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Receptor-associated protein (RAP) has two high-affinity binding sites for the low-density lipoprotein receptor-related protein (LRP): consequences for the chaperone functions of RAP

Jensen, Jan K., Dolmer, Klavs, Schar, Christine, Gettins, Peter G. W.·2009·Biochemical Journal
DOI 10.1042/bj20090175PMC PMC2708935
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Abstract

RAP (receptor-associated protein) is a three domain 38 kDa ER (endoplasmic reticulum)-resident protein that is a chaperone for the LRP (low-density lipoprotein receptor-related protein). Whereas RAP is known to compete for binding of all known LRP ligands, neither the location, the number of binding sites on LRP, nor the domains of RAP involved in binding is known with certainty. We have systematically examined the binding of each of the three RAP domains (D1, D2 and D3) to tandem and triple CRs (complement-like repeats) that span the principal ligand-binding region, cluster II, of LRP. We found that D3 binds with low nanomolar affinity to all (CR)2 species examined. Addition of a third CR domain increases the affinity for D3 slightly. A pH change from 7.4 to 5.5 gave only a 6-fold increase in Kd for D3 at 37 °C, whereas temperature change from 22 °C to 37 °C has a similar small effect on affinity, raising questions about the recently proposed D3-destabilization mechanism of RAP release from LRP. Surprisingly, and in contrast to literature suggestions, D1 and D2 also bind to most (CR)2 and (CR)3 constructs with nanomolar affinity. Although this suggested that there might be three high-affinity binding sites in RAP for LRP, studies with intact RAP showed that only two binding sites are available in the intact chaperone. These findings suggest a new model for RAP to function as a folding chaperone and also for the involvement of YWTD domains in RAP release from LRP in the Golgi.

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Figure 1 Fluorescence determination of affinities of CR fragments for RAP D3 Examples of emission spectra and fluorescence determination of affinities of CR fragments for RAP and its fragments. (A) Emission spectra of intrinsic tryptophan fluorescence excited at 295 nm of (■) 200 nM CR45, (●) 200 nM RAP-D3, (▲) mixture, (♦) difference between mixture and RAP-D3 and (▼) CR45 bound–unbound difference spectrum. (B) Change in tryptophan fluorescence followed at 338 nm for 200 nM CR45 titrated with 0–350 nM RAP-D3, and corrected for D3 fluorescence. Normalized raw data (n=3) are shown, with a best fit to a 1:1 binding model.
Figure 1 Fluorescence determination of affinities of CR fragments for RAP D3 Examples of emission spectra and fluorescence determination of affinities of CR fragments for RAP and its fragments. (A) Emission spectra of intrinsic tryptophan fluorescence excited at 295 nm of (■) 200 nM CR45, (●) 200 nM RAP-D3, (▲) mixture, (♦) difference between mixture and RAP-D3 and (▼) CR45 bound–unbound difference spectrum. (B) Change in tryptophan fluorescence followed at 338 nm for 200 nM CR45 titrated with 0–350 nM RAP-D3, and corrected for D3 fluorescence. Normalized raw data (n=3) are shown, with a best fit to a 1:1 binding model.CC BY-NC · PubMed Central
Figure 2 Gel-shift assay of binding of D1, D2 and D3 to CR56 Binding of CR56 to RAP-D1, D2, and D3 by non-denaturing gel-shift assay. The Figure shows the migration of 2μg of the indicated RAP-domain (D1, D2 or D3) incubated with 0, 1 or 2 molar ratio of CR56 on an 8% non-denaturing PAGE. The migration of 3 μg of CR56 alone is shown in the far-right lane.
Figure 2 Gel-shift assay of binding of D1, D2 and D3 to CR56 Binding of CR56 to RAP-D1, D2, and D3 by non-denaturing gel-shift assay. The Figure shows the migration of 2μg of the indicated RAP-domain (D1, D2 or D3) incubated with 0, 1 or 2 molar ratio of CR56 on an 8% non-denaturing PAGE. The migration of 3 μg of CR56 alone is shown in the far-right lane.CC BY-NC · PubMed Central
Figure 3 Gel-shift assay of binding of CRx to intact RAP Binding titration of CR56, CR345 and CR456 to RAP by non-denaturing gel-shift assay. The Figure show the migration of 2.5 μg of RAP either alone (RAP) or incubated with the indicated molar ratios of CR by 8% non-denaturing PAGE. (A) RAP incubated with 1–7 molar ratios of CR56. (B) RAP incubated with 1–8 molar ratios of CR345. (C) RAP incubated with 1–8 molar ratios of CR456. The migration of 7 μg of the indicated CR alone is shown in the far-left lane of each respective gel.
Figure 3 Gel-shift assay of binding of CRx to intact RAP Binding titration of CR56, CR345 and CR456 to RAP by non-denaturing gel-shift assay. The Figure show the migration of 2.5 μg of RAP either alone (RAP) or incubated with the indicated molar ratios of CR by 8% non-denaturing PAGE. (A) RAP incubated with 1–7 molar ratios of CR56. (B) RAP incubated with 1–8 molar ratios of CR345. (C) RAP incubated with 1–8 molar ratios of CR456. The migration of 7 μg of the indicated CR alone is shown in the far-left lane of each respective gel.CC BY-NC · PubMed Central
Figure 4 Fluorescence titration of CR into RAP Change in intrinsic tryptophan fluorescence followed at 338 nm. (A) RAP (100 nM) titrated with 0–500 nM CR56 and (B) 1 μM RAP titrated with 0–2.7μM CR456. Normalized raw data with a best fit to a two-binding site model is shown.
Figure 4 Fluorescence titration of CR into RAP Change in intrinsic tryptophan fluorescence followed at 338 nm. (A) RAP (100 nM) titrated with 0–500 nM CR56 and (B) 1 μM RAP titrated with 0–2.7μM CR456. Normalized raw data with a best fit to a two-binding site model is shown.CC BY-NC · PubMed Central
Figure 5 Size-exclusion chromatographic separation of (CR)x–RAP complexes Purification of RAP–CR56 and RAP–CR345 complexes by size-exclusion chromatography. The profiles show the separation of RAP-CR56 (●) and RAP-CR345 (○) complexes from 1:4.8 RAP:CR mixtures. As confirmed by SDS/PAGE (results not shown), RAP–CR complexes elute close to 70 ml, whereas un-complexed CR56 and CR345 elute at 112 ml and 98 ml respectively. RAP alone in an equivalent amount is shown as a solid line.
Figure 5 Size-exclusion chromatographic separation of (CR)x–RAP complexes Purification of RAP–CR56 and RAP–CR345 complexes by size-exclusion chromatography. The profiles show the separation of RAP-CR56 (●) and RAP-CR345 (○) complexes from 1:4.8 RAP:CR mixtures. As confirmed by SDS/PAGE (results not shown), RAP–CR complexes elute close to 70 ml, whereas un-complexed CR56 and CR345 elute at 112 ml and 98 ml respectively. RAP alone in an equivalent amount is shown as a solid line.CC BY-NC · PubMed Central
Figure 6 Sedimentation velocity analysis of (CR)x–RAP complexes Analysis of RAP–CR56 and RAP–CR345 complexes by analytical ultracentrifugation. Overlay of sedimentation velocity profiles for CR (thin line), RAP (thick line), and purified RAP–CR complex (●) are shown. (A) CR56. (B) CR345. The respective global fits for extinction coefficient-based stoichiometry determination are shown as small insets in top right corner of (A and B) [CR (thin line), RAP (thick line)].
Figure 6 Sedimentation velocity analysis of (CR)x–RAP complexes Analysis of RAP–CR56 and RAP–CR345 complexes by analytical ultracentrifugation. Overlay of sedimentation velocity profiles for CR (thin line), RAP (thick line), and purified RAP–CR complex (●) are shown. (A) CR56. (B) CR345. The respective global fits for extinction coefficient-based stoichiometry determination are shown as small insets in top right corner of (A and B) [CR (thin line), RAP (thick line)].CC BY-NC · PubMed Central
Figure 7 ITC analysis of binding of CR56 to RAP and RAP fragments (A) Titration of D1 into 1.8 μM CR56; (B) titration of D2 into 4.6 μM CR56; (C) titration of D3 into 1.8 μM CR56; (D) titration of CR56 into 3 μM RAP. The upper part of each panel gives the raw ITC data for the titration prior to correction for dilution of titrant, and the lower portion gives the best fit of the data to a single site model (A–C) or a two-site model (D).
Figure 7 ITC analysis of binding of CR56 to RAP and RAP fragments (A) Titration of D1 into 1.8 μM CR56; (B) titration of D2 into 4.6 μM CR56; (C) titration of D3 into 1.8 μM CR56; (D) titration of CR56 into 3 μM RAP. The upper part of each panel gives the raw ITC data for the titration prior to correction for dilution of titrant, and the lower portion gives the best fit of the data to a single site model (A–C) or a two-site model (D).CC BY-NC · PubMed Central
Figure 8 Model of RAP–cluster II complex Model of RAP binding to CR stretches. The Low energy solution 2 from a RAP structure modelling study by Lee et al. [28a], using individual NMR structures of RAP domain 1, 2 and 3 (PDB ID 2P01), is shown in a grey ribbon diagram. D1 is aligned with the D1 from a D1-CR56 complex based on NMR data by Jensen et al. [15] (PDB ID 2FYL). D3 is aligned with D3 from the X-ray crystal structure of D3 in complex with LA34 from the LDLR by Fisher et al. [21] (PDB ID 2FCW). CR5 and CR6 are shown in red and blue space-fill respectively. LA3 and LA4 are shown in yellow and green space-fill respectively. N- and C-termini of the CR and LA tandem repeats and the identity of the RAP domains are indicated. Top and bottom structures are related by a 90° rotation around a horizontal axis through the middle of the Figure. An outline trace of the CR56 space-filling structure in the bottom structure has been positioned between the end of the tandem domain bound to D1 and the tandem domain bound to D3 (shown in red dashes), to represent a possible location of an additional CR tandem repeat. The numbering CR3 through CR8 below the CR domains in the bottom representation indicates how the (CR)6 species CR3–CR8 might bind.
Figure 8 Model of RAP–cluster II complex Model of RAP binding to CR stretches. The Low energy solution 2 from a RAP structure modelling study by Lee et al. [28a], using individual NMR structures of RAP domain 1, 2 and 3 (PDB ID 2P01), is shown in a grey ribbon diagram. D1 is aligned with the D1 from a D1-CR56 complex based on NMR data by Jensen et al. [15] (PDB ID 2FYL). D3 is aligned with D3 from the X-ray crystal structure of D3 in complex with LA34 from the LDLR by Fisher et al. [21] (PDB ID 2FCW). CR5 and CR6 are shown in red and blue space-fill respectively. LA3 and LA4 are shown in yellow and green space-fill respectively. N- and C-termini of the CR and LA tandem repeats and the identity of the RAP domains are indicated. Top and bottom structures are related by a 90° rotation around a horizontal axis through the middle of the Figure. An outline trace of the CR56 space-filling structure in the bottom structure has been positioned between the end of the tandem domain bound to D1 and the tandem domain bound to D3 (shown in red dashes), to represent a possible location of an additional CR tandem repeat. The numbering CR3 through CR8 below the CR domains in the bottom representation indicates how the (CR)6 species CR3–CR8 might bind.CC BY-NC · PubMed Central

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Metadata

Authors
Jensen, J. K., Dolmer, K., Schar, C., & Gettins, P. G. W.
Affiliations
Department of Biochemistry and Molecular Genetics, University of Illinois at Chicago, 900 S. Ashland, M/C 669, Chicago, IL 60607, U.S.A.
Venue
Biochemical Journal
Publisher
Portland Press Ltd.
Published
2009-06-26
Volume
421
Issue
2
Pages
273-282
ISSN
  • 0264-6021
  • 1470-8728
DOI
10.1042/bj20090175
PMC
PMC2708935
Cited by
39
References
1
Language
en
Licence
creativecommons.org/licenses/by-nc/2.5/ (vor, 3681 d)

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CoChat. (2026). Citation check: Receptor-associated protein (RAP) has two high-affinity binding sites for the low-density lipoprotein receptor-related protein (LRP): consequences for the chaperone functions of RAP. https://app.cochat.ai/papers/10.1042/bj20090175
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Jensen, J. K., Dolmer, K., Schar, C., & Gettins, P. G. W. (2009). Receptor-associated protein (RAP) has two high-affinity binding sites for the low-density lipoprotein receptor-related protein (LRP): consequences for the chaperone functions of RAP. Biochemical Journal. https://doi.org/10.1042/bj20090175