Semisynthesis and segmental isotope labeling of the apoE3 N-terminal domain using expressed protein ligation

Apolipoprotein E (apoE) is an exchangeable apolipoprotein that functions as a ligand for members of the LDL receptor family, promoting lipoprotein clearance from the circulation. Productive receptor binding requires that apoE adopt an LDL receptor-active conformation through lipid association, and studies have shown that the 22 kDa N-terminal (NT) domain (residues 1–183) of apoE is both necessary and sufficient for receptor interaction. Using intein-mediated expressed protein ligation (EPL), a semisynthetic apoE3 NT has been generated for use in structure-function studies designed to probe the nature of the lipid-associated conformation of the protein. Circular dichroism spectroscopy of EPL-generated apoE3 NT revealed a secondary structure content similar to wild-type apoE3 NT. Likewise, lipid and LDL receptor binding studies revealed that EPL-generated apoE3 NT is functional. Subsequently, EPL was used to construct an apoE3 NT enriched with 15N solely and specifically in residues 112–183. 1H-15N heteronuclear single quantum correlation spectroscopy experiments revealed that the ligation product is correctly folded in solution, adopting a conformation similar to wild-type apoE3-NT. The results indicate that segmental isotope labeling can be used to define the lipid bound conformation of the receptor binding element of apoE as well as molecular details of its interaction with the LDL receptor. Apolipoprotein E (apoE) is an exchangeable apolipoprotein that functions as a ligand for members of the LDL receptor family, promoting lipoprotein clearance from the circulation. Productive receptor binding requires that apoE adopt an LDL receptor-active conformation through lipid association, and studies have shown that the 22 kDa N-terminal (NT) domain (residues 1–183) of apoE is both necessary and sufficient for receptor interaction. Using intein-mediated expressed protein ligation (EPL), a semisynthetic apoE3 NT has been generated for use in structure-function studies designed to probe the nature of the lipid-associated conformation of the protein. Circular dichroism spectroscopy of EPL-generated apoE3 NT revealed a secondary structure content similar to wild-type apoE3 NT. Likewise, lipid and LDL receptor binding studies revealed that EPL-generated apoE3 NT is functional. Subsequently, EPL was used to construct an apoE3 NT enriched with 15N solely and specifically in residues 112–183. 1H-15N heteronuclear single quantum correlation spectroscopy experiments revealed that the ligation product is correctly folded in solution, adopting a conformation similar to wild-type apoE3-NT. The results indicate that segmental isotope labeling can be used to define the lipid bound conformation of the receptor binding element of apoE as well as molecular details of its interaction with the LDL receptor. Apolipoprotein E (apoE) is a 299-amino acid protein that is a well-characterized ligand of the LDL receptor. X-ray crystallography of the N-terminal (NT) domain of apoE revealed that, in the absence of lipid, it adopts an elongated four-helix bundle conformation that is stabilized by interhelical hydrophobic interactions (1Wilson C. Wardell M.R. Weisgraber K.H. Mahley R.W. Agard D.A. Three-dimensional structure of the LDL receptor-binding domain of human apolipoprotein E.Science. 1991; 252: 1817-1822Crossref PubMed Scopus (601) Google Scholar). While this structure provides detailed information about the organization of apoE NT in a lipid-free state, lipid association is required for adoption of an LDL receptor competent conformation. Thus, it is assumed that apoE NT undergoes a significant conformational change in the presence of lipid to impart LDL receptor binding capability. The LDL receptor recognition sequence has been localized to a region of the protein that encompasses residues 130–172 in the NT domain and includes a sequence element with high positive charge density (2Morrow J.A. Arnold K.S. Dong J. Balestra M.E. Innerarity T.L. Weisgraber K.H. Effect of arginine 172 on the binding of apolipoprotein E to the low density lipoprotein receptor.J. Biol. Chem. 2000; 275: 2576-2580Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 3Weisgraber K.H. Apolipoprotein E: structure-function relationships.Adv. Protein Chem. 1994; 45: 249-302Crossref PubMed Google Scholar). An X-ray crystal structure of the extracellular portion of the LDL receptor at endosomal pH has been reported (4Rudenko G. Henry L. Henderson K. Ichtchenko K. Brown M.S. Goldstein J.L. Deisenhofer J. Structure of the LDL receptor extracellular domain at endosomal pH.Science. 2002; 298: 2353-2358Crossref PubMed Scopus (385) Google Scholar). From this structure, a model for LDL receptor function was proposed in which electrostatic interactions between receptor ligand binding repeats and apoE/B-containing lipoproteins facilitate internalization of the receptor/ligand complex at neutral pH (5Rudenko G. Deisenhofer J. The low-density lipoprotein receptor: ligands, debates and lore.Curr. Opin. Struct. Biol. 2003; 13: 683-689Crossref PubMed Scopus (54) Google Scholar). Upon receptor internalization and subsequent pH change in the early endosome, the receptor is proposed to release its lipoprotein ligand in response to a pH-induced molecular rearrangement that displaces bound ligand from the receptor. Two important gaps in our knowledge of this system include a detailed understanding of the receptor-active, lipid-associated conformation of the apoE ligand as well as the molecular details of its binding interaction with the receptor. In an effort to pursue high-resolution studies of lipid-bound apoE, we employed intein-mediated expressed protein ligation (EPL) (6Schwarzer D. Cole P.A. Protein semisynthesis and expressed protein ligation: chasing a protein's tail.Curr. Opin. Chem. Biol. 2005; 9: 561-569Crossref PubMed Scopus (71) Google Scholar, 7Muralidharan V. Muir T.W. Protein ligation: an enabling technology for the biophysical analysis of proteins.Nat. Methods. 2006; 3: 429-438Crossref PubMed Scopus (304) Google Scholar, 8David R. Richter M.P. Beck-Sickinger A.G. Expressed protein ligation. Method and applications.Eur. J. Biochem. 2004; 271: 663-677Crossref PubMed Scopus (126) Google Scholar). This approach, which allows for increasingly detailed analysis of structural elements within proteins, involves reconstruction of an intact protein from fragments generated separately. In this study, we used EPL to produce a semisynthetic apoE3-NT comprising the first 183 residues of apoE. The results reveal an efficient and novel approach for elucidation of the conformation of lipid-associated apoE3 NT that potentially provides a means to characterize molecular details of apoE-LDL receptor interactions. Taking advantage of the lone cysteine at position 112 in human apoE3 NT together with the requirement that the desired EPL reaction includes a substrate polypeptide possessing an NT cysteine, apoE(1-111) was cloned and expressed as a Saccharomyces cerevisiae-derived vacuolar ATPase intein domain and chitin binding domain fusion protein using the pTYB1 vector (New England Biolabs) as previously reported (9Hauser P.S. Ryan R.O. Expressed protein ligation using an N-terminal cysteine containing fragment generated in vivo from a pelB fusion protein.Protein Expr. Purif. 2007; 54: 227-233Crossref PubMed Scopus (16) Google Scholar). To facilitate optimal intein-mediated fusion protein cleavage (10Hackeng T.M. Griffin J.H. Dawson P.E. Protein synthesis by native chemical ligation: expanded scope by using straightforward methodology.Proc. Natl. Acad. Sci. USA. 1999; 96: 10068-10073Crossref PubMed Scopus (567) Google Scholar), valine 111 was mutated to alanine using the QuikChange method (Stratagene) according to the manufacturer's instructions. Expression and purification procedures for apoE(1-111) followed standardized protocols previously established for generating intein-mediated thioester-adducted proteins (11Xu M.Q. Evans Jr, T.C. Intein-mediated ligation and cyclization of expressed proteins.Methods. 2001; 24: 257-277Crossref PubMed Scopus (116) Google Scholar). Briefly, saturated overnight cultures of ER2566 Escherichia coli cells harboring the vector encoding the apoE(1-111) fusion protein were inoculated into 2xYT media containing 50 µg/ml ampicillin, grown to OD600 = 0.6, and induced with 1 mM isopropyl thiogalactopyranoside. After 6 h at 30°C the cells were pelleted by centrifugation (8,000 g for 15 min), solubilized with buffer A (20 mM Tris, 150 mM NaCl, and 1 mM EDTA, pH 8.0) containing 1% Triton X-100, and stored at −20°C. Dissolved cell pellets were combined, passed through a microfluidizer, sonicated, and centrifuged at 12,000 g for 20 min. Isolated clarified cell extract was passed over a chitin bead column preequilibrated with buffer A containing 1% Triton X-100. The column was washed with 10 column volumes of detergent-free buffer A and fusion protein cleavage induced by addition of 2-mercaptoethanesulfonic acid (MESNA) to a final concentration of 60 mM. Flow was arrested for 16–24 h at 22°C and eluted with two bed volumes of buffer A containing 5 mM MESNA. The sample was dialyzed against deionized water, lyophilized, and stored at −20°C. ApoE(1-111)-MESNA was further purified by semipreparative C8 reversed-phase HPLC on a Perkin-Elmer Series 200 HPLC. Human apoE(112-183) polypeptide was prepared from engineered apoE(1-183) starting material by cyanogen bromide (CNB)r-induced cleavage. Recombinant apoE3(1-183) containing engineered mutations at positions 111 (Val→Met) and 125 (Met→Ala) was expressed and purified as described elsewhere (12Fisher C.A. Wang J. Francis G.A. Sykes B.D. Kay C.M. Ryan R.O. Bacterial overexpression, isotope enrichment, and NMR analysis of the N-terminal domain of human apolipoprotein E.Biochem. Cell Biol. 1997; 75: 45-53Crossref PubMed Scopus (40) Google Scholar). Briefly, saturated overnight cultures were inoculated into M9 minimal media supplemented with 13.3 mM glucose, 0.1 mM CaCl2, 2 mM MgSO4, and 50 µg/ml ampicillin. At OD600 = 0.6, the culture was induced with 2 mM isopropyl thiogalactopyranoside. After 6 h at 30°C, bacteria were pelleted by centrifugation at 8,000 g for 15 min and the culture supernatant collected, concentrated by ultrafiltration, and subjected to heparin affinity chromatography and semipreparative C8 reversed-phase HPLC. Where indicated, apoE3 NT was expressed in M9 medium supplemented with 15NH4Cl (99% purity; Cambridge Isotope Laboratories) as the sole nitrogen source. All subsequent processing and ligation steps were identical to those described above for the unlabeled protein. Isolated apoE(1-183) was then subjected CNBr digestion (13Raussens V. Mah M.K. Kay C.M. Sykes B.D. Ryan R.O. Structural characterization of a low density lipoprotein receptor-active apolipoprotein E peptide, ApoE3-(126–183).J. Biol. Chem. 2000; 275: 38329-38336Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar). ApoE3(1-183) was dissolved in 80% formic acid (5 mg/ml). CNBr was added to achieve a CNBr/methionine ratio and the sample in an for h in the were by the addition of a of deionized and to This was to formic digestion in in apoE(112-183) containing the desired NT was further purified by semipreparative C8 reversed-phase HPLC on a Perkin-Elmer Series 200 chromatography Protein ligation reaction was by using a a were with was on a as described previously (9Hauser P.S. Ryan R.O. Expressed protein ligation using an N-terminal cysteine containing fragment generated in vivo from a pelB fusion protein.Protein Expr. Purif. 2007; 54: 227-233Crossref PubMed Scopus (16) Google Scholar). was on were for h at 20 and with employed purified and apoE(112-183) protein ligation were in containing purified apoE(1-111) and mM apoE(112-183) in an ratio to the reaction to (9Hauser P.S. Ryan R.O. Expressed protein ligation using an N-terminal cysteine containing fragment generated in vivo from a pelB fusion protein.Protein Expr. Purif. 2007; 54: 227-233Crossref PubMed Scopus (16) Google Scholar). Protein fragments were dissolved in 20 mM 150 mM NaCl, 2 supplemented with and 10 mM pH were at for h with reaction was by and by of the using for processing with 2004; Scholar, Scholar). dichroism spectroscopy analysis was on a were between and in 20 mM and 20 mM pH at a protein concentration of in a structure content was as previously described J.A. Weisgraber K.H. in the human apolipoprotein E by the 2000; PubMed Scopus Google Scholar). apoE NT were overnight at a concentration to and was at in a 1 LDL receptor binding of a human LDL receptor NT residues J. Ryan R.O. of low density lipoprotein receptor ligand interactions by 2006; Full Text Full Text PDF PubMed Scopus Google in 20 mM 2 mM CaCl2, and mM was with apoE3 NT on with the probe and with to as above V. Ryan R.O. of the lipid binding of the N-terminal domain of human apolipoprotein J. Biochem. 2001; PubMed Scopus Google Scholar). between apoE3 and was by between residues in the and the to apoE3 J. Ryan R.O. of low density lipoprotein receptor ligand interactions by 2006; Full Text Full Text PDF PubMed Scopus Google Scholar). wild-type apoE3 and EPL apoE3 NT were with lipid to protein as described by and Ryan V. Ryan R.O. of the lipid binding of the N-terminal domain of human apolipoprotein J. Biochem. 2001; PubMed Scopus Google and employed as in receptor binding In this the of the unlabeled to is as a function of in 10 min of at sample were on a Perkin-Elmer NMR experiments were on mM apoE3(1-183) segmental (residues apoE3 NT in buffer mM 50 mM NaCl, 20 mM and EDTA, pH containing mM and mM as chemical 1H-15N heteronuclear single quantum correlation were at 30°C using the sequence from the to a NMR with the and complex for 15N and The were in 15N and in The positions were at and in 15N and were using G. J. a processing system on PubMed Scopus Google and using NMR A for the and analysis of NMR 1994; PubMed Scopus Google Scholar). of residues was by of the chemical of in apoE3 NT C. D. Wang J. A of the apolipoprotein E LDL receptor binding 2005; PubMed Scopus Google Scholar). The of the semisynthesis was to an apoE3 NT protein specifically isotope in the receptor recognition of the protein residues The is in EPL requires an NT cysteine to protein ligation (11Xu M.Q. Evans Jr, T.C. Intein-mediated ligation and cyclization of expressed proteins.Methods. 2001; 24: 257-277Crossref PubMed Scopus (116) Google Scholar, T.W. of proteins by expressed protein Biochem. 2003; PubMed Scopus Google Scholar), the of apoE which a lone cysteine at position is well for To we generated a apoE(1-111) fragment and an apoE(112-183) This the for of a potentially cysteine elsewhere in the protein and employed an apoE fragment residues that encompasses the LDL receptor recognition To the apoE(1-111) intein-mediated cleavage of a protein was (9Hauser P.S. Ryan R.O. Expressed protein ligation using an N-terminal cysteine containing fragment generated in vivo from a pelB fusion protein.Protein Expr. Purif. 2007; 54: 227-233Crossref PubMed Scopus (16) Google Scholar). the CNBr cleavage of a intact apoE3 NT generated the apoE(112-183) of fragments to intact apoE3 with a between residues 111 and 112 and the fragments in the presence of to the reaction into the and of the native chemical ligation Chem. 2006; PubMed Scopus Google Scholar). ApoE(1-111)-MESNA was in high and the fragment was and in a of at high The of the kDa apoE(112-183) ligation substrate fragment is Upon of a of a product with identical to that of apoE3 NT was apoE(112-183) was ligation to have been reported for that two fragments kDa V. J. L. Muir T.W. of into Chem. 2004; PubMed Scopus Google Scholar). was by and the reaction was by the sample was to substrate A of heparin affinity chromatography and reversed-phase HPLC a purified semisynthetic apoE3 NT analysis of the ligation product and apoE3 NT well with the of proteins on acid and To the ligation product in to adopt a conformation similar to native apoE3 spectroscopy was and apoE3 NT were with at and to an content of with on apoE3 NT (12Fisher C.A. Wang J. Francis G.A. Sykes B.D. Kay C.M. Ryan R.O. Bacterial overexpression, isotope enrichment, and NMR analysis of the N-terminal domain of human apolipoprotein E.Biochem. Cell Biol. 1997; 75: 45-53Crossref PubMed Scopus (40) Google Scholar, V. G. The structure of human apolipoprotein and in organization with the of apoE Chem. 2006; PubMed Scopus Google Scholar, Weisgraber K.H. Human apolipoprotein in of the and Biol. Chem. Full Text PDF PubMed Google Scholar). studies revealed that apoE3 NT and ligation product were in solution, native to in the of on we that, EPL generated apoE3 NT in to adopt a conformation of apoE3 of on the secondary structure content of apoE3 NT. induced of apoE3 NT and EPL apoE3 NT Protein was by the function of in at with of apoE3 NT and semisynthetic apoE3 NT were with to V. Ryan R.O. of the lipid binding of the N-terminal domain of human apolipoprotein J. Biochem. 2001; PubMed Scopus Google Scholar). analysis revealed of and apoE3 NT To the of semisynthetic apoE3 NT to as an LDL receptor a binding J. Ryan R.O. of low density lipoprotein receptor ligand interactions by 2006; Full Text Full Text PDF PubMed Scopus Google was shown in both apoE3 and EPL apoE3 induced a in that is of binding to Thus, apoE3 NT and EPL apoE3 NT to with apoE3 for binding to receptor binding of apoE3 NT binding was by in of apoE3 and EPL apoE3 to a of and apoE3 g were at and at A of apoE3 NT semisynthesis is the of a segmental protein for heteronuclear NMR studies in our have shown that a fragment of apoE3 NT to residues to adopt secondary structure in buffer an in the presence of the lipid V. C.M. Ryan R.O. Sykes B.D. NMR structure and of a receptor-active apolipoprotein E Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar, V. C.M. Sykes B.D. Ryan R.O. structure of an apolipoprotein Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). In the approach, we to this by the structure of this region of the protein within the of an intact NT coli in minimal media containing 15NH4Cl as the sole nitrogen efficient labeling was (12Fisher C.A. Wang J. Francis G.A. Sykes B.D. Kay C.M. Ryan R.O. Bacterial overexpression, isotope enrichment, and NMR analysis of the N-terminal domain of human apolipoprotein E.Biochem. Cell Biol. 1997; 75: 45-53Crossref PubMed Scopus (40) Google Scholar). apoE3 NT was then used as starting material for of ligation with in of a semisynthetic apoE3 NT that 15N isotope in residues 112–183. 1H-15N were for and segmental apoE3 NT at pH In this the the chemical of with the nitrogen for of the in the polypeptide From this it can be that segmental semisynthetic apoE3 NT with a of by apoE3 significant of the NMR for EPL-generated apoE3 NT. The for segmental apoE3 NT were well with minimal the region of the In apoE3 NT a density from with a of residues in this on and position in the it is that and 15N the of protein. of the using NMR the nature of of in the and 15N region between the segmental and proteins of which this that of the 10 in apoE3 NT from the of segmental apoE3 NT. This is the apoE3 NT sequence in the from and in the from 112–183. of that in the two and and that between the two and The high of between segmental and apoE3 NT the of the two proteins and the by segmental isotope A similar is in the region to the to 15N to a in the of from in to in apoE3 NT. of the the of for the in apoE3 NT is The of this was to EPL to a apoE3 NT for structural analysis of lipid-bound apoE by NMR This approach a by Weisgraber and reported an X-ray structure of bound to at 10 C.A. Weisgraber K.H. of apolipoprotein E bound to Biol. Chem. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar). While the lipid reported in this were LDL receptor the secondary structure of the detailed of the receptor conformation. The in this apoE fragments together a to an intact NT domain that is similar to apoE3 NT. the LDL receptor binding for apoE3 and EPL apoE3 were with binding fragment residues was generated intein-mediated cleavage of a protein CNBr cleavage of a apoE3 NT a fragment residues 112–183. In the final ligation two were to facilitate the was to to intein-mediated cleavage of the and was to to CNBr cleavage at that studies of the ligation product that semisynthetic apoE3 NT folded in to adopt an bundle that lipid binding and is to function as an LDL receptor The of ligation product was to this method can be used to of specifically protein. The of the EPL approach to the to in within a protein the portion by of D. Cole P.A. Protein of containing Chem. 2001; PubMed Scopus Google Scholar, J. J. at position of a probe of Chem. 2003; PubMed Scopus Google Scholar), biophysical Cole P.A. and expressed protein ligation to Biochem. 2003; PubMed Scopus Google Scholar), J.L. Cole P.A. of the induced by Struct. Biol. 2003; PubMed Scopus Google Scholar), isotope J.A. T.M. D.A. D. Muir T.W. of a by using segmental labeling and Natl. Acad. Sci. USA. 2002; PubMed Scopus Google Scholar, R. Muir T.W. and of the Chem. 2002; PubMed Scopus Google Scholar), into EPL can be used to structural and of proteins that be to EPL for the of of proteins have for to of proteins with that be to achieve by protein purification T.W. of proteins by expressed protein Biochem. 2003; PubMed Scopus Google Scholar). both fragments to EPL can produce proteins that that for the of protein function as it to conformational V. J. L. Muir T.W. of into Chem. 2004; PubMed Scopus Google Scholar, Expressed protein ligation to probe of in the Biol. 1999; Full Text PDF PubMed Scopus Google Scholar), M.E. Muir T.W. of a protein and its in 2007; PubMed Scopus Google Scholar), and interactions V. J. L. Muir T.W. of into Chem. 2004; PubMed Scopus Google Scholar, Cole P.A. and expressed protein ligation to Biochem. 2003; PubMed Scopus Google Scholar). that the of EPL the presence of an cysteine at position 1 of the fragment and a on the NT fragment (11Xu M.Q. Evans Jr, T.C. Intein-mediated ligation and cyclization of expressed proteins.Methods. 2001; 24: 257-277Crossref PubMed Scopus (116) Google Scholar), elsewhere in the fragments be to the protein product that for studies to be In this study, our was to a segmental apoE3 NT. The to specifically into the receptor binding region of this protein for detailed structural apoE3 NT is in a lipid-associated state, characterization of its structural in this is While studies by NMR the of significant with lipid J. D. Sykes B.D. Ryan R.O. NMR for a conformational of lipid Cell Biol. PubMed Scopus Google Scholar). While NMR spectroscopy studies generated a of lipid-free apoE3 NT C. D. Wang J. A of the apolipoprotein E LDL receptor binding 2005; PubMed Scopus Google Scholar), with lipid requires an approach for details of the LDL receptor conformation. In to we a to specifically the region of A similar approach has been employed to apoE conformational using a domain C. Wang J. An efficient expressed protein ligation to segmental labeling of human apolipoprotein Sci. PubMed Scopus Google Scholar). This from the of this study, which to conformational of the receptor binding region of the NT domain association with lipid to LDL receptor binding In we a fragment of apoE to residues this was in the of the intact NT it to adopt a native conformation in (13Raussens V. Mah M.K. Kay C.M. Sykes B.D. Ryan R.O. Structural characterization of a low density lipoprotein receptor-active apolipoprotein E peptide, ApoE3-(126–183).J. Biol. Chem. 2000; 275: 38329-38336Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar). structural were in the presence of a V. C.M. Ryan R.O. Sykes B.D. NMR structure and of a receptor-active apolipoprotein E Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google a lipid V. C.M. Sykes B.D. Ryan R.O. structure of an apolipoprotein Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar), it is the absence of the of the domain the of the Thus, in to the structure of this of the protein within the of the intact NT the EPL approach was required for intact apoE NT in the presence of lipid is that and and high-resolution structure J. G. Structure and of human apolipoprotein Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, C.A. The structure and interactions of human apolipoprotein in 2004; PubMed Scopus Google Scholar). is from the characterization studies that the EPL-generated apoE3 NT adopts a native conformation. the segmental apoE3 NT to an that with the of apoE3 NT. of the residues comprising the fragment with residues in apoE3 that the two proteins adopt similar and similar chemical for residues to residues in the chemical of the protein between in protein conformation to in pH and can in the which the in in of the and be to to mutations at positions 111 and 125 to facilitate While this the chemical for it that the two acid in EPL-generated apoE3 NT to the chemical structural of the protein. the results indicate EPL has been to of an apoE3 NT. it is that segmental isotope labeling a approach to NMR of apoE3 NT for of its receptor conformation. to segmental apoE3 NT in complex with is a at the for apolipoprotein dichroism expressed protein ligation heteronuclear single quantum correlation 2-mercaptoethanesulfonic acid N-terminal high density lipoprotein low density lipoprotein receptor wild-type

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