Dynamic α-Helix Structure of Micelle-bound Human Amylin

Amylin is an endocrine hormone that regulates metabolism. In patients afflicted with type 2 diabetes, amylin is found in fibrillar deposits in the pancreas. Membranes are thought to facilitate the aggregation of amylin, and membrane-bound oligomers may be responsible for the islet β-cell toxicity that develops during type 2 diabetes. To better understand the structural basis for the interactions between amylin and membranes, we determined the NMR structure of human amylin bound to SDS micelles. The first four residues in the structure are constrained to form a hairpin loop by the single disulfide bond in amylin. The last nine residues near the C terminus are unfolded. The core of the structure is an α-helix that runs from about residues 5–28. A distortion or kink near residues 18–22 introduces pliancy in the angle between the N- and C-terminal segments of the α-helix. Mobility, as determined by 15N relaxation experiments, increases from the N to the C terminus and is strongly correlated with the accessibility of the polypeptide to spin probes in the solution phase. The spin probe data suggest that the segment between residues 5 and 17 is positioned within the hydrophobic lipid environment, whereas the amyloidogenic segment between residues 20 and 29 is at the interface between the lipid and solvent. This orientation may direct the aggregation of amylin on membranes, whereas coupling between the two segments may mediate the transition to a toxic structure. Amylin is an endocrine hormone that regulates metabolism. In patients afflicted with type 2 diabetes, amylin is found in fibrillar deposits in the pancreas. Membranes are thought to facilitate the aggregation of amylin, and membrane-bound oligomers may be responsible for the islet β-cell toxicity that develops during type 2 diabetes. To better understand the structural basis for the interactions between amylin and membranes, we determined the NMR structure of human amylin bound to SDS micelles. The first four residues in the structure are constrained to form a hairpin loop by the single disulfide bond in amylin. The last nine residues near the C terminus are unfolded. The core of the structure is an α-helix that runs from about residues 5–28. A distortion or kink near residues 18–22 introduces pliancy in the angle between the N- and C-terminal segments of the α-helix. Mobility, as determined by 15N relaxation experiments, increases from the N to the C terminus and is strongly correlated with the accessibility of the polypeptide to spin probes in the solution phase. The spin probe data suggest that the segment between residues 5 and 17 is positioned within the hydrophobic lipid environment, whereas the amyloidogenic segment between residues 20 and 29 is at the interface between the lipid and solvent. This orientation may direct the aggregation of amylin on membranes, whereas coupling between the two segments may mediate the transition to a toxic structure. Type 2 diabetes affects over 100 million people worldwide (1Amos A. F. McCarty D. J. Zimmet P. Diabetes Med. 1997; 14: 1-85PubMed Google Scholar) and is thought to cost upward of 130 billion dollars a year to treat in the United States alone (2Hogan P. Dall T. Nikolov P. Diabetes Care. 2003; 26: 917-932Crossref PubMed Scopus (1370) Google Scholar). The endocrine hormone amylin (also known as islet amyloid polypeptide) appears to have key roles in diabetes pathology (3Cooper G. J. Endocr. Rev. 1994; 15: 163-201Crossref PubMed Scopus (271) Google Scholar, 4Jayasinghe S. A. Langen R. Biochim. Biophys. Acta. 2007; 1768: 2002-2009Crossref PubMed Scopus (157) Google Scholar, 5Zdrojewicz Z. Belowska-Bién K. Diabetologia Doœswiadczalna i Kliniczna. 2006; 6: 169-172Google Scholar). The normal functions of amylin include the inhibition of glucagon secretion, slowing down the emptying of the stomach, and inducing a feeling of satiety through the actions of the hormone on neurons of the hypothalamus in the brain (5Zdrojewicz Z. Belowska-Bién K. Diabetologia Doœswiadczalna i Kliniczna. 2006; 6: 169-172Google Scholar). The effects of amylin are exerted in concert with those of insulin and reduce the level of glucose in the blood (3Cooper G. J. Endocr. Rev. 1994; 15: 163-201Crossref PubMed Scopus (271) Google Scholar, 5Zdrojewicz Z. Belowska-Bién K. Diabetologia Doœswiadczalna i Kliniczna. 2006; 6: 169-172Google Scholar). Circulating amylin levels increase in a number of pathological conditions, including obesity, syndrome X, pancreatic cancer, and renal failure (3Cooper G. J. Endocr. Rev. 1994; 15: 163-201Crossref PubMed Scopus (271) Google Scholar). Amylin levels together with insulin are raised initially in type 2 diabetes but fall as the disease progresses to a stage where the pancreatic islets of Langerhans β-cells that synthesize amylin no longer function (3Cooper G. J. Endocr. Rev. 1994; 15: 163-201Crossref PubMed Scopus (271) Google Scholar). One of the hallmarks of type 2 diabetes, found in 90% of patients, is the formation of extracellular amyloid aggregates composed of amylin (3Cooper G. J. Endocr. Rev. 1994; 15: 163-201Crossref PubMed Scopus (271) Google Scholar, 4Jayasinghe S. A. Langen R. Biochim. Biophys. Acta. 2007; 1768: 2002-2009Crossref PubMed Scopus (157) Google Scholar, 5Zdrojewicz Z. Belowska-Bién K. Diabetologia Doœswiadczalna i Kliniczna. 2006; 6: 169-172Google Scholar). The amyloid deposits accumulate in the interstitial fluid between islet cells and are usually juxtaposed with the β-cell membranes (3Cooper G. J. Endocr. Rev. 1994; 15: 163-201Crossref PubMed Scopus (271) Google Scholar). Aggregates of amylin are toxic when added to cultures of β-cells, so that the amyloid found in situ may be responsible for β-cell death as type 2 diabetes progresses (6Janson J. Ashley R. H. Harrison D. McIntyre S. Butler P. C. Diabetes. 1999; 48: 491-498Crossref PubMed Scopus (518) Google Scholar, 7Lorenzo A. Razzaboni B. Weir G. C. Yankner B. A. Nature. 1994; 368: 756-760Crossref PubMed Scopus (733) Google Scholar). Genetic evidence that amylin is directly involved in pathology includes a familial S20G mutation that leads to early onset of the disease (8Sakagashira S. Sanke T. Hanabusa T. Shimomura H. Ohagi S. Kumagaye K. Y. Nakajima K. Nanjo K. Diabetes. 1996; 45: 1279-1281Crossref PubMed Scopus (142) Google Scholar) and produces an amylin variant that aggregates more readily (9Ma Z. Westermark G. T. Sakagashira S. Sanke T. Gustavsson A. Sakamoto H. Engstrom U. Nanjo K. Westermark P. Amyloid. 2001; 8: 242-249Crossref PubMed Scopus (52) Google Scholar). As with all amyloids it is unclear whether fibrillar structures or soluble oligomers are responsible for pathology. A recurrent theme for amyloidogenic proteins is that toxicity appears to be exerted through membrane-bound oligomers that form pores and disrupt ion balance across membranes (4Jayasinghe S. A. Langen R. Biochim. Biophys. Acta. 2007; 1768: 2002-2009Crossref PubMed Scopus (157) Google Scholar, 10Chiti F. Dobson C. M. Annu. Rev. Biochem. 2006; 75: 333-366Crossref PubMed Scopus (5181) Google Scholar, 11Ferreira S. T. Vieira M. N. De Felice F. G. IUBMB Life. 2007; 59: 332-345Crossref PubMed Scopus (291) Google Scholar, 12Lansbury P. T. Lashuel H. A. Nature. 2006; 443: 774-779Crossref PubMed Scopus (578) Google Scholar, 13Ross C. A. Poirier M. A. Nat. Med. 2004; 10: S10-S17Crossref PubMed Scopus (2503) Google Scholar). Experimental evidence for such oligomers has been found for the amyloid-β (Aβ) 2The abbreviations used are: Aβ, amyloid-β; αS, α-synuclein; r. m. s. d. , root mean square deviation; NOE, nuclear Overhauser effect; NOESY, nuclear Overhauser effect spectroscopy; HSQC, heteronuclear single quantum coherence; TOCSY, total correlation spectroscopy. peptides (14Chimon S. Shaibat M. A. Jones C. R. Calero D. C. Aizezi B. Ishii Y. Nat. Struct. Mol. Biol. 2007; 14: 1157-1164Crossref PubMed Scopus (464) Google Scholar), which cause Alzheimer disease, and for α-synuclein (αS), the protein involved in Parkinson disease (15Lashuel H. A. Hartley D. Petre B. M. Walz T. Lansbury Jr. , P. T. Nature. 2002; 418: 291Crossref PubMed Scopus (1136) Google Scholar), a particular interest of our laboratory. The similar toxic effects exerted by these amyloidogenic molecules may have a common structural and physical basis. Detailed structural models are available for Aβ (16Coles M. Bicknell W. Watson A. A. Fairlie D. P. Craik D. J. Biochemistry. 1998; 37: 11064-11077Crossref PubMed Scopus (476) Google Scholar) and αS (17Ulmer T. S. Bax A. J. Biol. Chem. 2005; 280: 43179-43187Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar) bound to SDS micelle mimetics of membranes. For amylin there are models of peptide fragments 1–19 (18Nanga J. A. Biochemistry. PubMed Scopus (157) Google Scholar), A. F. U. A. 2003; PubMed Scopus Google Scholar), and D. A. F. D. 2007; PubMed Scopus Google Scholar) bound to but as of no of the This to be as the between structure and in amylin to when the we the solution structure of human amylin bound to SDS micelles. the structure with on and on the of amylin micelles. human amylin number and number from The peptides in and from human amylin by an C SDS from and from NMR of used for all heteronuclear NMR by of in a 90% solution of 100 to a amylin of used for NMR structure and relaxation 2 by of amylin of the solution to a of 2 and used for the with and by of amylin at in 100 and used for NMR NMR on a with a from the A of used for all NMR on and and first by type and K. Chem. 2003; PubMed Scopus Google Scholar) for from an as in the P. B. R. R. J. 1997; 10: PubMed Scopus Google Scholar). for on of the of in M. Bax A. J. Scholar) and and in 1994; PubMed Scopus Google Scholar). for amylin have been with the number of the used to the NMR structures of amylin and of the to the of the structures is in four and on the of in the angle of for residues with Bax A. J. Chem. Scopus Google Scholar) for residues on coupling data and data from 1994; PubMed Scopus Google for the amylin are as the over the structures structures no or The the A for and Scholar). function with interactions and to States D. J. S. M. J. Chem. Scopus Google the function States D. J. S. M. J. Chem. Scopus Google Scholar). a are as the over the structures structures no or The the A for and Scholar). function with interactions and to States D. J. S. M. J. Chem. Scopus Google Scholar). the function States D. J. S. M. J. Chem. Scopus Google Scholar). α-helix bond for 17 residues with and in the α-helix the 17 be in NMR as for to when amylin in in and For the on in structures with bond for the structures from to for residues and from to for residues the there no in and the NMR with the of within the of the with that the of all 17 bond is by the and coupling data that structures for the residues in and by the of the with structures these The NMR structure from with with the A for and Scholar) to a A. M. P. T. PubMed Scopus Google Scholar). The structures with no or for have been with the 15N of Amylin relaxation data correlation from the on Biochemistry. PubMed Scopus Google Scholar). relaxation data from with relaxation of and To for relaxation of and of 2 used between and determined from of the as a function of relaxation to where is the for relaxation is the and is the for or in the and as the of the as the of in an with for to a where the with an as in in the as in where and the root mean square in the with and D. J. Mol. Biol. 1994; PubMed Scopus Google Scholar). the of J. Chem. Scopus Google Scholar) with the P. M. D. J. PubMed Scopus Google Scholar) (17Ulmer T. S. Bax A. J. Biol. Chem. 2005; 280: 43179-43187Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar) for the To a for the correlation for we our to residues which and with α-helix structure. of we a correlation of for which used to for of Amylin in SDS on amylin in 100 SDS with and as a 100 solution in The solution used to the of 2 to in The aggregation number of SDS is about M. J. U. A. Biochemistry. 2001; PubMed Scopus Google Scholar), so that a 100 SDS solution to a micelle of or spin probe In a the of raised to to spin probes with at of and to of and of the ion to amylin. used to For a to in the of on an with at a of from to on in a The amylin in and of Amylin an in the of SDS human amylin in A. D. P. Biochemistry. 2005; PubMed Scopus Google Scholar), it is soluble and for at 2 in 100 SDS at an of The of amylin these is in of amylin with of SDS at In the of the of amylin that for an at and together with the of a at are more of a of and α-helix The α-helix structure in the of SDS to that in by NMR for amylin 2007; PubMed Scopus Google Scholar). In the of 5 there is α-helix structure in the the of our the micelle of SDS in is about 2 K. Chem. Google Scholar). As the SDS is raised from 5 to the of α-helix structure to and there are in the between and 100 SDS 100 SDS for our to a micelle of number of SDS molecules micelle is to be with the amylin 100 SDS an of to peptide so that micelle have of amylin NMR D. J. 2004; PubMed Scopus Google Scholar) a for amylin in with SDS that within the as that for the between SDS and a peptide of similar to the amylin data This that Aβ J. J. P. M. A. J. 2007; PubMed Scopus Google Scholar), amylin to as a at the 100 SDS used for In to at we data at in the of SDS the in amylin that in the between and The between to between the two In the the at the with α-helix structure and to by aggregation at the of from an Bax A. J. Chem. Scopus Google Scholar). of in the α-helix for all residues between 5 and and the of between residues 5 and 17 is that for residues the between A. J. PubMed Scopus Google Scholar) and the of amylin. The for the between residues 5 and are of α-helix structure. The a of with α-helix structure M. A. M. J. J. J. Biochem. PubMed Scopus Google Scholar, R. S. A. T. J. 1997; 6: PubMed Scopus Google Scholar). is for the and type of has been to α-helix F. Craik D. J. Biochemistry. 1994; PubMed Scopus Google Scholar, J. Chem. Scopus Google Scholar). The which are the to to be on a from residues to The data suggest a for α-helix structure in the of amylin. the for amylin. The are of α-helix structure. is a in the near residues with a in the α-helix structure. there is of within segment that the of of for an α-helix structure such as and are or between residues 5 and and are the two with with a from α-helix structure in in the α-helix are for the segment with the NMR of NMR structure of amylin is in on used to the NMR structure and to the of the structures are in the N residues are constrained to a hairpin by a disulfide bond between and The of the disulfide is in the α-helix and is a of The is The last residues between and are The core of the structure of an α-helix from residues 5 to of the of the NMR have a kink or in the α-helix between residues and the NMR is on the mean of the α-helix the root mean square of is and we a for that the r. m. s. d. , and we found that the of the structure the segments and are segment 18–22 the with that for residues This that the when the is is of an of the of the and segments in the of the used the R. M. K. J. Mol. 1996; 14: PubMed Scopus Google Scholar) to a of for the angle between segments and The of the amylin structure is of that with αS and the Alzheimer In the of SDS αS an α-helix hairpin structure (17Ulmer T. S. Bax A. J. Biol. Chem. 2005; 280: 43179-43187Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar, S. J. R. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). The peptide when bound to SDS an structure with a kink between residues and (16Coles M. Bicknell W. Watson A. A. Fairlie D. P. Craik D. J. Biochemistry. 1998; 37: 11064-11077Crossref PubMed Scopus (476) Google Scholar). The two segments and the kink but have to with an angle of (16Coles M. Bicknell W. Watson A. A. Fairlie D. P. Craik D. J. Biochemistry. 1998; 37: 11064-11077Crossref PubMed Scopus (476) Google Scholar). have been in the effects of amylin and Aβ, which may to J. PubMed Scopus Google Scholar). the two α-helix residues a more structure residues and and This is with the of the and which suggest the α-helix structure is more in the As amylin a of with the segment to the of the polypeptide The structural and of amylin with the in amylin the of the of human amylin in a A. A. J. Z. W. D. J. 1997; PubMed Scopus Google Scholar) the data A. B. S. 2004; PubMed Scopus Google Scholar). The of amylin, which has the for membranes M. S. A. Langen R. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), the The C-terminal of amylin, which in human amylin is the amyloidogenic M. S. A. Langen R. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, D. P. Biochemistry. 1999; PubMed Scopus Google Scholar), has the of 15N relaxation data for amylin. The relaxation and the suggest a of from the N to the C terminus of amylin. to a correlation for with the P. M. D. J. PubMed Scopus Google Scholar) data for the segment between residues 5 and The (17Ulmer T. S. Bax A. J. Biol. Chem. 2005; 280: 43179-43187Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar) a correlation of For SDS have a correlation of at K. T. J. Chem. 2002; Scopus Google Scholar), and the correlation of a between SDS and a peptide similar in to the amylin J. J. P. A. Biochemistry. 1997; PubMed Scopus Google Scholar). the is we that the correlation of SDS on a number of including and the molecules to the micelle A. D. M. J. Chem. Scopus Google Scholar). the correlation is with amylin to the SDS as a The P. M. D. J. PubMed Scopus Google Scholar) used to which the of on the to The data are in and suggest a of amylin The segment from residues to has the with all have between and the last residues have two residues and to for the relaxation of Amylin in the SDS 5 the of used to the interactions of amylin with SDS micelles. a probe of accessibility to a of to amylin, the C-terminal of the protein is The which is the The more the a of to amylin, of the from residues are The segment at of it is in the micelle C and the segment at of of the of and has at In to we at with This lipid has a spin near the of the so the probe be near the hydrophobic of the In to probe for residues and the at data suggest that amylin is the of the it to In we NMR the solution and of amylin when bound to SDS micelles. SDS are to structure by the structural models available for the amyloidogenic proteins Aβ (16Coles M. Bicknell W. Watson A. A. Fairlie D. P. Craik D. J. Biochemistry. 1998; 37: 11064-11077Crossref PubMed Scopus (476) Google Scholar) and α-synuclein (17Ulmer T. S. Bax A. J. Biol. Chem. 2005; 280: 43179-43187Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar) in from with SDS micelles. that the structure of amylin in the for with more mimetics or of the amylin NMR structure to and are in a of the NMR structure to the from 15N relaxation data amylin an α-helix from about 5 to The structure is but a of the C terminus from for to for form the of the and increases from residues to are constrained to form a hairpin by the disulfide and residues are unfolded. that in to the peptide amylin in that in an C is for amylin (3Cooper G. J. Endocr. Rev. 1994; 15: 163-201Crossref PubMed Scopus (271) Google Scholar), to the interactions of the hormone with the last residues in the amylin structure are we it is that has a in the of the peptide with membranes. The α-helix of amylin has a kink or near residues have that in the of amyloidogenic or from α-helix with the of in the fibrillar structures Scholar). in Aβ the first residues are and a kink in the α-helix in the (16Coles M. Bicknell W. Watson A. A. Fairlie D. P. Craik D. J. Biochemistry. 1998; 37: 11064-11077Crossref PubMed Scopus (476) Google Scholar). The first residues are in Aβ and a between residues and T. M. D. B. H. D. R. U. S. A. 2005; PubMed Scopus Google Scholar). The of α-synuclein of a with an of near residues (17Ulmer T. S. Bax A. J. Biol. Chem. 2005; 280: 43179-43187Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). This to a between and in the and more of α-synuclein with (17Ulmer T. S. Bax A. J. Biol. Chem. 2005; 280: 43179-43187Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar) in the structure J. Chem. Scopus Google Scholar, P. M. D. J. PubMed Scopus Google Scholar, M. J. U. A. Biochemistry. 2001; PubMed Scopus Google Scholar, A. D. P. Biochemistry. 2005; PubMed Scopus Google Scholar, 2007; PubMed Scopus Google Scholar, K. Chem. Google Scholar, D. J. 2004; PubMed Scopus Google Scholar, J. J. P. M. A. J. 2007; PubMed Scopus Google Scholar, to with between in a of α-synuclein M. T. D. R. H. R. U. S. A. PubMed Scopus Google Scholar). For amylin, residues and are thought to the of in amyloid of the peptide U. J. Mol. Biol. 2005; PubMed Scopus Google Scholar). The to the of amylin in the and the in the α-helix structure near residues 18–22 to a in the structure. suggest that may have roles in in the The of α-helix structure in amylin is with A NMR structure of a of human amylin in α-helix structure between residues and 17 (18Nanga J. A. Biochemistry. PubMed Scopus (157) Google Scholar). The from our structure at the C terminus is to effects in the A in SDS a structure of a of A. F. U. A. 2003; PubMed Scopus Google Scholar). A NMR and evidence for α-helix structure D. A. F. D. 2007; PubMed Scopus Google Scholar). the that on structure as as on the and of the A for amylin in is available from M. S. A. Langen R. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). This α-helix structure from residues to The 5 in our structure. This may from the of the at 2 and with for the the at in the in the data are with our structure of amylin. The and accessibility of the spin amylin M. S. A. Langen R. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). residues are in an α-helix to the NMR data and accessibility are in with the As for α-synuclein (17Ulmer T. S. Bax A. J. Biol. Chem. 2005; 280: 43179-43187Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar), protein are correlated with the of of the of segments of the polypeptide bound to is a common theme in bound proteins Biophys. J. 37: Full Text PDF PubMed Scopus Google Scholar). amylin to the micelle the of from to on accessibility to The of amylin are correlated with the data at with an of This that of the in be by the in of amylin. The correlation the of the micelle of in the polypeptide (4Jayasinghe S. A. Langen R. Biochim. Biophys. Acta. 2007; 1768: 2002-2009Crossref PubMed Scopus (157) Google Scholar). in the micelle increase the of α-helix structure and our data with that of the in amylin are at from by the A the of residues is in The in are hydrophobic and the of the whereas the be the M. S. A. Langen R. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). that the of the in the NMR is similar for the two of the which with the data that suggest residues in the are in the that the of the α-helix of amylin is the lipid with the from the N to the C terminus of the are with an of the of amylin lipid membranes H. B. J. J. Mol. Biol. 2006; PubMed Scopus Google Scholar). A our on the of amylin to SDS is in The in an SDS The in the NMR are to spin probe with and The segment between residues 5 and 17 is the of the structure and the of The disulfide residues to form a hairpin that near the of the micelle to a in a hydrophobic The of amylin as a the hormone to the amylin to as a H. B. J. J. Mol. Biol. 2006; PubMed Scopus Google Scholar), we are of such an in the normal function of amylin. the amylin to to A. 2005; PubMed Scopus Google Scholar) and to the A. 2005; PubMed Scopus Google Scholar) so the hydrophobic of the of the hormone facilitate those the of the residues a of from and these residues are at the interface between the micelle and solvent. residues are in the of the to the segment with an angle of The of the N terminus to membranes facilitate the of amylin toxic in the to increase the of amylin and reduce the cost of by molecules within the of the lipid (4Jayasinghe S. A. Langen R. Biochim. Biophys. Acta. 2007; 1768: 2002-2009Crossref PubMed Scopus (157) Google Scholar). The of the segment and at the interface between and may the of the amylin is but we have an of SDS to the peptide with of or solution conditions, membranes amylin aggregation J. Mol. Biol. PubMed Scopus Google Scholar, S. A. Langen R. Biochemistry. 2005; PubMed Scopus Google Scholar, J. Mol. Biol. 2004; PubMed Scopus Google Scholar). The in the correlation of the when are over the and the of a that the suggest that there is in the orientation of the segments and structures This is our a for amylin to membranes, a toxic composed of amylin have to the lipid aggregation of the amyloidogenic segment in the of a to the segment that it to the coupling between the amyloidogenic and segments facilitate the transition to a toxic when membrane-bound amylin NMR of the the to that structural on the membrane-bound aggregates and on the by which for with spin probe and for the available to and to and are for about