Conformational states of a bacterial α2-macroglobulin resemble those of human complement C3.

α(2) macroglobulins (α(2)Ms) are broad-spectrum protease inhibitors that play essential roles in the innate immune system of eukaryotic species. These large, multi-domain proteins are characterized by a broad-spectrum bait region and an internal thioester, which, upon cleavage, becomes covalently associated to the target protease, allowing its entrapment by a large conformational modification. Notably, α(2)Ms are part of a larger protein superfamily that includes proteins of the complement system, such as C3, a multi-domain macromolecule which is also characterized by an internal thioester-carrying domain and whose activation represents the pivotal step in the complement cascade. Recently, α(2)M/C3-like genes were identified in a large number of bacterial genomes, and the Escherichia coli α(2)M homolog (ECAM) was shown to be activated by proteases. In this work, we have structurally characterized ECAM by electron microscopy and small angle scattering (SAXS) techniques. ECAM is an elongated, flexible molecule with overall similarities to C3 in its inactive form; activation by methylamine, chymotrypsin, or elastase induces a conformational modification reminiscent of the one undergone by the transformation of C3 into its active form, C3b. In addition, the proposed C-terminus of ECAM displays high flexibility and different conformations, and could be the recognition site for partner macromolecules. This work sheds light on a potential bacterial defense mechanism that mimics structural rearrangements essential for activation of the complement cascade in eukaryotes, and represents a possible novel target for the development of antibacterials.

Glycosylation patterns of human alpha2-macroglobulin: analysis of lectin binding by electron microscopy.

Human alpha2-macroglobulin (alpha 2M) is a 720 kDa glycoprotein that presents two ultrastructural conformations: slow (S-alpha 2M) and fast (F-alpha 2M). alpha 2M acts mainly as a proteinase scavenger, but an immunomodulatory role was also proposed. This work studies the effect of desialylation and deglycosylation on the structure patterns of alpha 2M by ultrastructural analysis of lectin-induced aggregates, which represents a new approach that had never been previously used. Transmission electron microscopy (TEM) analysis showed the loss of S-alpha 2M conformation after deglycosylation, indicating that glycosidic side-chains contribute to the molecular stability of S-alpha 2M. TEM proved to be an important tool to analyze the effect of biochemical changes on alpha 2M, yielding an objective qualitative control of its morphological state. Certain carbohydrate residues did not vary between the alpha 2M conformations, since both bound similarly ConA and WGA lectins. However, the binding of PNA and BSI-B(4) was slightly lower in F-alpha 2M than in S-alpha 2M. Among the neuraminidases used to desialylate both conformations of alpha 2M that from Arthrobacter ureafaciens was the most effective. Incubation with the lectins ConA or SNA, respectively specific for mannosyl and sialyl residues, led to dose-dependent patterns of aggregation of alpha 2M molecules, mediated by lectin binding and clearly visualized by TEM.

The three-dimensional structure of the human alpha 2-macroglobulin dimer reveals its structural organization in the tetrameric native and chymotrypsin alpha 2-macroglobulin complexes.

Three-dimensional electron microscopy reconstructions of the human alpha(2)-macroglobulin (alpha(2)M) dimer and chymotrypsin-transformed alpha(2)M reveal the structural arrangement of the two dimers that comprise native and proteinase-transformed molecules. They consist of two side-by-side extended strands that have a clockwise and counterclockwise twist about their major axes in the native and transformed structures, respectively. This and other studies show that there are major contacts between the two strands at both ends of the molecule that evidently sequester the receptor binding domains. Upon proteinase cleavage of the bait domains and subsequent thiol ester cleavages, which occur near the central region of the molecule, the two strands separate by 40 A at both ends of the structure to expose the receptor binding domains and form the arm-like extensions of the transformed alpha(2)M. During the transformation of the structure, the strands untwist to expose the alpha(2)M central cavity to the proteinase. This extraordinary change in the architecture of alpha(2)M functions to completely engulf two molecules of chymotrypsin within its central cavity and to irreversibly encapsulate them.

The structure of the C949S mutant human alpha(2)-macroglobulin demonstrates the critical role of the internal thiol esters in its proteinase-entrapping structural transformation.

A three-dimensional reconstruction of a protein-engineered mutant alpha(2)-macroglobulin (alpha(2)M) in which a serine residue was substituted for the cysteine 949 (C949S), making it unable to form internal thiol ester moieties, was compared with native and methylamine-transformed alpha(2)Ms. The native alpha(2)M structure consists of two oppositely oriented Z-shaped strands. Thiol ester cleavage following an encounter with a proteinase or a nucleophilic attack by methylamine causes a structural transformation in which the strands assume an opposite handedness and a significant portion of the protein density migrates from the distal ends of the molecule toward the center. The C949S mutant showed a protein density distribution very similar to that of transformed alpha(2)M, with a compact central region of protein density connected to two receptor-binding arms on each end of the molecule. Since no particle shapes characteristic of native or half-transformed alpha(2)Ms were seen in electron micrographs and the C949S mutant and alpha(2)M-methylamine structures are highly similar, we conclude that the intact thiol esters maintain native alpha(2)M in a quasi-stable state. In their absence, alpha(2)M folds into the more stable transformed structure, which displays the functionally important receptor-binding domains and contains the proteinase-entrapping internal cavity.

Structural details of proteinase entrapment by human alpha2-macroglobulin emerge from three-dimensional reconstructions of Fab labeled native, half-transformed, and transformed molecules.

Three-dimensional electron microscopy reconstructions of native, half-transformed, and transformed alpha2-macroglobulins (alpha2Ms) labeled with a monoclonal Fab Fab offer new insight into the mechanism of its proteinase entrapment. Each alpha2M binds four Fabs, two at either end of its dimeric protomers approximately 145 A apart. In the native structure, the epitopes are near the base of its two chisel-like features, laterally separated by 120 A, whereas in the methylamine-transformed alpha2M, the epitopes are at the base of its four arms, laterally separated by 160 A. Upon thiol ester cleavage, the chisels on the native alpha2M appear to split with a separation and rotation to give the four arm-like extensions on transformed alpha2M. Thus, the receptor binding domains previously enclosed within the chisels are exposed. The labeled structures further indicate that the two protomeric strands that constitute the native and transformed molecules are related and reside one on each side of the major axes of these structures. The half-transformed structure shows that the two Fabs at one end of the molecule have an arrangement similar to those on the native alpha2M, whereas on its transformed end, they have rotated. The rotation is associated with a partial untwisting of the strands and an enlargement of the openings to the cavity. We propose that the enlarged openings permit the entrance of the proteinase. Then cleavage of the remaining bait domains by a second proteinase occurs with its entrance into the cavity. This is followed by a retwisting of the strands to encapsulate the proteinases and expose the receptor binding domains associated with the transformed alpha2M.

Three-dimensional structure of the human plasmin alpha2-macroglobulin complex.

The three-dimensional reconstructions of the human plasmin alpha2-macroglobulin binary complex were computed from electron microscopy images of stain and frozen-hydrated specimens. The structures show excellent agreement and reveal a molecule with approximate dimensions of 170 (length) x 140 (width) x 140 A (depth). The asymmetric plasmin structure imparts significant asymmetry to the plasmin alpha2-macroglobulin complex not seen in the structures resulting from the reaction of alpha2-macroglobulin with methylamine or chymotrypsin. The structure shows, when combined with other studies, that the C-terminal catalytic domain of the rod-shaped plasmin molecule is entrapped inside of the alpha2-macroglobulin cavity, whereas its N-terminal kringle domains protrude outside one end between the two arm-like features of the transformed alpha2-macroglobulin structure. This arrangement ensures that the catalytic site of plasmin is prevented from degrading plasma proteins. The internalized C-terminal portion of the plasmin structure resides primarily on the major axis of alpha2-macroglobulin, suggesting that after the initial cleavage of the two bait domains and the thiol esters, the rod-shaped plasmin molecule enters the alpha2-macroglobulin cavity through the large openings afforded by the half-transformed structure. This mode of entrapment requires the untwisting and the separation of the two strands that constitute the alpha2-macroglobulin structure.

On the structural changes of native human alpha2-macroglobulin upon proteinase entrapment. Three-dimensional structure of the half-transformed molecule.

The reconstructions of an intermediate form of human alpha2-macroglobulin (half-transformed alpha2M) in which two of its four bait regions and thiol ester sites were cleaved by chymotrypsin bound to Sepharose were obtained by three-dimensional electron microscopy from stain and frozen-hydrated specimens. The structures show excellent agreement and reveal a structure with approximate dimensions of 195 (length) x 135 (width) and 130 A (depth) with an internal funnel-shaped cavity. The structure shows that a chisel-shaped body is connected to a broad base at the opposing end by four stands. Four approximately 45 A diameter large openings in the body of the structure result in a central cavity that is more accessible to the proteinase than those associated with the native or fully transformed structures. The dissimilarity in the shapes between the two ends of alpha2M half-transformed and the similarity between its chisel-shaped body and that of native alpha2M indicate that the chymotrypsin has cleaved both bait regions in the bottom-half of the structure. Consequently, its functional division lies on the minor axis. The structural organization is in accord with biochemical studies, which show that the half-transformed alpha2M migrates on native polyacrylamide gels at a rate intermediate to the native and fully transformed alpha2M and is capable of trapping 1 mol of proteinase. Even though its upper portion is similar to the native molecule, significant differences in their shapes are apparent and these differences may be related to its slower reaction with a proteinase than the native structure. These structural comparisons further support the view that the transformation of alpha2M involves an untwisting of its strands with an opening of the cavity for entrance of the proteinase and a retwisting of the strands around the proteinase resulting in its encapsulation.

Similar architectures of native and transformed human alpha2-macroglobulin suggest the transformation mechanism.

The refined three-dimensional structure of native human alpha2-macroglobulin (alpha2M) has been determined by cryoelectron microscopy and three-dimensional reconstruction. New features corresponding to "sigmoid arches," "basal bodies," and "apical connections" were observed in the molecule. Since similar elements are found in the architecture of transformed alpha2M, the 2 volumes were aligned in three dimensions. In their common orientations, they show many similarities except near the openings of the central chamber. In the native conformation, these apertures are fully opened, allowing the proteases to access the central chamber of the molecule, while in the transformed structure, they are partially closed. These structures suggest that alpha2M conformational change involves a strong lateral compression and a vertical stretching of the native particle seen in its four-petaled flower view to produce the H view of the transformed form. A model of structural transformation, in which all the parts of the alpha2M molecule seem involved in the entrapment of the proteinases is proposed.

Imaging biological structures with the cryo atomic force microscope.

It has long been recognized that one of the major limitations in biological atomic force microscopy (AFM) is the softness of most biological samples, which are easily deformed or damaged by the AFM tip, because of the high pressure in the contact area, especially from the very sharp tips required for high resolution. Another is the molecular motion present at room temperature due to thermal fluctuation. Using an AFM operated in liquid nitrogen vapor (cryo-AFM), we demonstrate that cryo-AFM can be applied to a large variety of biological samples, from immunoglobulins to DNA to cell surfaces. The resolution achieved with cryo-AFM is much improved when compared with AFM at room temperature with similar specimens, and is comparable to that of cryo-electron microscopy on randomly oriented macromolecules. We will also discuss the technical problems that remain to be solved for achieving even higher resolution with cryo-AFM and other possible applications of this novel technique.

The novel three-dimensional structure of native human alpha 2-macroglobulin and comparisons with the structure of the methylamine derivative.

A three-dimensional reconstruction of alpha 2-macroglobulin (alpha 2 M) was computed from stain images. The structure appears to have point group symmetry 222 and, as also revealed by a tilt experiment, has the gross shape of a oval that displays a approximately 90 degrees twist in the body of the molecule. The reconstruction reveals a novel structure that consists of two Z-shaped components arranged in opposite orientation. These shapes are interconnected by two bridges at the elbow bends of the Z and by two archlike features that join their ends. The molecule has dimensions of approximately 190 x 125 x 120 A that encloses a 90 degrees twisted ellipsoidal shaped central cavity of 70 x 35 A. The cavity has four small openings arranged in a staggered configuration that extend to the outside. Serial slices of alpha 2 M and alpha 2 M-methylamine show that the bodies of the structures appear to be twisted in the opposite orientation. It is proposed that the four thioester bonds in the native molecule are responsible for maintaining its twisted configuration and that their cleavage with methylamine results in the structure becoming twisted in the opposite orientation. A comparison of average images derived from unstained particles of monoclonal Fab-labeled alpha 2 M and alpha 2 M-methylamine is consistent with this proposal. This unusual change in the handedness of alpha 2 M may have an important role in the encapsulation of the proteinase.

Differences in the binding of transforming growth factor beta 1 to the acute-phase reactant and constitutively synthesized alpha-macroglobulins of rat.

Human alpha 2-macroglobulin (alpha 2M) is a proteinase inhibitor and carrier of certain growth factors, including transforming growth factor beta 1 (TGF-beta 1). The constitutively synthesized homologue of human alpha 2M in the adult rat is alpha 1M. Rat alpha 2M is an acute-phase reactant, expressed at high levels in experimental trauma, pregnancy and in certain pathological conditions. The physiological role of rat alpha 2M is not known. In this investigation, we demonstrated that rat alpha 1M and rat alpha 2M bind TGF-beta 1. The equilibrium dissociation constants (KD) for the binding of TGF-beta 1 to the native forms of alpha 1M and alpha 2M were 257 and 109 nM respectively. alpha 1M underwent conformational change when it reacted with methylamine. The resulting product bound TGF-beta 1 with higher affinity (32 nM). Methylamine-treated rat alpha 2M did not undergo conformational change and did not bind TGF-beta 1 with increased affinity. Previous studies suggest that the native conformation may be the principal form responsible for the cytokine-carrier activity of alpha 2M in plasma and serum-supplemented cell culture medium. To confirm that native rat alpha 2M is a more efficient TGF-beta 1 carrier than native alpha 1M, fetal bovine heart endothelial cell (FBHE) proliferation assays were performed. TGF-beta 1 (5 pM) inhibited FBHE proliferation, and native alpha 2M (0.3 microM) counteracted this activity whereas alpha 1M (0.3 microM) had almost no effect. Rat alpha 2M underwent conformational change when it reacted with plasmin incorporating 1.1 mol of plasmin/mol. alpha 2M-plasmin bound TGF-beta 1; the KD (61 nM) was lower (P < 0.01) than that determined for the native alpha 2M-TGF-beta 1 interaction. These studies demonstrate that both rat alpha-macroglobulins are carriers of TGF-beta 1. The native form of rat alpha 2M probably has a predominant role, compared with native alpha 1M, as a TGF-beta 1 carrier in the plasma during the acute-phase response.

Architecture of native human alpha 2-macroglobulin studied by cryoelectron microscopy and three-dimensional reconstruction.

The architecture of the native human alpha 2-macroglobulin was studied by cryoelectron microscopy and image processing techniques. The lip, padlock, doughnut, and four-petaled flower views of this homotetrameric proteinase inhibitor were observed in the frozen-hydrated specimen, and a new view, termed eye view, was also characterized. The present three-dimensional reconstruction demonstrates that all these electron microscope views derive from a single three-dimensional structure. The molecule is composed of two horizontal bodies and of two oblique arches, which border a large central cavity. The polymorphism and the flexibility of the native alpha 2-macroglobulin are discussed.

An approach to the intramolecular localization of the thiol ester bonds in the internal cavity of human alpha 2-macroglobulin based on correspondence analysis.

The plasma proteinase inhibitor alpha 2-macroglobulin (alpha 2M) can trap small proteins including cytokines. With the four internal thiol ester bonds being involved in the covalent binding of proteinases and other ligands, it was of interest to precisely localize these active groups in the alpha 2M molecule. This was approached by comparing methylamine-transformed human alpha 2M (alpha 2M-MA) and alpha 2M-MA chemically coupled to cytochrome c (Cyt c). This enzyme was chosen because of its size, which is close to that of cytokines, and because of the easy quantification of the reaction stoichiometry. The two molecular species were first mixed in a single sample that was deposited on a carbon-coated grid, negatively stained, and imaged in the electron microscope. The aligned images of the two macromolecular species were then separated by correspondence analysis and hierarchical ascendant classification and compared through the calculation of a subtraction image. The interest of this approach is that prior to the separation of the images, the two molecular species are subjected to rigorously identical treatments. The subtraction images between the average images of alpha 2M-MA and Cyt c-alpha 2M-MA allowed an unambiguous localization of the Cyt c molecules in the internal cavity of the alpha 2M molecule. The internal cavity is gradually filled when the Cyt c/alpha 2M ratio increases. However, it is not yet clear whether the thiol groups are located in the median portion of the wall or in the interwall (paddle) structure.

Three-dimensional architecture of human alpha 2-macroglobulin transformed with methylamine.

A frozen-hydrated sample embedded in vitreous ice of human alpha 2-macroglobulin transformed by methylamine was imaged by cryoelectron microscopy and reconstructed in three dimensions. In the reconstruction, the cage-like architecture of this protease inhibitor is fully revealed with a clear visualization of two lozenge-shaped lateral walls connected by thin bridges. The shape and dimensions of the internal cavity normally containing the trapped protease(s) is described. The possible locations of the thiol ester sites and inter-subunit connections are also discussed.

Entrapment and inhibition of human immunodeficiency virus proteinase by alpha 2-macroglobulin and structural changes in the inhibitor.

The inhibitory effect of alpha 2-macroglobulin (alpha 2M), a major plasma proteinase inhibitor, on human immunodeficiency virus (HIV) proteinase was investigated. The activity of HIV proteinase toward the Moloney murine sarcoma virus-derived gag protein (a high-molecular-mass substrate) was found to be inhibited by alpha 2M at pH 5.5-7.4. On the other hand, the activity toward the B chain of oxidized insulin (a low-molecular-mass substrate) was scarcely inhibited. The complex of alpha 2M and HIV proteinase was isolated by gel filtration and the enzyme was shown to be significantly protected by the complex formation from autoinactivation under nonreducing conditions. The stoichiometry of the complex formation was found to be 2:1 (enzyme: alpha 2M, mol/mol). These results demonstrate the entrapment and concomitant inhibition of HIV proteinase by alpha 2M.