Partial amino acid sequences of rabbit C-reactive protein, a peptide derived from human C-reactive protein by cyanogen bromide cleavage, and the Cit subcomponent of the human complement component CI have been determined. Extensive sequence homology between these proteins establish their evolutionary relationships. In addition, examination of C-reactive proteins by negative-stain electron microscopy revealed that the protein is composed of five subunits arranged in cyclic symmetry. This structure is similar to that reported for both Cit and the amyloid P-component. The extensive structural relationship suggests similar or overlapping functions and the term pentraxin is proposed to describe these homologous proteins.Activation of the primary complement (C) pathway is one of the principle biological activities of immunoglobulins. The C pathway sequence is initiated via an interaction between the Fc region of certain immunoglobulins and the C1 macromolecular complex, which leads to an internal activation of C1 and the generation of its proteolytic activity (1). During recent years several alternative mechanisms for the activation of the C system have been described. Most of these activators operate through one or another of the complex maze of interactions known as the properdin or alternative pathway (2). However, one of these agents, C-reactive protein (CRP), would appear to act in a manner entirely analogous to immunoglobulin. It has been found that upon interaction with a variety of substances, CRP can activate the C cascade and initiate both the opsonic and lytic potentials of this system through an activation of the primary C pathway (3-6). CRP has been described as being comprised of probably identical subunits, ca 23,000 daltons, noncovalently linked to form an oligomer of molecular weight 120,000-140,000 (7,8 During preliminary presentations (15, 16) of some of the data described here, an extensive amino acid-sequence homology (ca 50%) between CRP and Cit was noted. Although there are no a priori reasons to preclude such an extensive homology between an activator of C1 and one of its subcomponents, this was unexpected. We report here partial amino acid sequences of human and rabbit CRP and human Cit that establish the evolutionary relatedness of these proteins. In addition, we present a detailed ultrastructural analysis of both human and rabbit CRP by negative stain electron microscopy that shows it to be assembled as a cyclic pentamer, in a manner similar to both Cit and amyloid P-component. The term pentraxin (Greek. revrE, five and 'pat, berry) is proposed to describe these related proteins.MATERIALS AND METHODS C-Reactive Proteins. Human CRP was isolated by affinity chromatography of human serous fluids on agarose beads (Bio-Gel A-15m) to which pneumococcal C-polysaccharide was covalently linked (5). Subunits and aggregates of CRP were removed from the native protein by gel filtration in Trisbuffered saline on Bio-Gel A-0.5m. The purity of such preparations of CRP has been previously established...

BiochemistryConformational flexibility in the active sites of aspartyl proteinases revealed by a pepstatin fragment binding to penicillopepsin ( Communicated by Joseph S. Fruton, June 21, 1982 ABSTRACT Crystals of the molecular complex between the esterified tripeptide fragment of pepstatin and the aspartyl proteinase penicillopepsin are isomorphous with crystals of native penicillopepsin. The difference electron-density map at 1.8-A resolution, computed by using the amplitude differences and refined phases of reflections from the crystal of native penicillopepsin, unambiguously showed the binding mode of isovaleryl Pepstatin is a naturally occurring inhibitor of aspartyl proteinases (1); it has the amino acid composition isovaleryl (Iva)-ValVal-Sta-Ala-StaOH, in which Sta is the residue of statine [(4S,3S)-4-amino-3-hydroxyl-6-methylheptanoic acid] (2). The interaction ofpepstatin with aspartyl proteases is characterized by small dissociation constants [i.e., 4.6 x 10-11 M with porcine pepsin (3) and 1.5 x 10-10 M with penicillopepsin (unpublished results)]. It Structure I Penicillopepsin is an aspartyl proteinase isolated from the mold Penicillium janthinellum. Its crystal structure (6, 7) has been refined at 1.8-A resolution (8) to a conventional R-factor § of 0.136 for those reflections with I, the intensity of Bragg reflections, equal to or greater than the estimated standard deviation of the intensity measurement derived from counting statistics [I 2 o-(I)]. The aspartyl proteinases are characterized by a long binding cleft that can accommodate 7 or 8 amino acid residues of an oligopeptide substrate in an extended conformation. The two catalytically important aspartyl residues, Asp&jand Asp-213, are centrally located in this binding cleft (Asp32and Asp-215 in the porcine pepsin sequence numbering).Fluorescence measurements on the aspartyl proteinases with specific substrates have indicated that conformational mobility of groups in the active site may play an important role in the mechanism of these enzymes (9). MATERIALS AND METHODSThe enzyme, penicillopepsin, was prepared, purified, and crystallized as described (6,7,10). Crystals of the native enzyme The pepstatin derivative Iva-Val-Val-StaOEt was synthesized by the following procedure. t-Butoxycarbonyl (Boc)-StaOEt (12) was deprotected with 4 M HCl in dioxane and sequentially coupled with Boc-L-valine, Boc-L-valine, and isovaleryl anhydride by using procedures previously described for-the synthesis of pepstatin analogues (13) 6137The publication costs ofthis article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.

The polypeptide chain of the acid protease penicillo pepsin folds via an 18-stranded mixed beta-sheet into two distinct lobes separated by a 30-A long groove which is the extended substrate binding site. The catalytic residues Asp-32 and Asp-215 are located in this groove and their carboxyl groups are in intimate contact. Alignment of the amino acid sequence with that of pepsin shows regions of high homology.

Comparison of the three-dimensional structures of native endothiapepsin (EC 3.4.23.6) and 15 endothiapepsin oligopeptide inhibitor complexes defined at high resolution by X-ray crystallography shows that endothiapepsin exists in two forms differing in the relative orientation of a domain comprising residues 190-302. There are relatively few interactions between the two parts of the enzyme; consequently, they can move as separate rigid bodies. A translational, librational, and screw analysis of the thermal parameters of endothiapepsin also supports a model in which the two parts can move relative to each other. In the comparison of different aspartic proteinases, the rms values are reduced by up to 47% when the two parts of the structure are superposed independently. This justifies description of the differences, including those between pepsinogen and pepsin (EC 3.4.34.1), as a rigid movement of one part relative to another although considerable distortions within the domains also occur. The consequence of the rigid body movement is a change in the shape of the active site cleft that is largest around the S3 pocket. This is associated with a different position and conformation of the inhibitors that are bound to the two endothiapepsin forms. The relevance of these observations to a model of the hydrolysis by aspartic proteinases is briefly discussed.