A role in the decrease of oxy-myoglobin functionality

This work is focused at understanding the interaction of H₂S with Myoglobin (Mb), in particular the Sulfmyoglobin (SMb) product, whose physiological role is controversial and not well understood. The scattering curves, Guinier, Kratky, Porod and P(r) plots were analyzed for oxy-Mb and oxy-Hemoglobin I (oxyHbI) in the absence and presence of H₂S, using Small and Wide Angle X-ray Scattering (SAXS/WAXS) technique. Three dimensional models were also generated from the SAXS/WAXS data. The results show that SMb formation, produced by oxyMb and H₂S interaction, induces a change in the protein conformation where its envelope has a very small cleft and the protein is more flexible, less rigid and compact. Based on the direct relationship between Mb's structural conformation and its functionality, we suggest that the conformational change observed upon SMb formation plays a contribution to the protein decrease in O₂ affinity and, therefore, on its functionality.     

The maturation-dependent conformational change of the major capsid protein of bacteriophage T4 involves a substantial change in secondary structure

We have investigated the conformational basis of the expansion transformation that occurs upon maturation of the bacteriophage T4 prohead, by using laser Raman spectroscopy to determine the secondary structure of the major capsid protein in both the precursor and the mature states of the surface lattice. This transformation involves major changes in the physical, chemical, and immunological properties of the capsid and is preceded in vivo by processing of its major protein, gp23 (56 kDa), to gp23* (49 kDa), by proteolysis of its N-terminal gp23-delta domain. The respective secondary structures of gp23 in the unexpanded state, and of gp23* in the expanded state, were determined from the laser Raman spectra of polyheads, tubular polymorphic variants of the capsid. Similar measurements were also made on uncleaved polyheads that had been expanded in vitro and, for reference, on thermally denatured polyheads. We find that, with or without cleavage of gp23, expansion is accompanied by substantial changes in secondary structure, involving a major reduction in alpha-helix content and an increase in beta-sheet. The beta-sheet contents of gp23* or gp23 in the expanded state of the surface lattice, and even of gp23 in the unexpanded state, are sufficient for a domain with the "jellyroll" fold of antiparallel beta-sheets, previously detected in the capsid proteins of other icosahedral viruses.(ABSTRACT TRUNCATED AT 250 WORDS)     

The pH-dependent conformational change of diphtheria toxin

Labeling by a hydrophobic photoactivatable reagent and limited proteolysis have been used to study conformational changes of diphtheria toxin related to its pH-dependent membrane insertion and translocation. TID (3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine) labels diphtheria toxin at pH 5 much more efficiently than at pH 7, both in the presence and absence of lipid vesicles. In the absence of membranes, the extent of labeling is greater and the pH dependence is stronger. As analyzed on sodium dodecyl sulfate-polyacrylamide gels and by high pressure liquid chromatography, both the A- and B-subunits and most of the cyanogen bromide fragments of the toxin are labeled by TID at acid pH. The products of trypsin cleavage of diphtheria toxin at pH 5 are different from those seen at neutral pH. Trypsin-susceptible sites were identified by gel electrophoresis of the trypsin fragments, combined with electrophoresis and high pressure liquid chromatography of CNBr digests of trypsin-treated toxin. At neutral pH, the main sites of digestion are at the junction between the A- and B-fragments and near the NH2 terminus of the A-fragment. At pH 5.2, these sites are less efficiently cut, and new sites appear near the NH2 terminus of the B-fragment, in an amphipathic portion of the sequence. Thus, even in the absence of membranes, acid pH induces a significant conformational change in diphtheria toxin. This change involves burial of some previously accessible sites, exposure of previously inaccessible sites, and the formation of hydrophobic regions over an extensive portion of the polypeptide chain.     

The role of water structure in conformational changes of nucleic acids in ambient and high-pressure conditions.

This review describes and summarizes data on the structure and properties of water under normal conditions, at high salt concentration and under high pressure. We correlate the observed conformational changes in nucleic acids with changes in water structure and activity, and suggest a mechanism of conformational transitions of nucleic acids which accounts for changes in the water structure. From the biophysical, biochemical and crystallographic data we conclude that the Z-DNA form can be induced only at low water activity produced by high salt concentrations or high pressure, and accompanied by the stabilizing conjugative effect of the cytidine O4' electrons of the CG base pairs.     

The secondary structure of calcineurin regulatory region and conformational change induced by calcium/calmodulin binding

The protein serine/threonine phosphatase calcineurin (CN) is activated by calmodulin (CaM) in response to intracellular calcium mobilization. A widely accepted model for CN activation involves displacement of the CN autoinhibitory peptide (CN(467-486)) from the active site upon binding of CaM. However, CN activation requires calcium binding both to the low affinity sites of CNB and to CaM, and previous studies did not dissect the individual contributions of CNB and CaM to displacement of the autoinhibitory peptide from the active site. In this work we have produced separate CN fragments corresponding to the CNA regulatory region (CNRR(381-521), residues 381-521), the CNA catalytic domain truncated at residue 341, and the CNA-CNB heterodimer with CNA truncated at residue 380 immediately after the CNB binding helix. We show that the separately expressed regulatory region retains its ability to inhibit CN phosphatase activity of the truncated CN341 and CN380 and that the inhibition can be reversed by calcium/CaM binding. Tryptophan fluorescence quenching measurements further indicate that the isolated regulatory region inhibits CN activity by occluding the catalytic site and that CaM binding exposes the catalytic site. The results provide new support for a model in which calcium binding to CNB enables CaM binding to the CNA regulatory region, and CaM binding then instructs an activating conformational change of the regulatory region that does not depend further on CNB. Moreover, the secondary structural content of the CNRR(381-521) was tentatively addressed by Fourier transform infrared spectroscopy. The results indicate that the secondary structure of CNRR(381-521) fragment is predominantly random coil, but with significant amount of beta-strand and alpha-helix structures.     

Use of NMR to study serpin function

Two-dimensional heteronuclear [1H,15N] single quantum correlation NMR spectra of serpins show dramatic changes between native and loop-inserted conformations, making them very sensitive reporters of the serpin conformation. Much of the spectral overlap that arises when all amides are 15N labelled can be removed by use of selective labelling of a single type of amino acid, such as alanine. The method allows ready determination of whether loop insertion is present, and to what extent, as well as providing information on motional freedom of components of the complex and of the reactive center loop. With label introduced separately into the proteinase, information can also be obtained on the conformational changes brought about in that moiety by complex formation. In addition, with the use of cryoprobes, high field spectrometers, TROSY-based signal detection and deuteration, samples as small as 1-2mg can easily be examined, making it applicable to a wide range of serpins, including those that can only be expressed in mammalian cells.