alpha1-Proteinase inhibitor forms initial non-covalent and final covalent complexes with elastase analogously to other serpin-proteinase pairs, suggesting a common mechanism of inhibition

Despite several concordant structural studies on the initial non-covalent complex that serpins form with target proteinases, a recent study on the non-covalent complex between the serpin alpha(1)-proteinase inhibitor (alpha(1)PI) and anhydroelastase concluded that translocation of the proteinase precedes cleavage of the reactive center loop and formation of the acyl ester. Because this conclusion is diametrically opposite to those of the other structural studies on serpin-proteinase pairs, we proceeded to examine this specific serpin-proteinase complex by the same successful NMR approach used previously on the alpha(1)PI-Pittsburgh-S195A trypsin pair. Both non-covalent complex with anhydroelastase and covalent complex with active elastase were made with (15)N-alanine-labeled wild-type alpha(1)PI. The heteronuclear single quantum correlation spectroscopy (HSQC) NMR spectrum of the non-covalent complex showed that the entire reactive center loop remained exposed, and the serpin body maintained a conformation indistinguishable from that of native alpha(1)PI, indicating no movement of the proteinase and no insertion of the reactive center loop into beta-sheet A. In contrast, the HSQC NMR spectrum of the covalent complex showed that the reactive center loop had fully inserted into beta-sheet A, indicating that translocation of the proteinase had occurred. These results agree with previous NMR, fluorescence resonance energy transfer, and x-ray crystallographic studies and suggest that a common mechanism is employed in formation of serpin-proteinase complexes. We found that preparations of anhydroelastase that are not appropriately purified contain material that can regenerate active elastase over time. It is likely that the material used by Mellet and Bieth contained such active elastase, resulting in mistaken attribution of the behavior of covalent complex to that of the non-covalent complex.     

Kinetic analysis of binding interaction between the subgroup A Rous sarcoma virus glycoprotein SU and its cognate receptor Tva: calcium is not required for ligand binding

Tva is the receptor for subgroup A Rous sarcoma virus, and it contains a single LDL-A module which is the site of virus interaction. In this study, we expressed the entire extracellular region of Tva (referred to as Ecto-Tva) as a GST fusion protein and characterized its refolding properties. We demonstrated that the correct folding of the Ecto-Tva protein, like that of the Tva LDL-A module, is calcium dependent. We used the IAsys system to measure the kinetics of binding between the surface (SU) subunit of the viral glycoprotein and Tva in real time. We found that the Ecto-Tva protein and the Tva LDL-A module displayed similar affinities for SU, providing direct evidence that the LDL-A module of Tva is the only viral interaction domain of the receptor. Furthermore, misfolded Tva proteins displayed lower binding affinities to SU, largely due to a decrease in their association rates, suggesting that a high association rate between SU and Tva is crucial for efficient virus-host interaction. Furthermore, we found that calcium did not influence the overall binding affinity between Tva and SU. These results indicate that, although calcium is important in facilitating correct folding of the LDL-A module of Tva, it is not essential for ligand binding. Thus, these results may have broad implications for the mechanism of protein folding and ligand recognition of the LDL receptor and other members of the LDL receptor superfamily.     

Solution structure of the viral receptor domain of Tva and its implications in viral entry

Tva is the cellular receptor for subgroup A avian sarcoma and leukosis virus (ASLV-A). The viral receptor function of Tva is determined by a 40-residue, cysteine-rich motif called the LDL-A module. Here we report the solution structure of the LDL-A module of Tva, determined by nuclear magnetic resonance (NMR) spectroscopy. Although the carboxyl terminus of the Tva LDL-A module has a structure similar to those of other reported LDL-A modules, the amino terminus adopts a different conformation. The LDL-A module of Tva does not contain the signature antiparallel beta-sheet observed in other LDL-A modules, and it is more flexible than other reported LDL-A modules. The LDL-A structure of Tva provides mechanistic insights into how a simple viral receptor functions in retrovirus entry. The side chains of H38 and W48 of Tva, which have been identified as viral contact residues by mutational analysis, are solvent exposed, suggesting that they are directly involved in EnvA binding. However, the side chain of L34, another potential viral contact residue identified previously, is buried inside of the module and forms the hydrophobic core with other residues. Thus L34 likely stabilizes the Tva structure but is not a viral interaction determinant. In addition, we propose that the flexible amino-terminal region of Tva plays an important role in determining specificity in the Tva-EnvA interaction.     

Characterization of the LDL-A module mutants of Tva,the subgroup A Rous sarcoma virus receptor,and the implications in protein folding

Tva is the cellular receptor for subgroup A Rous sarcoma virus (RSV-A), and the viral receptor function is solely determined by a 40-residue motif called the LDL-A module of Tva. In this report, an integral approach of molecular, biochemical, and biophysical techniques was used to examine the role of a well-conserved tryptophan of the LDL-A module of Tva in protein folding and ligand binding. We show that substitution of tryptophan by glycine adversely affected the correct folding of the LDL-A module of Tva, with only a portion giving a calcium-binding conformation. Furthermore, we show that the misfolded LDL-A conformations of Tva could not efficiently bind to its ligand. These results indicate that this conserved tryptophan in the LDL-A module of Tva plays an important role in correct protein folding and ligand recognition. Furthermore, these results suggest that the familial hypercholesterolemia (FH) French Canadian-4 mutation is likely caused by protein misfolding of low-density lipoprotein receptor, thus explaining the defect for this class of FH.     

Structural similarity of the covalent complexes formed between the serpin plasminogen activator inhibitor-1 and the arginine-specific proteinases trypsin, LMW u-PA, HMW u-PA, and t-PA: use of site-specific fluorescent probes of local environment

We have used two fluorescent probes, NBD and dansyl, attached site-specifically to the serpin plasminogen activator inhibitor-1 (PAI-1) to address the question of whether a common mechanism of proteinase translocation and full insertion of the reactive center loop is used by PAI-1 when it forms covalent SDS-stable complexes with four arginine-specific proteinases, which differ markedly in size and domain composition. Single-cysteine residues were incorporated at position 119 or 302 as sites for specific reporter labeling. These are positions approximately 30 A apart that allow discrimination between different types of complex structure. Fluorescent derivatives were prepared for each of these variants using both NBD and dansyl as reporters of local perturbations. Spectra of native and cleaved forms also allowed discrimination between direct proteinase-induced changes and effects solely due to conformational change within the serpin. Covalent complexes of these derivatized PAI-1 species were made with the proteinases trypsin, LMW u-PA, HMW u-PA, and t-PA. Whereas only minor perturbations of either NBD and dansyl were found for almost all complexes when label was at position 119, major perturbations in both wavelength maximum (blue shifts) and quantum yield (both increases and decreases) were found for all complexes for both NBD and dansyl at position 302. This is consistent with all four complexes having similar location of the proteinase catalytic domain and hence with all four using the same mechanism of full-loop insertion with consequent distortion of the proteinase wedged in at the bottom of the serpin.     

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.     

The High Affinity Binding Site on Plasminogen Activator Inhibitor-1 (PAI-1) for the Low Density Lipoprotein Receptor-related Protein (LRP1) Is Composed of Four Basic Residues

Plasminogen activator inhibitor 1 (PAI-1) is a serpin inhibitor of the plasminogen activators urokinase-type plasminogen activator (uPA) and tissue plasminogen activator, which binds tightly to the clearance and signaling receptor low density lipoprotein receptor-related protein 1 (LRP1) in both proteinase-complexed and uncomplexed forms. Binding sites for PAI-1 within LRP1 have been localized to CR clusters II and IV. Within cluster II, there is a strong preference for the triple CR domain fragment CR456. Previous mutagenesis studies to identify the binding site on PAI-1 for LRP1 have given conflicting results or implied small binding contributions incompatible with the high affinity PAI-1/LRP1 interaction. Using a highly sensitive solution fluorescence assay, we have examined binding of CR456 to arginine and lysine variants of PAI-1 and definitively identified the binding site as composed of four basic residues, Lys-69, Arg-76, Lys-80, and Lys-88. These are highly conserved among mammalian PAI-1s. Individual mutations result in a 13–800-fold increase in K_d values. We present evidence that binding involves engagement of CR4 by Lys-88, CR5 by Arg-76 and Lys-80, and CR6 by Lys-69, with the strongest interactions to CR5 and CR6. Collectively, the individual binding contributions account quantitatively for the overall PAI-1/LRP1 affinity. We propose that the greater efficiency of PAI-1·uPA complex binding and clearance by LRP1, compared with PAI-1 alone, is due solely to simultaneous binding of the uPA moiety in the complex to its receptor, thereby making binding of the PAI-1 moiety to LRP1 a two-dimensional surface-localized association.     

S-ovalbumin, an ovalbumin conformer with properties analogous to those of loop-inserted serpins.

Most serpins are inhibitors of serine proteinases and are thought to undergo a conformational change upon complex formation with proteinase that involves partial insertion of the reactive center loop into a beta-sheet of the inhibitor. Ovalbumin, although a serpin, is not an inhibitor of serine proteinases. It has been proposed that this deficiency arises from the presence of a charged residue, arginine, at a critical point (P14) in the reactive center region, which prevents loop insertion into the beta-sheet and thereby precludes inhibitory properties. To test whether loop insertion is prevented in ovalbumin we have examined the properties of two forms of ovalbumin: the native protein and S-ovalbumin, a form that forms spontaneously from native ovalbumin and has increased stability. Calorimetric measurements showed that S-ovalbumin was more stable than ovalbumin by about 3 kcal mol-1. CD spectra, which indicated that S-ovalbumin had less alpha-helix than native ovalbumin, and 1H NMR spectra, which indicated very similar overall structures, suggest limited conformational differences between the two forms. From comparison of the susceptibility of the reactive center region of each protein to proteolysis by porcine pancreatic elastase and by subtilisin Carlsberg, we concluded that the limited native-to-S conformational change specifically affected the reactive center region. These data are consistent with a structure for S-ovalbumin in which part of the reactive center loop has inserted into beta-sheet A to give a more stable structure, analogously to other serpins. However, the rate of loop insertion appears to be very much lower than for inhibitory serpins.(ABSTRACT TRUNCATED AT 250 WORDS)     

NMR and ESR studies on human pregnancy zone protein. Comparison with human alpha 2-macroglobulin.

NMR and ESR spectroscopies have been used to examine the plasma protease inhibitor pregnancy zone protein (PZP) and its complex with chymotrypsin. The 1H NMR spectrum of PZP shows relatively few sharp resonances, which, by analogy with human alpha 2-macroglobulin, probably arise from the proteolytically sensitive bait region. Upon reaction with chymotrypsin to form a 1:1 protease.PZP tetramer complex, there is a large increase in the intensity of sharp resonances due to an increase in mobility of these residues. 35Cl NMR has been used to follow binding of zinc and manganese to apo-PZP. Zinc binding causes a linear broadening of the bulk Cl-, consistent with access of Cl- to PZP-bound zinc. Since zinc in the two highest affinity sites in human alpha 2-macroglobulin causes no broadening of Cl-, it is concluded that these zinc sites are absent from PZP. The mobility of chymotrypsin in the PZP.chymotrypsin complex was examined by covalently attaching a nitroxide spin label at the serine residue in the active site of the enzyme and examining the appearance of the ESR spectrum. The chymotrypsin is rigidly held by the PZP to which it is covalently bound. In an analogous experiment performed previously on alpha 2-macroglobulin, chymotrypsin, bound in the presence of methylamine and therefore largely noncovalently bound, was found to be free to rotate inside the cage formed by the protease inhibitor.