Ligand-assisted Aggregation of Proteins

A comprehensive series of solution and crystallographic studies reveal how simple, achiral, bivalent ligands of the cyclic pyruvate of glycerol promote face-to-face complex formation of the pentraxin, serum amyloid P component (SAP) into decamers. SAP, a protein of the human innate immune system, is universally present in amyloids, including cerebral amyloid deposits found in the brain of Alzheimer disease patients. Removal of SAP through a specific aggregation mechanism mediated by multivalent ligands appears to provide therapeutic benefit in the progression of this disease. Crystallographic studies reveal that in our novel series of ligands only the methyl and carboxylate moieties of the pyruvate ketal directly interact with the protein, but the geometric constraints imposed by the tether dictate which of two chair conformations are adopted by the pyruvate dioxane ring. Solution studies, as interpreted through a simple thermodynamic model, account for the distribution of pentameric and decameric bound states at different ligand concentrations and indicate that differences in the flexibility of the tether determine the geometry and stability of the specific aggregates formed between SAP and two different bivalent ligands. The factors affecting the design of ligands promoting face-to-face protein dimerization as well as potential biological implications are discussed. A comprehensive series of solution and crystallographic studies reveal how simple, achiral, bivalent ligands of the cyclic pyruvate of glycerol promote face-to-face complex formation of the pentraxin, serum amyloid P component (SAP) into decamers. SAP, a protein of the human innate immune system, is universally present in amyloids, including cerebral amyloid deposits found in the brain of Alzheimer disease patients. Removal of SAP through a specific aggregation mechanism mediated by multivalent ligands appears to provide therapeutic benefit in the progression of this disease. Crystallographic studies reveal that in our novel series of ligands only the methyl and carboxylate moieties of the pyruvate ketal directly interact with the protein, but the geometric constraints imposed by the tether dictate which of two chair conformations are adopted by the pyruvate dioxane ring. Solution studies, as interpreted through a simple thermodynamic model, account for the distribution of pentameric and decameric bound states at different ligand concentrations and indicate that differences in the flexibility of the tether determine the geometry and stability of the specific aggregates formed between SAP and two different bivalent ligands. The factors affecting the design of ligands promoting face-to-face protein dimerization as well as potential biological implications are discussed. The design, synthesis, and characterization of tailored multivalent ligands is currently a rapidly expanding frontier for novel pharmaceuticals (1Boas U. Heegaard P.M. Chem. Soc. Rev. 2004; 33: 43-63Crossref PubMed Scopus (1088) Google Scholar). In our design of inhibitors for the radially symmetric, pentameric bacterial toxins of Escherichia coli 0157, we observed that tailored multivalent ligands could provide a dramatic amplification in avidity over monovalent ligands (2Kitov P.I. Sadowska J.M. Mulvey G. Armstrong G.D. Ling H. Pannu N.S. Read R.J. Bundle D.R. Nature. 2000; 403: 669-672Crossref PubMed Scopus (802) Google Scholar). This design principle was subsequently validated for the structurally related cholera toxin and heat-labile enterotoxin (3Fan E.K. Zhang Z.S. Minke W.E. Hou Z. Verlinde C.L.M.J. Hol W.G.J. J. Am. Chem. Soc. 2000; 122: 2663-2664Crossref Scopus (242) Google Scholar, 4Zhang Z. Merritt E.A. Ahn M. Roach C. Hou Z. Verlinde C.L. Hol W.G. Fan E. J. Am. Chem. Soc. 2002; 124: 12991-12998Crossref PubMed Scopus (124) Google Scholar). Besides inhibition enhancements, aggregation of receptors is essential for triggering a variety of biological responses; e.g. dimerizer-regulated gene expression offers pharmacological regulation to gene therapies (5Pollock R. Clackson T. Curr. Opin. Biotechnol. 2002; 13: 459-467Crossref PubMed Scopus (94) Google Scholar). Although the multivalency effect caused by ligand chelation can be readily rationalized in general (6Kitov P.I. Bundle D.R. J. Am. Chem. Soc. 2003; 125: 16271-16284Crossref PubMed Scopus (374) Google Scholar, 7Gargano J.M. Ngo T. Kim J.Y. Acheson D.W. Lees W.J. J. Am. Chem. Soc. 2001; 123: 12909-12910Crossref PubMed Scopus (109) Google Scholar), the structural and design principles of the aggregation mechanism are not well understood. Serum amyloid P component (SAP) 1The abbreviations used are: SAP, serum amyloid P component(s); MOβDG, methyl 4,6-(1-carboxyethylidene)-β-d-galactoside; DLS, dynamic light scattering; GFC, gel filtration chromatography; ELISA, enzyme-linked immunosorbent assay. belongs to the pentraxin family of circulating serum proteins and is characterized by 5-fold radial symmetry of the identical, noncovalently associated subunits. A part of the innate immune system, SAP binds a variety of ligands, including proteins, carbohydrates, and nucleic acids, in a Ca2+-dependent manner. SAP seems to provide marginal advantage in fighting certain infections, but its primary role is believed to be the disposal of cellular debris released by apoptotic and necrotic cells, thereby preventing autoimmunity (8Noursadeghi M. Bickerstaff M.C. Gallimore J.R. Herbert J. Cohen J. Pepys M.B. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 14584-14589Crossref PubMed Scopus (122) Google Scholar, 9Bickerstaff M.C. Botto M. Hutchinson W.L. Herbert J. Tennent G.A. Bybee A. Mitchell D.A. Cook H.T. Butler P.J. Walport M.J. Pepys M.B. Nat. Med. 1999; 5: 694-697Crossref PubMed Scopus (439) Google Scholar, 10Gillmore J.D. Hutchinson W.L. Herbert J. Bybee A. Mitchell D.A. Hasserjian R.P. Yamamura K. Suzuki M. Sabin C.A. Pepys M.B. Immunology. 2004; 112: 255-264Crossref PubMed Scopus (64) Google Scholar). As a universal component of abnormal amyloid deposits, including the cerebral amyloid beta of Alzheimer disease, P component plays a role in protecting fibrils from proteolysis and promoting amyloid deposition both in vitro and in vivo (11Tennent G.A. Lovat L.B. Pepys M.B. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4299-4303Crossref PubMed Scopus (338) Google Scholar, 12Botto M. Hawkins P.N. Bickerstaff M.C. Herbert J. Bygrave A.E. McBride A. Hutchinson W.L. Tennent G.A. Walport M.J. Pepys M.B. Nat. Med. 1997; 3: 855-859Crossref PubMed Scopus (214) Google Scholar). Inhibition of SAP has recently been proposed as a pharmaceutical target, and bivalent d-proline derivatives were reported to show therapeutic efficacy in dramatically reducing serum SAP levels as well as shrinking amyloid deposits in mice (13Pepys M.B. Herbert J. Hutchinson W.L. Tennent G.A. Lachmann H.J. Gallimore J.R. Lovat L.B. Bartfai T. Alanine A. Hertel C. Hoffmann T. Jakob-Roetne R. Norcross R.D. Kemp J.A. Yamamura K. Suzuki M. Taylor G.W. Murray S. Thompson D. Purvis A. Kolstoe S. Wood S.P. Hawkins P.N. Nature. 2002; 417: 254-259Crossref PubMed Scopus (460) Google Scholar). Despite the low intrinsic affinity of d-proline for the Ca2+-binding site, the bivalent derivatives bridge two pentameric SAP with submicromolar activity. To extend our current understanding of the use of multivalent ligands in the treatment of amyloid diseases, we have synthesized a series of simple but potent bivalent inhibitors of SAP that incorporate linkers of different flexibility. The interactions of two of the most potent ligands with SAP have been thoroughly characterized through solution studies including dynamic light scattering, crystal structures of SAP-ligand complexes, and a thermodynamic model for binding. These studies reveal several striking structural and thermodynamic implications of linker flexibility in multivalent binding and pose intriguing questions regarding the factors that determine the stability of ligand-induced face-to-face protein complexes. Gel Filtration Chromatography (GFC)—GFC was carried out using a PerSeptive Biosystems BioCAD Sprint liquid chromatography system. For most experiments, the running buffer was TN buffer (10 mm Tris, pH 8.0, 140 mm NaCl, and 20 mm CaCl2). Typically, 20 μl of protein solution (1 mg/ml SAP) in buffer A (10 mm Tris-HCl, pH 8.0, 140 mm NaCl, 1 mm EDTA) was mixed with 20 mm CaCl2 and either 2′-dAMP, compound 1, or compound 3 (each at 20 mg/ml; compounds shown in Scheme 1). The protein-ligand mixtures were equilibrated overnight at 4 °C before loading onto a Superose 6 GL 10/300 column (Amersham Biosciences) or a Zorbax GF-250 9.4/250 column (Agilent Technologies). The flow rate was 1.0 ml/min, the temperature was 25 °C, and elution was monitored by measuring absorbance at 280 nm. Dynamic Light was carried out at 25 °C using a SAP and of compounds (10 compound 1 (10 or compound 3 were equilibrated in buffer A 20 mm CaCl2 overnight at 4 °C to of SAP with compounds 1 and was carried out over a of SAP (1 and mm of compounds 1 or were equilibrated in buffer mm pH mm overnight at 4 °C before were only the light rate was to and the was of the were in was buffer A and to mg/ml to of the were by the (1 μl of protein and 1 μl of ligand and 1 μl of equilibrated 1 of at crystal were in the of compound A at a ligand of mm with a solution of mm pH mm overnight and to for a of mm mm at a ligand of mm with a solution of mm pH mm several of mm mm in the of compound at a ligand of mm with a solution of mm pH mm 3 or 4 and to for several of mm mm to the in the of compound were to a solution by to a mm pH mm and and 1 of well solution mm pH mm and were from a using a and with a were using at the Light The were and using Z. 1997; PubMed Scopus Google Scholar), and from PubMed Scopus Google Scholar). and for the different SAP are in from the are in from the are in from the are in from the are in from the are in from the are in is the of a and is the of of for the of the used in for the of from of and from temperature factors factors component as by factors component as by factors component as by The from the are in is the of a and is the of of for the of the used in for the of from factors component as by in a The protein from the SAP of the complex were used as the model for E. Hutchinson W.L. Pepys M.B. Wood S.P. J. 1997; PubMed Scopus Google Scholar). The of SAP with compound 1 at mm or crystal was using A. J. 1997; Scopus Google Scholar). A was in the to crystal and a of 2001; PubMed Scopus Google was used to out with of the as The of SAP with compound 1 at mm or crystal was using J. A. Scopus Google Scholar). were in the to crystal and a of was used to out with as a The of SAP with compound at mm was using W.L. J. M. Pannu N.S. Read R.J. T. PubMed Scopus Google Scholar). were in the to crystal and a of was used to out with as a was used for the of and temperature as well as for symmetry were only for the of SAP with compound of the low of J. 1999; 125: PubMed Scopus Google was used for and model Hutchinson J.M. PubMed Scopus Google and G. C. Nature. PubMed Scopus Google were used to in the The and for crystal structures are in and of has been shown to to a of bacterial and in a Ca2+-dependent M.B. D.R. Hutchinson W.L. Gallimore J.R. P.M. E. J. 1997; Scopus Google Scholar). The crystal of a complex of SAP and methyl recently how the cyclic pyruvate ketal in of can directly interact with the Ca2+-binding and of SAP D. Pepys M.B. Wood S. J. 2002; PubMed Scopus Google Scholar). this structural we that a of the cyclic pyruvate ketal of glycerol could be used to the binding of a novel series of bivalent inhibitors to face-to-face of SAP As part of a bivalent derivatives 1 and as well as monovalent 3 were and the SAP binding for compound was by a These compounds have a simple, and can be in from In this we our studies two compounds in the of the linker The and the of the linkers in compounds 1 and are identical, but the in compound thereby the flexibility of the As a the binding of compound to SAP, as by ELISA, is that of the 1, which in binds linker geometric for of the is a in the design of multivalent ligands. The in in this the studies 1). of SAP in directly the different states adopted by serum amyloid P component in the of bivalent ligands in and were As shown SAP in TN buffer at the in the of ligands and in the of W.L. E. Pepys M.B. Med. 2000; PubMed Google reported crystallographic and studies indicate that SAP two different decameric E. Hutchinson W.L. Pepys M.B. Wood S.P. J. 1997; PubMed Scopus Google Scholar, J. G. Pepys M.B. Wood S.P. Nature. PubMed Scopus Google Scholar, Gallimore J.R. Pepys M.B. J. 1997; PubMed Scopus Google Scholar). SAP in the of a monovalent either or compound at a the of a pentameric which is with crystallographic and solution studies D. Pepys M.B. Wood S. J. 2002; PubMed Scopus Google Scholar, Gallimore J.R. Pepys M.B. J. 1997; PubMed Scopus Google Scholar). SAP in the of compound at a with a decameric which with the and crystallographic studies filtration 6 in a and a of In TN SAP and SAP bound to of either or compound have 6 nm. In SAP bound to compound 3 in TN buffer or SAP in buffer have of and The in the is with the of a decameric as shown by GFC, solution scattering, and E. Hutchinson W.L. Pepys M.B. Wood S.P. J. 1997; PubMed Scopus Google Scholar, W.L. E. Pepys M.B. Med. 2000; PubMed Google light in in a was used to the in for of SAP equilibrated with concentrations of the bivalent compounds 1 and 1). As the concentrations of the bivalent compounds from to the of the in solution from a for a or and a a for a or As the of compound 1 is the to the for a In at the concentrations of compound that could be the at the for a To account for the in of and in solution as a of ligand a thermodynamic model the principles of was a to the The model to the that the two and ligand to be at of the concentrations of ligand and and are the stability for the formation of pentameric and decameric for pentameric and decameric in which the are as is the of ligands in a from 1 to of the model two intrinsic binding and K. The binding the of the between binding of the and a affinity of the A binding the of the binding of bivalent ligand in with binding of two For to be of in The for and for different bivalent ligands and can be by binding to the The of and can be from at pH the binding is for the binding affinity the pH used for the experiments, appears that the of and from are with by our model to the 1). between and and stability and The thermodynamic model and the crystallographic structures both to provide a for understanding the of the differences in the binding of compounds 1 and of the structural of ligand-induced SAP crystal structures were for formed between SAP and compound 1 at mm and at mm A and as well as for SAP bound to compound at mm The in the crystal structures are with the of observed by at different concentrations of compounds 1 and compound a in which two of the ligands for the complex are a complex with only bivalent ligands In of the complexes, the carboxylate of the cyclic pyruvate ketal in ligand directly to the two of a SAP interactions between and both are a of the characterized SAP with E. Hutchinson W.L. Pepys M.B. Wood S.P. J. 1997; PubMed Scopus Google Scholar), D. Pepys M.B. Wood S. J. 2002; PubMed Scopus Google Scholar), and a bivalent d-proline (13Pepys M.B. Herbert J. Hutchinson W.L. Tennent G.A. Lachmann H.J. Gallimore J.R. Lovat L.B. Bartfai T. Alanine A. Hertel C. Hoffmann T. Jakob-Roetne R. Norcross R.D. Kemp J.A. Yamamura K. Suzuki M. Taylor G.W. Murray S. Thompson D. Purvis A. Kolstoe S. Wood S.P. Hawkins P.N. Nature. 2002; 417: 254-259Crossref PubMed Scopus (460) Google Scholar). carboxylate directly to a from a ligand and and with the pyruvate ketal The only interactions between the protein and the ligand of between the ligand methyl and a formed by from and This is in the complex between SAP and D. Pepys M.B. Wood S. J. 2002; PubMed Scopus Google Scholar). In the decameric and are by bivalent ligands 4 and In the decameric complex the of the two are of Ca2+-binding are by ligand in the and the for of the of A is in the decameric complex formed with E. Hutchinson W.L. Pepys M.B. Wood S.P. J. 1997; PubMed Scopus Google of in the of In the decameric complex that the two are to of only of the Ca2+-binding are by with of by bivalent ligands and the of bound The from to in the of by bivalent ligands and are and for the two of structures of SAP bound to compound 1 crystal and compound The of the is the for in the and shown in the and The of for is in the as a ligand-induced aggregation to the of novel in a of and to protein and through multivalent and inhibitors the multivalent of interactions have been shown to provide in a of The of binding a of the SAP and the role of SAP in promoting amyloid formation in as Alzheimer disease and provide for the of novel multivalent ligands that promote the of amyloid The principles the design and mechanism of multivalent ligands for therapeutic and studies into the interactions between multivalent ligands and SAP are to The binding of multivalent ligands to binding to a complex of states that both the and binding of different multivalent ligands. To model binding states in this complex system, we have a simple, thermodynamic model that the distribution of different bound of in solution to from ELISA, DLS, and as a of bivalent ligand The in stability of the formed by compounds 1 and is with the of compound in the and with associated with the flexibility of compound 1 is to the of the and in principle of by is to be by a of The of the binding in be observed by ELISA, the decameric are with pentameric binding are with bound ligand at ligand These are and to to the The model that the used for ELISA, the observed inhibition with the of the formed by and can be by In and both indicate that SAP a pentameric As the concentrations of bivalent ligands that the of protein in solution which in a in the of and in the of 1). For compound 1, the of in solution a that is to that for a of over the of the appears to a in the of and in the of For compound the a over the of but concentrations could not be of the of the The in observed by for the distribution of states by the model 1). The of and used to the are with that were by is that indicate binding of both compounds 1 and at the pH used for and with binding at pH The decameric complex in formed in the of compound 1 at the pentameric complex in formed with compound 1 at and the decameric complex in formed with compound at mm are with the and the binding Despite interactions between the carboxylate and methyl of the ligand with the protein and compounds 1 or is striking that the dioxane different conformations in different In the the a chair in which the carboxylate is the methyl and linker are This appears to be the adopted by the but that a of conformations be In in both bivalent and chair is adopted in which the carboxylate is the methyl and linker are Although the with the carboxylate is the only observed by for the ligand in specific geometric constraints for face-to-face in a decameric complex to the chair of the dioxane ring. The conformations of the bound ligands indicate that the in binding caused by multivalency is by the associated with the of of the pyruvate ketal ring. This that design of ligands have to both conformations of the dioxane ring. The structures of the decameric and provide a for understanding the geometric for the formation of between two SAP and bivalent ligands. For the complex is not observed between different A ligand is bound to site, with well the dioxane but to the linker or which are and in the A structural of the and that the different linker structures of two bivalent ligands to the different of the two decameric complexes. The linker found in compound 1 appears to a of that binding to be by ligand in a In the linker found in compound a the of The geometric of the linkers a between the two the symmetry between The in compound 1 of with the found in compound The of is the in complexes, and a As a the binding interactions between ligand and Ca2+-binding site, in with the geometric in the linker the of to the In to the between pentameric the of in different complexes, the decameric complex the 5-fold In the complex with 2′-dAMP, the from are In the decameric formed with compounds 1 and show that the from are from The a of and of to with the decameric Although be of to a with the decameric complex formed between SAP and the bivalent d-proline (13Pepys M.B. Herbert J. Hutchinson W.L. Tennent G.A. Lachmann H.J. Gallimore J.R. Lovat L.B. Bartfai T. Alanine A. Hertel C. Hoffmann T. Jakob-Roetne R. Norcross R.D. Kemp J.A. Yamamura K. Suzuki M. Taylor G.W. Murray S. Thompson D. Purvis A. Kolstoe S. Wood S.P. Hawkins P.N. Nature. 2002; 417: 254-259Crossref PubMed Scopus (460) Google Scholar), the for that have not been in the is to that are interactions between in either the or the A to this in the complex between the of and is to model conformations for both that to a in the two of SAP in the two of can between of the between is between of SAP The of this is is for the of in the crystal to the of this be that this be to be at or pH that which is found in the crystal the only SAP in the the a to a E. Hutchinson W.L. Pepys M.B. Wood S.P. J. 1997; PubMed Scopus Google Scholar). from the potential between and be interactions between The low and of the complex not for of The for the complex to reveal well but to The model of from crystallographic in the complex not reveal a of well The for the between that the between a dynamic the of for or interactions between is that a of between are the and binding interactions of different ligands. is at present different of with differences in biological activity. the formation of SAP by bivalent be with from serum through is to different of be at different be that the binding ligand not be the that of in vivo studies to the therapeutic efficacy of compounds 1 and in amyloid as Alzheimer disease. crystal pose several questions regarding the distribution of bound states in solution and in the crystal that be at The most compound its the of only bivalent ligands in the face-to-face complex with this crystal the solution of compound with SAP, the for the of this compound is complex by the and the thermodynamic model used to binding or be in the of conformations of the ketal and determine the different of protein in the decameric complexes. the of in the decameric complex with compound be the of crystal as to intrinsic in the tether of compound compound a decameric complex with the in solution that is in to the decameric complex This is of the binding have binding thereby to or between as for the of compound A of this is that is the of crystal of a decameric complex into the complex with to and in which ligands are from binding The of the crystal are: and which are the for protein J. 33: PubMed Scopus Google Scholar). the of crystal interactions in the crystal not to a is of to that in the crystal of SAP by bivalent d-proline ligands (13Pepys M.B. Herbert J. Hutchinson W.L. Tennent G.A. Lachmann H.J. Gallimore J.R. Lovat L.B. Bartfai T. Alanine A. Hertel C. Hoffmann T. Jakob-Roetne R. Norcross R.D. Kemp J.A. Yamamura K. Suzuki M. Taylor G.W. Murray S. Thompson D. Purvis A. Kolstoe S. Wood S.P. Hawkins P.N. Nature. 2002; 417: 254-259Crossref PubMed Scopus (460) Google Scholar), the tether is not and a appears to be In that as is the of the tether is by the geometric constraints the or crystal are To the solution of decameric by compound be to determine the of ligand binding in formed in solution using a that studies have reported the distribution of SAP states in solution J.A. J. 2003; PubMed Google Scholar), but ligand binding studies pose a has been used to determine ligand binding in with a of P.I. Bundle D.R. 2001; PubMed Scopus Google Scholar). the of the decameric at a to the currently achiral, bivalent ligands the pyruvate ketal of glycerol to submicromolar binding with SAP and the formation of face-to-face decameric and Solution studies using DLS, crystal and a thermodynamic model that interactions determine the of the the of protein is by tether and that the of the complex can the of the pyruvate ketal ring. This a of principles for the design of novel compounds that can the thermodynamic of multivalent binding at the the formation of specific aggregates with the geometric for therapeutic

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