Klavs Dolmer

Disulfide bridges in human complement component C3b

The disulfide bridges of human complement component C3b, derived from C3 by removal of the 77‐residue C3a, have been determined. The 10 bridges are Cys537‐Cys794, Cys605‐Cys640, Cys851‐Cys1491, Cys1079‐Cys1169, Cys1336‐Cys1467, Cys1367‐Cys1436, Cys1448‐Cys1489, Cys1496‐Cys1568, Cys1515‐Cys1639, and Cys1615‐Cys1624. Including the 3 bridges in C3a (Cys670‐Cys698, Cys672‐Cys705, and Cys685‐Cys706) previously determined by high‐resolution X‐ray crystallography [Hoppe‐Seylers Z. Physiol. Chem. 361 (1980) 1389‐1399] all disulfide bridges of C3 are localized. C3 and the strongly related C4 and C5 are members of the α2‐macroglobulin superfamily. The predicted bridge patterns of C4 and C5 are discussed and compared with that of α2‐macroglobulin.

How the Serpin α1-Proteinase Inhibitor Folds

Background: Functional serpins uniquely adopt a metastable conformation by an unknown folding pathway. Results: Ability of constituent α1-proteinase inhibitor (α1PI) peptides to associate reveals the order of folding. Conclusion: Metastability results from the inability of sheet A strand 4 to efficiently insert before completion of C-terminal sheet B. Significance: Pathway helps explains the nature of the polymerogenic intermediate of the Z variant of α1PI. Serpins are remarkable and unique proteins in being able to spontaneously fold into a metastable conformation without the aid of a chaperone or prodomain. This metastable conformation is essential for inhibition of proteinases, so that massive serpin conformational change, driven by the favorable energetics of relaxation of the metastable conformation to the more stable one, can kinetically trap the proteinase-serpin acylenzyme intermediate. Failure to direct folding to the metastable conformation would lead to inactive, latent serpin. How serpins fold into such a metastable state is unknown. Using the ability of component peptides from the serpin α1PI to associate, we have now elucidated the pathway by which this serpin efficiently folds into its metastable state. In addition we have established the likely structure of the polymerogenic intermediate of the Z variant of α1PI.

Specificity of Binding of the Low Density Lipoprotein Receptor-related Protein to Different Conformational States of the Clade E Serpins Plasminogen Activator Inhibitor-1 and Proteinase Nexin-1

The low density lipoprotein receptor-related protein (LRP) is the principal clearance receptor for serpins and serpin-proteinase complexes. The ligand binding regions of LRP consist of clusters of cysteine-rich ∼40-residue complement-like repeats (CR), with cluster II being the principal ligand-binding region. To better understand the specificity of binding at different sites within the cluster and the ability of LRP to discriminate in vivo between uncomplexed and proteinase-complexed serpins, we have systematically examined the affinities of plasminogen activator inhibitor-1 (PAI-1) and proteinase nexin-1 (PN-1) in their native, cleaved, and proteinase-complexed states to (CR)2 and (CR)3 fragments of LRP cluster II. A consistent blue shift of the CR domain tryptophan fluorescence suggested a common mode of serpin binding, involving lysines on the serpin engaging the acidic region around the calcium binding site of the CR domain. High affinity binding of non-proteinase-complexed PAI-1 and PN-1 occurred to all fragments containing three CR domains (3–59 nm) and most that contain only two CR domains, although binding energies to different (CR)3 fragments differed by up to 18% for PAI-1 and 9% for PN-1. No detectable difference in affinity was seen between native and cleaved serpin. However, the presence of proteinase in complex with the serpin enhanced affinity modestly and presumably nonspecifically. This may be sufficient to give preferential binding of such complexes in vivo at the relevant physiological concentrations.

Three Complement-like Repeats Compose the Complete α2-Macroglobulin Binding Site in the Second Ligand Binding Cluster of the Low Density Lipoprotein Receptor-related Protein

Given the importance of the low density lipoprotein receptor-related protein (LRP) as an essential endocytosis and signaling receptor for many protein ligands, and of α2-macroglobulin (α2M)-proteinase complexes as one such set of ligands, an understanding of the specificity of their interaction with LRP is an important goal. A starting point is the known role of the 138-residue receptor binding domain (RBD) in binding to LRP. Previous studies have localized high affinity α2M binding to the eight complement repeat (CR)-containing cluster 2 of LRP. In the present study we have identified the minimum CR domains that constitute the full binding site for RBD and, hence, for α2M on LRP. We report on the ability of the triple construct of CR3-4-5 to bind RBD with an affinity (Kd = 130 nm) the same as for isolated RBD to intact LRP. This Kd is 30-fold smaller than for RBD to CR5-6-7, demonstrating the specificity of the interaction with CR3-4-5. Binding requires previously identified critical lysine residues but is almost pH-independent within the range of pH values encountered between extracellular and internal compartments, consistent with an earlier proposed model of intracellular ligand displacement by intramolecular YWTD domains. The present findings suggest a model to explain the ability of LRP to bind a wide range of structurally unrelated ligands in which a nonspecific ligand interaction with the acidic region present in most CR domains is augmented by interactions with other CR surface residues that are unique to a particular CR cluster.

Localisation of the major reactive lysine residue involved in the selfcrosslinking of proteinase-activated Limulus α2-macroglobulin

When α 2‐macroglobulin (α 2M) from the American horseshoe crab, Limulus polyphemus, reacts with proteinases, its thiol esters, like those of other α‐macroglobulins, become activated, leading to the formation of covalently crosslinked species that can be detected as high molecular weight bands in reducing SDS‐PAGE. While other α‐macroglobulins extensively form crosslinks to the reacting proteinase, Limulus α 2M does not. It rather becomes internally crosslinked. It was found from N‐terminal sequence analysis of purified [14C]carboxymethylated peptides from Limulus α 2M‐trypsin complexes that an isopeptide bond formed in approx. 60% yield from the thiol esterified Gln‐1002 specifically to Lys‐254 in the opposing monomer of the disulphide bridged dimer is the main cause of the internal crosslinking.

Crystallisation and preliminary X-ray analysis of the receptor-binding domain of human and bovine α2-macroglobulin

The receptor‐binding domains (RBDs) of human and bovine α 2‐macroglobulin (α 2M) have been isolated after limited proteolysis of methylamine‐treated α 2M with papain. Single crystals of the RBDs have been grown by vapour diffusion. Crystals of human RBD are very thin plates unsuited for data collection. However, crystals of RBD from bovine α 2M give diffraction patterns suitable for X‐ray analysis, and a complete dataset with a maximum resolution of 2.3 Å has been collected with synchrotron radiation at cryogenic temperature. The crystals belong to spacegroup P3121 or P3221 with cell parameters , .