Probing Solvent Accessibility of Transthyretin Amyloid by Solution NMR Spectroscopy

Abstract

The human plasma protein transthyretin (TTR) may form fibrillar protein deposits that are associated with both inherited and idiopathic amyloidosis. The present study utilizes solution nuclear magnetic resonance spectroscopy, in combination with hydrogen/deuterium exchange, to determine residue-specific solvent protection factors within the fibrillar structure of the clinically relevant variant, TTRY114C. This novel approach suggests a fibril core comprised of the six β-strands, A-B-E-F-G-H, which retains a native-like conformation. Strands C and D are dislocated from their native edge region and become solvent-exposed, leaving a new interface involving strands A and B open for intermolecular interactions. Our results further support a native-like intermolecular association between strands F-F′ and H-H′ with a prolongation of these β-strands and, interestingly, with a possible shift in β-strand register of the subunit assembly. This finding may explain previous observations of a monomeric intermediate preceding fibril formation. A structural model based on our results is presented. The human plasma protein transthyretin (TTR) may form fibrillar protein deposits that are associated with both inherited and idiopathic amyloidosis. The present study utilizes solution nuclear magnetic resonance spectroscopy, in combination with hydrogen/deuterium exchange, to determine residue-specific solvent protection factors within the fibrillar structure of the clinically relevant variant, TTRY114C. This novel approach suggests a fibril core comprised of the six β-strands, A-B-E-F-G-H, which retains a native-like conformation. Strands C and D are dislocated from their native edge region and become solvent-exposed, leaving a new interface involving strands A and B open for intermolecular interactions. Our results further support a native-like intermolecular association between strands F-F′ and H-H′ with a prolongation of these β-strands and, interestingly, with a possible shift in β-strand register of the subunit assembly. This finding may explain previous observations of a monomeric intermediate preceding fibril formation. A structural model based on our results is presented. Amyloidosis represents a group of disorders in which endogenous proteins or peptides adopt anomalous folds and aggregate into elongated amyloid fibrils. At least 20 different human disease conditions are associated with protein deposits, of which Alzheimer's disease and prion-associated disorders are among the most well known examples (1Sipe J.D. Cohen A.S. J. Struct. Biol. 2000; 130: 88-98Crossref PubMed Scopus (840) Google Scholar). Because the macroscopic properties of amyloid, in terms of aggregation and solubility, prevent the use of single crystal x-ray crystallography, as well as solution NMR spectroscopy, the atomic resolution of the molecular structure of amyloid fibrils is poorly understood. So far, structural information has mainly arisen from fiber diffraction studies (2Kirschner D.A. Abraham C. Selkoe D.J. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 503-507Crossref PubMed Scopus (481) Google Scholar), cryoelectron microscopy (3Jimenez J.L. Guijarro J.I. Orlova E. Zurdo J. Dobson C.M. Sunde M. Saibil H.R. EMBO J. 1999; 18: 815-821Crossref PubMed Scopus (445) Google Scholar), mass spectrometry (4Kheterpal I. Zhou S. Cook K.D. Wetzel R. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 13597-13601Crossref PubMed Scopus (165) Google Scholar), and solid state NMR spectroscopy (5Tycko R. Annu. Rev. Phys. Chem. 2001; 52: 575-606Crossref PubMed Scopus (108) Google Scholar). From these data a general picture has emerged implying the occurrence of parallel or antiparallel β-sheets, in which the β-strands lie perpendicular to the direction of the long fibril axis. Familial amyloidotic polyneuropathy is a hereditary form of amyloidosis caused by one of more than 70 different single point mutations in the 13.7-kDa human plasma protein transthyretin (TTR) 1The abbreviations used are: TTRtransthyretinAFMatomic force microscopyBMEβ-mercaptoethanolddeuteratedDTTdithiothreitolH/Dhydrogen/deuteriumHSQCheteronuclear single quantum coherence.1The abbreviations used are: TTRtransthyretinAFMatomic force microscopyBMEβ-mercaptoethanolddeuteratedDTTdithiothreitolH/Dhydrogen/deuteriumHSQCheteronuclear single quantum coherence. (6Connors L.H. Richardson A.M. Theberge R. Costello C.E. Amyloid. 2000; 7: 54-69Crossref PubMed Scopus (121) Google Scholar). The clinical manifestation of familial amyloidotic polyneuropathy is predominantly peripheral polyneuropathy, affecting motor, sensory, and autonomic functions, with accumulation of amyloid deposits along the peripheral nerves and within the visceral organs. The TTR wild-type protein can also form amyloid in vivo and is associated with a senile form of disease affecting around 25% of the population above 80 years of age (7Westermark P. Sletten K. Johansson B. Cornwell III, G.G. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 2843-2845Crossref PubMed Scopus (604) Google Scholar). The native fold of TTR has long since been established through x-ray diffraction studies (8Blake C.C. Burridge J.M. Oatley S.J. Biochem. Soc. Trans. 1978; 6: 1114-1118Crossref PubMed Scopus (19) Google Scholar), and these show that native TTR adopts a tetrameric arrangement, where each monomeric subunit contains eight β-strands, denoted A–H, arranged in a characteristic sandwich of two four-stranded β-sheets (DAGH and CBEF). Two monomeric subunits are associated via F-F′ and H-H′ interactions to form a dimer that in turn interacts in a face-to-face manner with another dimer to form the tetramer. Crystal structures of several in vivo identified amyloidogenic mutants of TTR have been determined over the years. However, they show only subtle and minor variations in their three-dimensional structure compared with the native structure of wild-type TTR (9Hornberg A. Eneqvist T. Olofsson A. Lundgren E. Sauer-Eriksson A.E. J. Mol. Biol. 2000; 302: 649-669Crossref PubMed Scopus (178) Google Scholar). transthyretin atomic force microscopy β-mercaptoethanol deuterated dithiothreitol hydrogen/deuterium heteronuclear single quantum coherence. transthyretin atomic force microscopy β-mercaptoethanol deuterated dithiothreitol hydrogen/deuterium heteronuclear single quantum coherence. In this work we have focused on the characterization of the fibrillar structure of TTR. We have recently developed a general method using NMR spectroscopy in combination with hydrogen/deuterium (H/D) exchange to facilitate the identification of individual core residues within a fibril (10Ippel J.H. Olofsson A. Schleucher J. Lundgren E. Wijmenga S.S. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 8648-8653Crossref PubMed Scopus (85) Google Scholar). The technique relies on the partial solvent protection of hydrogen-bonded amide protons throughout the length of the fibril. Back-bone amide protons located on the exterior of the fibril are more solvent-accessible and thus experience a relatively higher solvent exchange rate compared with amide protons that are located inside the fibril core and shielded from the solvent. In practice, the method works by pulse labeling fully protonated fibrils in a D2O solution over a given period of time. The resulting partially deuterated fibrils are subsequently converted into an NMR detectable monomeric state by resolubilization using a denaturing agent at low pH. This treatment traps the H/D exchange pattern originally present in the fibril and which can be via the monomeric from the core residues of the the NMR method also for a of their protection A of the method is (10Ippel J.H. Olofsson A. Schleucher J. Lundgren E. Wijmenga S.S. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 8648-8653Crossref PubMed Scopus (85) Google Scholar). A approach recently for fibrils from M. K. Struct. Biol. 2002; PubMed Scopus Google Scholar). The present study the method above to the in vivo amyloidogenic variant, TTRY114C. This protein can be to form fibrils at conditions T. Olofsson A. T. S. J. Lundgren E. Sauer-Eriksson A.E. 2002; PubMed Scopus Google and a model for TTR The of fibrils into at a rate that is than the exchange rate within the monomeric state is by the of as a of the H/D exchange by using heteronuclear single quantum NMR This to a of solvent protection factors within the fibrillar state at individual and the structural in the fibril core on the data we a structural model of TTR amyloid and a possible for formation. and the into to or protein by in and as using the of of and for in the of by at for 20 The further using an exchange by using 20 and using a with a of from amyloid fibrils a solution in 20 at for of β-mercaptoethanol to prevent intermolecular formation. The solution into a which is a characteristic of amyloid fibrils. on a with a or in the of to as a of the in and further on a to to with and to for with and The with a using in A at and at an rate to The and in using at 20 using a with a protein of 20 in a between and and by of a The is as in to where is the C is the of is the length and is the of residues in the of the structure of the protein by using the P. 6: PubMed Scopus Google Scholar). in combination with H/D exchange used to study the exchange rate of protons within the fibrillar The fibrillar solution with a monomeric of in D2O and for of time. the solution through of of an of and The subsequently within 20 into a mass The of determined from the mass and the mass of a fully deuterated monomeric NMR and of TTR in at on a at and with a A NMR deuterated and 20 deuterated dithiothreitol in D2O at or in D2O at a of The NMR data using S. J. A. J. 6: PubMed Scopus Google or in for M. P. P. T. J. 2000; 18: PubMed Scopus Google Scholar). The resonance of TTR the resonance for using and three-dimensional and S. J. 1999; PubMed Scopus Google M. S. C. NMR 1999; Scopus Google Scholar). H/D of by H/D exchange on the amyloid fibrils by a of the amyloid which the of the The amyloid subsequently using D2O and the of and the two a of in which the of the exchange fibrils and through the of of D2O to of and 20 The protection of amide protons of the fibrils by a of the solution in with six and data be to the of the fibrils into a monomeric a to each between protons within the fibril and the monomeric a H/D on the A protein solution in and 20 in and by a in a D2O solution and 20 in and the of an of as and the H/D exchange of the fibrils in from by using and by the The of the solution determined from of the region in a NMR to each individual the of amide in the are for the monomeric at of the fibrils. The of these amide of each in the monomeric state to a single where is the H/D exchange rate and is the at The the of of amide that the exchange period with D2O in the fibril to resolubilization by The of protection within the fibrillar state thus be by a of in a fully the between in the two is in of H/D exchange and conditions of H/D model of the fibril structure of using the from wild-type TTR the determined protection and known intermolecular (3Jimenez J.L. Guijarro J.I. Orlova E. Zurdo J. Dobson C.M. Sunde M. Saibil H.R. EMBO J. 1999; 18: 815-821Crossref PubMed Scopus (445) Google C. M. 2001; PubMed Scopus Google C. M. Struct. Biol. 2002; PubMed Scopus Google Scholar). The monomeric structure in R. M. K. J. Mol. Scopus Google by the and residues in the crystal and by strands C and by an in 18: PubMed Scopus Google using the force The a on the structure to the that as a of the A structural model of the fibril in by the in with our protection factors and intermolecular as well as known intermolecular two interface a novel interface between the antiparallel β-strands and at residues and a native interface between the antiparallel β-strands F-F′ and H-H′ at residues and at an protein in E. and as a native T. Olofsson A. T. S. J. Lundgren E. Sauer-Eriksson A.E. 2002; PubMed Scopus Google Scholar). a of the the tetrameric fold of the protein is and an to form amyloid at and also the rate of In a a of at and the aggregation by native the as molecular at the of the The of amyloid fibrils by a of these fibrils. The in present in both wild-type TTR and as well as the in are of the of intermolecular within the fibrils C. J. E. Lundgren E. EMBO J. PubMed Scopus Google T. Olofsson A. T. S. J. Lundgren E. Sauer-Eriksson A.E. 2002; PubMed Scopus Google Scholar). of intermolecular the at This treatment intermolecular as by where fibrils in a solution previous of the protein The well that can be in to the of a within the TTR with the spectroscopy used to further the structural properties of TTRY114C. The for the native form of and TTR wild-type by the P. 6: PubMed Scopus Google and in an of and This is in with a recently crystal structure of only subtle from the native TTR wild-type T. Olofsson A. T. S. J. Lundgren E. Sauer-Eriksson A.E. 2002; PubMed Scopus Google Scholar). fibrils at a protein around 20 at by a that for structure by of spectroscopy with the P. 6: PubMed Scopus Google Scholar). The of the fibrillar fold is compared with the native to a of of in of a more The is in with a previous study on TTR wild-type amyloid A. P. Wijmenga S. Lundgren E. J. Biol. Chem. 2001; PubMed Scopus Google and is further that the structural of the amyloid state from that of the native by of amyloid in a the H/D exchange pattern of amide protons in the using our is to of the fibrils into a monomeric at a rate than the rate of H/D different for The in an solution as by where the fibrils are into a monomeric well in the H/D exchange pattern of the in solution to a of where the solvent exchange rate is at a is also known to which further the exchange within these of the A of fibrils in at to is in The of a at and to at is characteristic for a protein with structure and to by the P. 6: PubMed Scopus Google Scholar), the of these of in for the of the exchange pattern of the fibril is the of protein from the monomeric in The of a protein in and at at a with and a of an is for an protein in with a with a molecular mass of at least resonance using with a of three-dimensional resonance the of an amide group on the and the into a of amide be in a these the of residues and exchange in and to and and as of amide in the of and or be of exchange or In for the H/D exchange NMR of H/D The amyloid fibrils from at protein of around a and from the solution through The exchange from the solution and to a D2O solution and by the of a of D2O solution to the by and of the by The which and spectrometry that the of the solvent-exposed, amide protons by within from the of in that a of exchange The exchange are in with on 1999; PubMed Scopus Google Scholar). Our mass spectrometry data that around amide protons within the fibrillar state of of H/D by the fibrils to a period in D2O by into by the of a D2O solution and to a region of the resolubilization of the fibrils into amide to be from solvent a for the protection of each within the the exchange in the monomeric state through a of at the The of is in The of the amide of each to a single by to the amide at of fibril This represents the of protection each in the fibrillar A with a fully protonated the of protection within the fibrillar by a of in of H/D exchange and a of conditions of H/D a for residues and (10Ippel J.H. Olofsson A. Schleucher J. Lundgren E. Wijmenga S.S. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 8648-8653Crossref PubMed Scopus (85) Google Scholar), the rate of fibril is in fibrils to at the In the of a amyloid the of protection as a be the monomeric at the into a monomeric a NMR to each The of the by used to of the The that a and to the of the into a detectable From on a of with two different rate and The of protection the monomeric at each The amide protection for residues of the fibril is in and a structure of the native TTR that the protection to well with the native β-strands and and to β-strand the and strands C and D show or determined solvent protection factors of amyloid fibrils The of protection are as with exchange of the amide within of in D2O and protection exchange the time. residues that a detectable H/D exchange rate within are each the structure within the native form is residues are with and their protection are within the to an to the that the and for and solvent protection in a new Because the fibrillar fold from the in to that the amide protons as protection in are in the fibrillar state and the of an within the A H/D on the in this approach residues in with protection in the well within the monomeric state their within the fibril. The of the amyloid at atomic resolution has the of the of amyloid for a long as well as within the we have a new and method that structural information the amyloid fibril a H/D to solvent exchange of the fibrils by a into monomeric and using solution NMR The method to the amyloidogenic from the (10Ippel J.H. Olofsson A. Schleucher J. Lundgren E. Wijmenga S.S. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 8648-8653Crossref PubMed Scopus (85) Google Scholar). this work we have our technique and the fibrillar core structure of transthyretin amyloid, a protein associated with both inherited and idiopathic amyloidosis. The by which the native fold of TTR may be converted into elongated fibrillar has been and studies have that of the native tetrameric structure into a monomeric amyloid PubMed Scopus Google PubMed Scopus Google P. 2001; PubMed Scopus Google A. I. J. Biol. Chem. 2001; PubMed Scopus Google Scholar). of amyloidogenic TTR the by which amyloid is that each has a different on the structure and of the have that the amyloidogenic is by the rate of tetrameric than P. 2001; PubMed Scopus Google Proc. Natl. Acad. Sci. U. S. A. 2001; PubMed Scopus (121) Google Scholar). The clinically relevant protein may be converted into amyloid conditions and as a model for a TTR amyloid fibril. In fibrils into in solution conditions in which the solvent exchange rate is a of H/D exchange present in the amyloid fibril and by solution NMR The of in to using mass spectrometry in an approach to that by and (4Kheterpal I. Zhou S. Cook K.D. Wetzel R. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 13597-13601Crossref PubMed Scopus (165) Google Scholar). this the of as The to a exchange rate and to the fibrillar The conditions in the H/D exchange and by NMR The a with mass and a partial protection of around residues From the resonance possible to of residues using NMR and a of in at the residues of for which the protection factors be determined the protection factors the native monomeric The exchange data that well residues within to the native strands and that they the core of the fibril. The of residues in strands C and and D and as well as in the and and that this region is of the fibrillar This is by several of our previous on the structures of TTR fibrils where the both in vivo C. J. E. Lundgren E. EMBO J. PubMed Scopus Google and in K. Olofsson A. I. A. C. S. Lundgren E. PubMed Scopus Google an of strands C and D and their The of a dislocated edge region is further by the of two for the amyloid which show a to the strands C and K. Olofsson A. I. A. Lundgren E. Proc. Natl. Acad. Sci. U. S. A. 1999; PubMed Scopus Google Scholar). The that the C and D strands become and a more within the fibril is by the H/D exchange data A protection from solvent exchange can be for residues and of B. a protection be the protein has a native where B is antiparallel between strands C and on both However, as our data show that C is a novel to B be TTR fibrils using resonance to between to at in the protein C. M. Struct. Biol. 2002; PubMed Scopus Google Scholar). The results of these support a model where two B strands in an antiparallel manner on and form a novel This model is in with our data the H/D solvent protection pattern for residues has a at the In the native structure of is located in a four-stranded between strands B and in an antiparallel of the within this also to be in the fibrillar state a solvent protection is for residues and in as well as for residues and in is also that residues and their native residues and in the fibril. the solvent protection a native or native among strands and From the of amyloid the native between strands F-F′ and H-H′ a and an is that a native-like is also within in the fibrillar This has been by and from an resonance study on TTR fibrils a between two strands C. M. 2001; PubMed Scopus Google Scholar). In to the relatively residues in the dimer interface of the native structure K. P. J. Mol. Biol. 2000; PubMed Scopus Google Scholar), we a protection of residues and in the fibril also a of to in the of the native structure K. P. J. Mol. Biol. 2000; PubMed Scopus Google Scholar). The of and that they of the This protection pattern a of the subunit with a prolongation of the β-strand or a shift in register between the two The well with our data the two strands are on or of as in the native the native structure an antiparallel of two strands an interface between two through intermolecular via residues and Our results also support the of a native-like interface between the two strands within the fibril a protection of and our residues and are in the or only to a low in the native a that further the of a shift in register between the β-strands in the the two strands between and of as within the native the protection pattern which The shift in β-strand register between F-F′ and H-H′ in the dimer interface to a of the dimer into to the of the interface and the between and fibril formation. A shift in register further explain the finding by C. A.M. Biochem. J. 2000; PubMed Google Scholar), where the native register by in or and which in protection of the is for residues and which well with the native structure where they are hydrogen-bonded to the and residues and located between strands A and protection of residues and is This with the in the native structure in which residues and form to residues and of is hydrogen-bonded to of is in A and may be the of a native-like to Our results support a model of the fibril in which the native of the six β-strands and are and to into a native-like conformation. Our data further an interface region by an antiparallel of strands F-F′ and H-H′ with a subunit and a shift in register by the strands C and D are dislocated from their native and become leaving strands A and B protection of this novel edge as well as of a is by an intermolecular association via antiparallel strands and of the model is given in where the residues are in and within the core of the the residues are in and on the fibrillar different of the TTR amyloid have been diffraction studies of vivo TTR amyloid have that a of within the fibril C. PubMed Scopus Google Scholar). The data support a model in which β-strands a turn of the where or the This with our in which eight a sandwich a with a length along the direction of the long fibril to β-strands or the of the fibrillar core is in with data of a C. 1999; PubMed Scopus Google Scholar). Two of TTR amyloid have been based on crystal structures from amyloidogenic TTR The model from a amyloidogenic an within the crystal where form a involving within the A.M. J. Biol. Chem. PubMed Scopus Google Scholar). this a of a fibril with the β-strands arranged parallel to the fibril axis. The native fold of each monomeric subunit is within this This is in with our results the solvent protection pattern as well as only support a native The model is based on a study in which we a crystal structure of the amyloidogenic protein K. Olofsson A. I. A. C. S. Lundgren E. PubMed Scopus Google Scholar), where a of D T. K. Olofsson A. Lundgren E. Sauer-Eriksson A.E. Mol. 2000; 6: PubMed Google Scholar). This structural a novel region between strands C and D an intermolecular with the on an within the A of can be within the crystal a of This structure is also in with the solvent protection pattern from the within this However, the model in the structural in the edge of may an for of strands A and B and a to form fibrils. the structural preceding amyloid the of tetrameric TTR has been both native and conditions using H/D exchange in combination with NMR K. P. J. Mol. Biol. 2000; PubMed Scopus Google K. J. Mol. Biol. 2002; PubMed Scopus Google K. Struct. Biol. 2000; 7: PubMed Scopus Google Scholar). studies information the of TTR and to form The results show that at TTR a core of residues located in strands and also within the strands A and B K. J. Mol. Biol. 2002; PubMed Scopus Google Scholar). The H/D solvent protection pattern has also been amyloid conditions by at low an established method of fibrils from TTR K. Struct. Biol. 2000; 7: PubMed Scopus Google Scholar). The data show that the amide protons within the strands and become more they are In an solvent within the the that a of the is for the of amyloid formation. The protection pattern and the for tetrameric TTR is in with our model and structural The model suggests that strands C and D prevent of strands A and thus via This a a in the of amyloid formation. However, at low a monomeric is predominantly PubMed Scopus Google Scholar). this is an intermediate in the of fibril or represents an conditions where a is converted into fibrils to be A previous study support the of a where the of has been using amyloidogenic TTR mutants a low tetrameric conditions and low and where amyloid is A. P. Wijmenga S. Lundgren E. J. Biol. Chem. 2001; PubMed Scopus Google Scholar). This can further be to aggregate into fibrillar that is in the of amyloid formation. work the interface of the the and strands or is by novel within strands A and B. In we have and identified the fibrillar core structure of a TTR amyloid at a and a structural model for a fibril as well as a possible for fibril The results novel structural which from a point of is as for the of TTR amyloid formation.