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. 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. The low density lipoprotein receptor-related protein (LRP) 2The abbreviations used are: LRP, low density lipoprotein receptor-related protein; α2M, α2-macroglobulin; CR, complement-like repeat; GSH, reduced glutathione; GSSG, oxidized glutathione; LDL, low density lipoprotein; LDLR, LDL receptor; RAP, receptor-associated protein; RBD, receptor binding domain; TEV, tobacco etch virus NIa protease; GdnHCl, guanidine HCl; Ni-NTA, nickel-nitrilotriaceticacid;β-ME,β-mercaptoethanol;HPLC, high performance liquid chromatography; MES, 4-morpholineethanesulfonic acid. is an essential member (1Herz J. Clouthier D.E. Hammer R.E. Cell. 1992; 71: 411-421Abstract Full Text PDF PubMed Scopus (511) Google Scholar) 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 (2Herz J. Strickland D.K. J. Clin. Investig. 2001; 108: 779-784Crossref PubMed Scopus (897) Google Scholar). 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 (3Catterall C.F. Lyons A. Sim R.B. Day A.J. Harris T.J. Biochem. J. 1987; 242: 849-856Crossref PubMed Scopus (84) Google Scholar). 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 (4Herz J. Hamann U. Rogne S. Myklebost O. Gausepohl H. Stanley K.K. EMBO J. 1988; 7: 4119-4127Crossref PubMed Scopus (742) Google Scholar, 5Kristensen T. Moestrup S.K. Gliemann J. Bendtsen L. Sand O. Sottrup-Jensen L. FEBS Lett. 1990; 276: 151-155Crossref PubMed Scopus (256) Google Scholar, 6Strickland D.K. Ashcom J.D. Williams S. Burgess W.H. Migliorini M. Argraves W.S. J. Biol. Chem. 1990; 265: 17401-17404Abstract Full Text PDF PubMed Google Scholar). 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 (7Beglova N. North C.L. Blacklow S.C. Biochemistry. 2001; 40: 2808-2815Crossref PubMed Scopus (42) Google Scholar). Although the cysteines and calcium-coordinating residues are highly conserved, most of the remaining ∼30 residues are variable (8Simonovic M. Dolmer K. Huang W. Strickland D.K. Volz K. Gettins P.G.W. Biochemistry. 2001; 40: 15127-15134Crossref PubMed Scopus (54) Google Scholar). 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 ∼760-kDa α2M-protease complexes, various ∼100-kDa serpin-proteinase complexes such as plasminogen activator inhibitor 1-urokinase-like plasminogen activator (uPA), thrombin-antithrombin, and protease nexin-1-uPA, apolipoprotein E, and lipoprotein lipase, amyloid precursor protein, complement C3, and lactoferrin (2Herz J. Strickland D.K. J. Clin. Investig. 2001; 108: 779-784Crossref PubMed Scopus (897) Google Scholar). One of the best studied LRP ligands is α2M. α2Misan abundant, homo-tetrameric pan-proteinase inhibitor (9Sottrup-Jensen L. J. Biol. Chem. 1989; 264: 11539-11542Abstract Full Text PDF PubMed Google Scholar) that is also reported to bind various growth factors (10Crookston K.P. Webb D.J. LaMarre J. Gonias S.L. Biochem. J. 1993; 293: 443-450Crossref PubMed Scopus (67) Google Scholar) and that is evolutionarily related to the complement proteins C3, C4, and C5 (11Sottrup-Jensen L. Stepanik T.M. Kristensen T. Lqnblad P.B. Jones C.M. Wierzbicki D.M. Magnusson S. Domdey P.B. Wetsel R.A. Lundwall Å. Tack B.F. Fey G.H. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 9-13Crossref PubMed Scopus (138) Google Scholar). Binding of α2M to LRP requires the exposure of a previously hidden receptor binding domain (RBD) that the residues of each α2M of RBD from the that with LRP thus, between and α2M of α2M can only in internalization and of the but also in intracellular signaling ligand an important one to study J. M. K. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). is that the RBD of α2M, is known and of a by a W. Dolmer K. Gettins P.G.W. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google is responsible for binding of complexes to LRP T. Moestrup S.K. Gliemann J. Bendtsen L. Sand O. Sottrup-Jensen L. FEBS Lett. 1990; 276: 151-155Crossref PubMed Scopus (256) Google Scholar, 6Strickland D.K. Ashcom J.D. Williams S. Burgess W.H. Migliorini M. Argraves W.S. J. Biol. Chem. 1990; 265: 17401-17404Abstract Full Text PDF PubMed Google Scholar, H. Biochem. J. PubMed Scopus Google Scholar, L. Gliemann J. FEBS Lett. PubMed Scopus Google Scholar). intact α2M can bind to LRP with an affinity that high LRP isolated RBD binds with a Kd of L. Gliemann J. FEBS Lett. PubMed Scopus Google Scholar, Biochemistry. 1989; PubMed Scopus Google Scholar). The is to be to the of α2M interaction with or more LRP LRP is present high density or to an interaction with more than one cluster of CR domains within a LRP Thus, has reported that α2M can bind to both cluster 2 and cluster each cluster is as a A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). Given the importance both of LRP as an essential endocytosis and signaling receptor and of complexes as an LRP an understanding of the specificity of their interaction is a very goal. starting point is the known importance of RBD in the of lysine residues within RBD that are required for binding to LRP M. Moestrup S.K. Gliemann J. Sottrup-Jensen L. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google the of high affinity α2M binding to CR cluster 2 of LRP A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google and earlier that RBD can bind to the CR domain of cluster 2, with affinity K. Huang W. Gettins P.G.W. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). In the present study we have on and to the minimum CR domains that with constitute the full binding site for RBD and, hence, α2M on LRP. We report on the ability of the triple domain construct of CR3-4-5 to bind RBD with an affinity the same as for isolated RBD to intact LRP and to in a that requires the identified critical lysine in a that is almost pH-independent within the range of pH values encountered between extracellular and internal The the proposed model of intracellular ligand that displacement by YWTD domains to the ligand binding clusters from a in affinity of the YWTD domain for the CR cluster L. K. K. J. PubMed Scopus Google Scholar). and of and of α2M as previously W. Dolmer K. Gettins P.G.W. Sci. 7: PubMed Scopus Google Scholar). in to = and with isolated and in GdnHCl, pH RBD on a with GdnHCl, pH and with GdnHCl, pH and of RBD, an on N. J. PubMed Scopus Google The pH of an of RBD to by the of 2 and to with GdnHCl, pH RBD to of in a of of and in a with of pH the with of in pH and with of and in with 2 of pH RBD with pH RBD on the and, a with of to be to a of starting with the with as RBD on the be in pH RBD with for 2 to the The domain is the one used for studies W. Dolmer K. Gettins P.G.W. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) and is also the domain that is by of α2M. to and the pH to with 2 to The RBD pH The on a in pH and with a of A RBD from by a and to of type and of of LRP to a containing in to an = with and the to The protein as previously K. Huang W. Gettins P.G.W. Biochemistry. PubMed Scopus Google with and on from the by on a with a of in acid. and CR3-4-5 of LRP to a protein protein and a The CR3-4-5 protein in to an = in and with The protein on a to the with TEV, the the and the by as for to a and as for CR3-4-5 to a and a of the previously K. Huang W. Gettins P.G.W. Biochemistry. PubMed Scopus Google Scholar). Each protein in a of GdnHCl, pH and reduced with a of for Each with pH and with a of protein for a of complement repeat of The of containing and for with to by by of complement repeat the of pH and on a in the same a to to with a containing pH LRP by as In each the protein than the reduced protein CR3-4-5 and pH and a The protein with a of a to that only and, hence, protein CR3-4-5 with in the of and a with of CR3-4-5 of binding RBD with a containing of by on a with a of in pH each protein the the = of of of disulfide This has to be to within a of for most proteins T. Sci. PubMed Scopus Google Scholar). on a with on both the and RBD the residues in and be and RBD binding by the in The in pH containing to protein to the pH binding in containing or and of the of and CR5-6-7, in pH containing of Kd by to a binding site model by the be that LRP used the Kd for the binding that the for to a of to the Kd is and a and a for in pH to to the The in the with the the of the of and have previously that the complement-like repeat in the cluster of LRP, binds RBD with affinity (Kd to suggest that part of the RBD binding site on LRP K. Huang W. Gettins P.G.W. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). We and CR3-4-5 to the full affinity of RBD for LRP be by domains and to of the of domains to the binding each CR domain contains three disulfide of which are critical for the and, hence, in ligand binding of the domain S.C. Biol. PubMed Scopus Google we to that only used in binding This a particular the previously for only a that to be as by the in in the to the RAP, to be important for LRP and in Hammer R.E. J. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). in the of we used RAP, which of the to GSH, with the with In the of with an affinity RBD used to that bind with the that both and the and CR3-4-5 only that In of CR3-4-5 that the RBD affinity the of binding to the complement repeats is also of and can be by the in and CR3-4-5 in the and of a to binding with the ability of each domain to bind The in the of each which has for CR domains from both LRP and LDLR (8Simonovic M. Dolmer K. Huang W. Strickland D.K. Volz K. Gettins P.G.W. Biochemistry. 2001; 40: 15127-15134Crossref PubMed Scopus (54) Google Scholar, K. Huang W. Gettins P.G.W. Biochemistry. PubMed Scopus Google Scholar) and also a of internal that the of than the of from the isolated of and in the and of calcium are to that of CR3 as an important for RBD we also and with the only a on or binding a large in of domains. RBD Binding to and CR3-4-5 by a of the of and CR3-4-5 for RBD, RBD and the CR large in for a of and RBD, that the interaction and only a for the but the than and This that CR3-4-5 is from an in the of RBD to a the more RBD Binding to and by contains but each CR domain contains one with RBD, the a large in the of the is a of of the We to RBD binding to each of these LRP by the in each complement-like repeat and the in is three and CR5-6-7, for RBD binding and be to a binding site the of the Whereas CR3 previously to bind to RBD with a Kd of only to RBD much with a Kd of and CR3-4-5 with a Kd more than of smaller nm) The of the Kd is that the values reported for binding of RBD to intact LRP nm) and that CR3-4-5 the of the RBD binding site within LRP. In in a with a of its affinity for RBD than for the construct demonstrating the for RBD binding CR3-4-5 than other with a 30-fold Kd for binding CR3-4-5 than for RBD binding to LRP CR3-4-5 RBD binding to CR3-4-5. The LRP in each that is than the Kd for of RBD and α2M for CR and and receptor model 2 to to as = to to K. Huang W. Gettins P.G.W. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google L. Gliemann J. FEBS Lett. PubMed Scopus Google and Biochemistry. 1989; PubMed Scopus Google receptor model S. Gonias S.L. Biochem. PubMed Scopus Google Scholar, S.K. Gliemann J. J. Biol. Chem. Full Text PDF PubMed Google as = J. Biol. Chem. 1985; Full Text PDF PubMed Google Scholar). in a of and in RBD Binding to the of both isolated RBD and intact α2M, has that only of the within RBD that are highly conserved are important for binding to LRP M. Moestrup S.K. Gliemann J. Sottrup-Jensen L. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, S. Gonias S.L. Biochem. PubMed Scopus Google Scholar). These are and on the of the of We CR3-4-5 contains the full LRP binding site for the same as LRP for these for RBD RBD containing the and binding to CR3-4-5 by and by RBD containing the to a with CR3-4-5 to a on 2, and the in The of CR3-4-5 with a large of RBD a type of with the type RBD with smaller The to the of the interaction such that RBD to the binding A best to a a Kd of which a of the Kd by Given the of to the affinity of the RBD to pH of ligands from LRP and other LDL receptor family in and the receptors are to the with isolated LRP have a in affinity for complexes between pH and S.K. K. Sottrup-Jensen L. Gliemann J. J. Biol. Chem. 1990; 265: Full Text PDF PubMed Google Scholar). has that binding extracellular pH and pH for pH J. PubMed Scopus Google Scholar). of calcium affinity to CR domains in pH range such a has proposed on the of the extracellular of LDLR, pH that interactions between the domain and complement-like repeats and and in L. K. K. J. PubMed Scopus Google Scholar). The interactions bridges between lysine and residues in the and acidic residues in the complement repeats and one between a in and a in the This a model in which of residues low pH the intramolecular interaction between domains and ligand binding complement-like repeats and in and thus, displacement of the previously In such a of affinity between LRP and a ligand as the pH is be to the pH of binding for the of RBD with CR3-4-5 pH or to that reported pH with the in binding affinity of in a by and by we have a interaction between LRP the three CR domains from cluster 2 of LRP, and RBD, the domain from α2M that contains the full binding The of the Kd of 130 for interaction from for binding of RBD to intact LRP that CR3-4-5 the binding site within cluster 2 for This is also consistent with an earlier study LRP which that a that only CR3-4-5 from cluster 2 and the CR domains from cluster as as intact LRP in Migliorini M. Argraves K. M. Strickland D.K. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus (67) Google Scholar). The in affinity of the in the RBD lysine to be important for binding to LRP S. Gonias S.L. Biochem. PubMed Scopus Google and a of the importance of these residues for The of the CR3-4-5 binding site within cluster 2 is by the 30-fold Kd for site compared with that in the of pH of the interaction the pH from extracellular to the proposed of intracellular ligand that on affinity of an intramolecular YWTD domain to internalized protein Although the affinity for the the affinity of isolated RBD for LRP and, the full RBD binding site within LRP, that of α2M for LRP is important to that binding in both a of the interaction the reported affinity of α2M for LRP of the binding for the of the of the interaction affinity has only very high LRP which the α2M be of CR3-4-5 sites on or more LRP LRP which α2M be to bind to only one LRP, a much affinity of has reported S.K. Gliemann J. J. Biol. Chem. Full Text PDF PubMed Google Scholar). with such a the present binds with of the affinity Although is is only be that complexes have also reported to bind with affinity to cluster from LRP A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). one RBD in α2M to the present site of CR3-4-5 in cluster 2, a RBD from the same α2M to only a site within cluster to the of LRP is to have considerable and is known that four in α2M are for more likely that a from a interaction the than that of the other three bind to LRP and that the is by binding on the α2M surface which also be to be four the that the of RBD binding is to the same of the of the LRP the of Kd values and, and J. Biol. Chem. 1985; Full Text PDF PubMed Google Scholar) for RBD binding to and an to be of the from CR domains. the values from can be that CR3 of the binding and of these is a large and each be composed of a very In the of the of domains and from with the domain of the N. Blacklow S.C. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar) is of particular The that the of binding to be by a interaction between and on and the highly the calcium binding site of each CR This with studies of Strickland and M. S. Strickland D.K. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) identified these same as the only critical for binding and of in Kd for complexes with containing the Although are a number of other in and are in thus, that an binding site interaction may LDL receptor family ligand In with we have that lysine and in intact α2M, previously to be important for RBD and α2M binding to LRP, are in the interaction between CR3-4-5 and RBD, with a to binding of The of these to one in RBD and the that to is consistent with the of binding from a binding site residues in plasminogen activator inhibitor have in plasminogen and plasminogen complexes binding to LRP and the receptor L. Biochem. J. PubMed Scopus Google Scholar, S. S. Strickland D.K. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google and has to be for binding to LDLR A. H. M. J. Biol. Chem. 1988; Full Text PDF PubMed Google Scholar). One with the present findings is that are in with an earlier study on the affinity of of CR domains from cluster 2 of LRP to RBD, which that both and to RBD with low affinity of Moestrup S.K. M. Biochemistry. PubMed Scopus Google Scholar). Although we in the present we the interaction and a Kd than previously is is a between the may from the of an protein in the study that the affinity of RBD for CR3-4-5 as the pH is such as in the a model of displacement of ligands by the intramolecular interaction between the ligand binding complement-like repeats and the domains. has proposed that of residues as the that binding of the than the ligands L. K. K. J. PubMed Scopus Google Scholar). Although studies ligand binding clusters or of ligand binding cluster 2 have to of α2M Migliorini M. Argraves K. M. Strickland D.K. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus (67) Google Scholar, K. J. J. Biol. Chem. Full Text PDF PubMed Google Scholar, H. D.J. J. Sci. 2001; PubMed Google be that these studies have on receptors the domains between clusters and Given that RBD binds CR3-4-5 pH and the of the extracellular domain of LDLR low pH intramolecular interactions between the YWTD domain and complement-like repeats, is likely that one of the domains between clusters and 2 in intact LRP is responsible for the interaction with CR3-4-5 and the displacement of α2M from the A point from the present studies specificity and the of ligand binding sites within LRP. Thus, most ligands have to cluster 2 and, to a cluster cluster contains more (10Crookston K.P. Webb D.J. LaMarre J. Gonias S.L. Biochem. J. 1993; 293: 443-450Crossref PubMed Scopus (67) Google Scholar) of the CR domain than cluster 2 (8Simonovic M. Dolmer K. Huang W. Strickland D.K. Volz K. Gettins P.G.W. Biochemistry. 2001; 40: 15127-15134Crossref PubMed Scopus (54) Google Scholar). Given the importance of interactions between lysine residues and the calcium binding sites of CR domains, in the N. Blacklow S.C. Cell. Full Text Full Text PDF PubMed Scopus Google the of domain of with N. M. Biol. Scopus Google or the domain and complement-like repeats in the low pH of LDLR L. K. K. J. PubMed Scopus Google the of the calcium binding region may be critical in or ligands can Although four acidic residues the calcium through their other residues through the One of these is the of a In and the is in each This is likely to the of each of these calcium binding sites and the to on has that is required for binding to CR repeats from LRP Moestrup S.K. M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus (67) Google Scholar). that in clusters 2 and most ligand binding has an acidic is also that in 7 of and of 11 repeats, is only in of repeats in cluster and of the 2 CR domains in cluster In the that such interactions are and for ligand binding and in ligand binding sites within a cluster on the of we have in the present study that such are the CR3-4-5 binds with 30-fold Kd than each of the three CR domains in each have the 2, thus, has a high affinity binding site for α2M RBD that be composed of both and other interactions that the CR3-4-5 unique for ligand within cluster. such a for a high affinity ligand binding site on LRP and other LDLR family may for the ability of these receptors to bind with high affinity and specificity to many structurally unrelated We previously that the unique of CR domains for a of high affinity binding sites for ligands (8Simonovic M. Dolmer K. Huang W. Strickland D.K. Volz K. Gettins P.G.W. Biochemistry. 2001; 40: 15127-15134Crossref PubMed Scopus (54) Google Scholar). In of the present findings and of N. Blacklow S.C. Cell. Full Text Full Text PDF PubMed Scopus Google model be that a which can be by most CR domains, between acidic residues a CR domain binding site and a of residues on the can be augmented in a by interactions that of the of surface residues in a of CR domains. This high affinity binding sites to ligands, each with unique of such CR domain clusters to high high binding sites has proposed J. A. W. A. A. M. J. PubMed Scopus Google Scholar, PubMed Scopus Google Scholar). In we have that CR3-4-5 the binding site for RBD on LRP. Although α2M binding has to both clusters 2 and the between the affinity of RBD for CR3-4-5 and LRP the site in cluster being cluster 2, the CR3-4-5 site is with a Kd 30-fold than to We have that the interaction between RBD and that of RBD or α2M with intact LRP, on and The interaction between RBD and CR3-4-5 in the pH that one of the domains between clusters and 2 is in α2M from CR3-4-5 in the We for on the