Localization of basic residues required for receptor binding to the single α‐helix of the receptor binding domain of human α<sub>2</sub>‐macroglobulin

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Given the importance of the low density lipoprotein receptorrelated protein (LRP) as an essential endocytosis and signaling receptor for many protein ligands, and of ␣ 2 -macroglobulin (␣ 2 M)-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 ␣ 2 M 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 ␣ 2 M on LRP. We report on the ability of the triple construct of CR3-4-5 to bind RBD with an affinity (K d ‫؍‬ 130 nM) the same as for isolated RBD to intact LRP. This K d 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.The low density lipoprotein receptor-related protein (LRP) 2 is an essential member (1) of the low density lipoprotein (LDL) family of mosaic receptor proteins that are responsible for binding and internalization of a large number of protein ligands (2). Each of these receptors contains one or more clusters of ligand binding domains, known both as complement-like repeats (CR) and LDL receptor type A modules (3). Whereas LDLR contains only one such cluster of 7 CR domains, LRP has four clusters containing 2, 8, 10, and 11 repeats, counting from the N terminus (4 -6). Commonly used nomenclature, thus, describes cluster 2 as being composed of domains CR3 through CR10.Each CR domain contains three conserved disulfide bridges and a functionally required calcium binding site within 40 -42 residues. These domains are linked by variable length, flexible linkers that are likely to afford considerable orientational freedom to each CR domain with respect to others in the cluster (7). Although the cysteines and calcium-coordinating residues are highly conserved, most of the remaining ϳ30 residues are variable (8). This potentially gives LRP a wide variety of ligand recognition sites within its four clusters of 31 CR domains and may in part explain the very much broader range of ligands that can bind and be internalized by LRP compared with the much simpler LDLR. Thus, LRP binds ligands as diverse as ϳ760-kDa ␣ 2 M-protease complexes, various ϳ100-kDa serpin-proteinase complexes s...

Section: Expression Purification and Refolding Of Wild Type And Varmentioning

confidence: 99%

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

Dolmer

Gettins

2006

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“…Expression and Purification of Human RBD-Recombinant human RBD was expressed and purified as described previously (18). Briefly, RBD was expressed in E. coli as a 165 residue fusion protein that, in addition to RBD, contained an N-terminal 6-histidine tag, a thrombin cleavage site, and an extra 10 residues from ␣ 2 M at the N terminus that had been thought to be necessary for stability of RBD (19).…”

Section: Methodsmentioning

confidence: 99%

“…Assignments-The backbone assignments were obtained according to standard procedures and have been published (18). Three-dimensional FIG.…”

mentioning

confidence: 99%

NMR Solution Structure of the Receptor Binding Domain of Human α2-Macroglobulin

Huang

Dolmer

Liao

et al. 2000

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Human ␣ 2 -macroglobulin-proteinase complexes bind to their receptor, the low density lipoprotein receptorrelated protein (LRP), through a discrete 138-residue C-terminal receptor binding domain (RBD), which also binds to the ␤-amyloid peptide. We have used NMR spectroscopy on recombinantly expressed uniformly 13 C/ 15 Nlabeled human RBD to determine its three-dimensional structure in solution. Human RBD is a sandwich of two antiparallel ␤-sheets, one four-strand and one fivestrand, and also contains one ␣-helix of 2.5 turns and an additional 1-turn helical region. The principal ␣-helix contains two lysine residues on the outer face that are known to be essential for receptor binding. A calcium binding site (K d ϳ 11 mM) is present in the loop region at one end of the ␤-sandwich. Calcium binding principally affects this loop region and does not significantly perturb the stable core structure of the domain. The structure and NMR assignments will enable us to examine in solution specific binding of RBD to domains of the receptor and to ␤-amyloid peptide.Human ␣ 2 -macroglobulin (␣ 2 M) 1 is a highly abundant, very high molecular mass (ϳ720 kDa) plasma protein that is best known as a broad spectrum inhibitor of proteinases (1). ␣ 2 M is also reported to bind to certain growth factors (2) and to be involved in binding and clearance of the 42-residue ␤-amyloid peptide (3, 4) that is thought to contribute to the etiology of Alzheimer's disease through formation of fibrils. The essential nature of ␣ 2 M is indicated by the failure to find any individuals with ␣ 2 M deficiency (5). The means of inhibition and clearance of proteinases by ␣ 2 M is through a unique proteinase-induced conformational change in the ␣ 2 M that physically traps the proteinase within a cage-like interior cavity (6, 7). The massive extent of the conformational change and the nature of the sequestration have been likened to the action of a Venus fly trap closing around its prey. Because inhibition by ␣ 2 M involves sequestration of the proteinase from the surrounding milieu rather than direct and specific binding to the proteinase active site, the inhibitor is not limited to inhibition of one type or class of proteinase, but can instead, uniquely, inhibit proteinases of all four mechanistic classes.The trapping conformational change not only results in sequestration of the proteinase, but also exposure of a receptor binding region on ␣ 2 M that is solely responsible for mediating binding to the clearance receptor LRP (low density lipoprotein receptor-related protein) (8). It has been shown that the receptor binding region is located within a discrete domain that comprises the last 138 residues of the ␣ 2 M monomer (9, 10). This domain, termed the receptor binding domain (RBD), can be preparatively isolated from methylamine-transformed human ␣ 2 M by limited digestion with papain (9) or endo-Lys proteinase (10). Cleavage by either proteinase occurs between lysine and glutamate in the highly charged sequence Glu-LysGlu-Glu, which suggests that this ...

“…Reaction with proteases induces a major conformational change in ␣ 2 M so that the protease is physically trapped (2,3). The same conformational change reveals a cryptic recognition site for low density lipoprotein receptor-related protein (LRP-1) (4,5). Because of the function of LRP-1 as an endocytic receptor, ␣ 2 M-protease complexes are rapidly cleared from the bloodstream and probably other sites of generation (6).…”

mentioning

confidence: 99%

Molecular Dissection of the Human α2-Macroglobulin Subunit Reveals Domains with Antagonistic Activities in Cell Signaling

Mantuano

Mukandala

et al. 2008

␣ 2 -Macroglobulin (␣ 2 M) is a plasma protease inhibitor, which reversibly binds growth factors and, in its activated form, binds to low density lipoprotein receptor-related protein (LRP-1), an endocytic receptor with cell signaling activity. Because distinct domains in ␣ 2 M are responsible for its various functions, we hypothesized that the overall effects of ␣ 2 M on cell physiology reflect the integrated activities of multiple domains, some of which may be antagonistic. To test this hypothesis, we expressed the growth factor carrier site and the LRP-1 recognition domain (RBD) as separate GST fusion proteins (FP3 and FP6, respectively). 2 is a 718-kDa homotetrameric glycoprotein, found in the plasma and extracellular spaces, which was first recognized as a broad spectrum protease inhibitor (1). Reaction with proteases induces a major conformational change in ␣ 2 M so that the protease is physically trapped (2, 3). The same conformational change reveals a cryptic recognition site for low density lipoprotein receptor-related protein (LRP-1) (4, 5). Because of the function of LRP-1 as an endocytic receptor, ␣ 2 M-protease complexes are rapidly cleared from the bloodstream and probably other sites of generation (6).In addition to its role as a protease inhibitor, ␣ 2 M is an important carrier of specific growth factors, including transforming growth factor-␤ (TGF-␤), platelet-derived growth factor-BB (PDGF-BB), nerve growth factor-␤ (NGF-␤), and neurotrophin-4 (7, 8). ␣ 2 M-carrier interactions are principally reversible in nature (7). As a result, ␣ 2 M may inhibit growth factor activity (9, 10) or stabilize the growth factor for potential delivery to cell signaling receptors (11). There also is evidence that ␣ 2 M, which is "activated" by reaction with proteases, initiates cell signaling by binding to LRP-1 (12-15) or other receptors, such as glucose-regulated protein-78 (Grp 78) (16,17). However, in studies with intact ␣ 2 M, the possibility that cell signaling results from growth factors that are carried by ␣ 2 M must be considered (11,18).A structural model of ␣ 2 M has been developed, based on the crystal structure of complement component C3, which is homologous to ␣ 2 M (19). This model describes ␣ 2 M as a modular structure, consisting of multiple independently folded domains. To localize ␣ 2 M domains that are responsible for various activities, our laboratory generated a library of glutathione S-transferase (GST) fusion proteins, containing overlapping segments of the ␣ 2 M subunit (20 -22). Fusion protein-3 (FP3) includes residues 591-774 and contains the sequence in ␣ 2 M responsible for binding growth factors. FP6 contains amino acids 1242-1451 and the LRP-1-binding site, in which a single ␣-helix that includes Lys 1370 and Lys 1374 plays a central role (23,24). Assignment of ␣ 2 M activities to specific fusion proteins, such as FP3 and FP6, has been validated by mutagenesis of full-length recombinant ␣ 2 M (25, 26). Thus, the ␣ 2 M fusion proteins provide an opportunity to assess activities assigne...