Exploring the amyloid state of proteins by micro

The Amyloid State of Proteinsand Micro-X-ray DiffractionQuickTime and adecompressorare needed to see this picture.Meytal Landau, Michael Sawaya, Luki Goldschmidt, ArthurLaganowsky, Stuart Sievers, Howard Chang, Lin Jiang,Duilio Cascio, Jacques Colletier, Anni Zhao, BorisBrumshtein, Cong Liu, Poh Teng, Jiyong Park, MingleiZhao, Angie Soriaga, Heather McFarlane, Rebecca NelsonDavid Baker, John Karanicolas , Christian Riekel ,Roland Riek , James Nowick , Jan Munch

Micro X-ray Diffractionfor Structural BiologyThe biological problem and ourneed for micro-X-ray diffractionWhat micro-X-ray diffractionhas allowed us to findFuture opportunities for micro-and nano-X-ray diffraction in biology

Amyloid• Unbranched, elongated protein fibrils• Associated with varied diseases (e.g. CJDAlzheimer’s, Dialysis-related amyloidosis)• Cross-β diffraction pattern shows β strandsperpendicular to fiber axis: common spineKishimoto, Namba et al (2004)

Amyloid Fibril-Related ConditionsAmyloid (24) Prion (transmissible) Amyloid-likeAlzheimer’s Aβ [Psi+] Sup35Alzheimer’s Tau [Ure2] Ure3 Parkinson’s α-synucleinDiabetes IIAmylinaka IAPPLouGehrig’s(ALS)SuperoxideDismutaseTDP-43InjectionInsulinCJD, GSSPrPHIV SexualSEVIamyloidosisKurutransmissionDialysisamyloidosisβ2-microglobulinBSE, vCJD(mad cow)PrP Cancer p53SenileTrans-amyloidosisthyritin

My Scientific Dilemma in 2001Important biological problem—structure of amyloid5.4 M Alzheimer’s patients in US in 2010~19 M patients expected by 2050Economic burden:In 2010 ~$ 183B in US heath care costs~11 M people provide unpaid care of AD patientsEssentially no structural informationStructure-based design impossibleAmyloid crystals discovered but 30,000 times smallerthan crystals we had previously worked with

Short segments of fiber-forming proteinsform both amyloid fibers and microcrystalsGNNQQNYsinglefibril~80ÅBalbirnie et al. PNAS 2001singlefibril~80ÅNNQQNNQQIn both microcrystalsand fibrils,β-strands are normalto the long axis.NNQQNYGNNQQNY~280Å0.5mmGNNQQNY fibrils exhibit allproperties of amyloid fibrils:dye binding, cooperativeaggregation kinetics, stability,cross-β diffraction1μm1μmFibers seem to growfrom tips of crystals

10-100 mM < 24 hrGNNQQNY microcrystalsX-raysCrystal width = ~280 ÅBalbirnie et al. PNAS 2001Research of Ruben Diaz & Donald Caspar

Crystal unit cell dimensions and space groupdetermined from X-ray powder diffractionBalbirnie et al. PNAS 2001

Packing of GNNQQNY peptides in microcrystalsBalbirnie, Grothe, & Eisenberg PNAS 2001

Failed Approachesto the Structure2001-2005X-ray powder diffractionTextured X-ray powder diffractionElectron diffractionSolid-state NMR

Microfocus is required to reducebackground noiseChristian Riekel100 µm beam diameterStandard at home or synchrotronnly a fraction of incoming X-rays impinge crystal.High background obscures reflections1 µm beam diameterESRF ID13All X-rays impinge crystalLow background, good I/σ.

Microcrystals ofsegments of 15 amyloidformingproteins haveyielded >90 steric zippersX-ray beamCrystal50μGlass pin

GNNQQNY, a dry steric zipperFibers and microcrystal have 50,000 layersExtended strands, H-bonded 4.8Åapart into in register β-sheetsGln, Asn, Tyr sidechains also H-bondedTwo sheets, interdigitated, with tightlycomplementary sidechains, bondedby van der Waals forcesMore tightly complementary thanany previous structure in PDBDry between the β-sheetsNelson et al. Nature 2005

caView down the fibril axis shows selfcomplementaryinteractions betweenpaired beta sheets of the steric zipperand weak interactions between pairsOne unit cellNelson et al. Nature, 2005Nine unit cells

As of April 2011, ~90 X-raycrystal structures of fibrillikedry steric zippers from12 disease-relatedproteins:Sup35 and PrP prionsAβ and Tau - Alzheimer’sα-synuclein - Parkinson’sIAPP - Diabetes type 2SOD - ALSTransthyritin-familialamyloidosisLysozyme-lysozymeamyloidosisInsulin-injectionamyloidosisSEVI-HIV transmission

Structure-baseddesign of ablocker offibril formationfor TauSievers et al.Nature 2011

D-TLKIVW prevents fibril formation by Tau K12K12Fluorescent Units – 510 nmK12 + D-TLKIVWHoursResearch of Stuart Sievers & Howard Chang

Defining theAmyloid Pharmacophore

GGVVIA +JugloneMeytal LandauGGVVIA + R-(−)-ApomorphineGGVVIA + 1.2,2′-DihydroxybenzophenoneGGVVIA +CurcuminGGVVIA +Chicago SkyBlueGGVVIA +Phenol RedGGVVIA +BenserazideVQIVYK +RhodamineVQIVYK+PhenolRedVQIVYK +NeocuproineVQIVYK +JugloneVQIVYK +PerphenazineVQIVYK +AZETGGVVIA +EGCGKLVFFA +ThTVQIVYK +Phenol RedAbeta17-40+ FDDNPVQIVYK +ThTGVVEVD +Orange GVQIVYK +MethyleneBlueSSTNVG +Phenol redVQIVYK +Azure CVQIVYK +MeclocyclinesulfosalicylateVQIVYK + 1,2-NaphthoquinoneVQIVYK+RhodamineVQIVYK+Hexadecyltrimethylammonium bromideVQIVYK+ApomorphineVQIVYK +RolitetracyclineGGVVIA+ThTGGVVIA +RhodamineGGVVIA +Rifamycin SVGGVVIA + 1,2-NaphthoquinoneSSTNVG+Phenol RedVQIVYK +Chicago skyblue 6BVQIVYK+DobutamineGGVVIA+Chicago SkyBlueGGVVIA +Orange GGGVVIA+Azure CGGVVIA +Trimethyl(tetradecyl)ammoniumbromideGGVVIA + CreosolGGVVIA +Eosin YVQIVYK+FDDNP

KLVFFA (from Amyloid Beta) + Orange G

Structure of KLVFFA with Orange GKLVFFA + Orange G 5-10:1mM10-30% w/v Polyethylene glycol 1,500,20-30% v/v GlycerolRmerge = 18.4%; Resolution=1.8A;Completeness=96.4%C2;a, b, c 43.64 26.85 9.55Å;β 91.55 °Rwork/Rfree(%)= 22.6/27.6The asymmetric unit contains:Two peptide segmentsOne Orange GSix water moleculesLandau et al. PLoS BiologyIn press

Towards atomicprotein structures fromnano-crystals

Amyloid-like crystalsof KLIMY from PAPgrown by Anni ZhaoX-ray beam1 µm diameter4

A 3-sheet prionstructure?Only nanocrystalsavailable

Nano-crystals oflonger (11-residue) segmentPerhaps from the toxic segmentof alpha-synuclein(Parkinson’s disease))

Towards determiningstructures of crystals andother ordered proteinaggregatesfound in human and otheranimal cells

10 micron Mictigen micromesh in cryostream

2Drosophilablood cells1

Drosophila crystal cellon 10 µm mesh10umThe diffraction pattern revealspowder rings at 50, 38, 26, 13,4.2, and 3.7 Å spacingsMichael Sawaya, Mari Gingery, Duilio CascioGroup of Utpal Banerjee, UCLAJacques Colletier, Christian Riekel, ESRF-IBS

Eosinophile (type of white blood cell) granulesCharacterized by George Palade et al. (1965) by EMUnit cell dimensions tentatively determined byAlice Soragni, Jacques Colletier,Manfred Brunner, Christian Riekel

Cell Types Containing Intracellular Crystalline InclusionsSpecies Cell type Description of crystals Protein ReferenceDrosophila Crystal cells Intracellular inclusions [lamellar] Prophenol oxidase (Shrestha & Gateff 1982);T. M. Rizki & R. M. Rizki 1980)HumanRatGuinea pigMouseEosinophil leukocytesMembrane-bound granules [0.3-1.2 um]Granule cores [lamellar]? (Miller et al. 1966)Human B cell lymphomas ER-bound crystal rods [lamellar] Ig (Peters et al. 1984)Human Abnormal mitochondria inmuscle myopathiesCrystal rods in outer mitochondrialmembrane compartmentMitochondrial creatine kinase (Stadhouders et al. 1994)Human Kidney mitochondria Helical crystals in outer mitochondrial ? Jasmin 1978compartment; linear and flexuous crystalsin matrixHumanDogMonkeyLiver mitochondria Intramitochondrial [lamellar] ? Wills 1965Human Ad5-infected KB cells Intranuclear adenovirus-induced inclusions heteromeric capsid protein (Franqueville et al. 2008);formed of penton base and (Carstens et al. 1975)fiber subunitsFrog Oocyte mitochondria Intramatrix & intracristae inclusions[lamellar]? Spornitz 1972Armadillo Epididymus Single membrane-bound cytoplasmiccrystalline rods [lamellar]? (Edmonds et al. 1973)Earthworm Spermatazoa Intranuclear inclusions [lamellar] ? (Anderson et al. 1968)Tomato Young leaf mesophyll Intracellular inclusions [Cubic] ? Singh 1976Helicobacter pyloriCausative agent of gastricdiseasesCytoplasmic paracrystalline inclusions Pfr, bacterial ferritin (Frazier et al. 1993)Photorhabdus luminescens Entomopathogenic bacteria Intracellular inclusions cipAcipB(Bintrim et al. 1998)Bacillus thuringiensis Insecticidal bacteria parasporal crystals Cry (Hofte et al. 1989)Paenibacillus popilliae Insecticidal bacteria parasporal crystals ? Weiner 1978Brevibacillus laterosporus Mosquitocidal bacteria parasporal crystals ? (Smirnova et al. 1996); (Orlovaet al. 1998)

Summary•Micro-X-ray diffraction has enabled the determination ofthe atomic structures of the amyloid state, includingdesign of inhibitors and partial definition of the amyloidpharmacophor•The structures of still smaller crystals are needed tounderstand the toxic mechanism of amyloid perhapswith LCLS• LCLS offers the possibility of learning the atomicstructure of ordered aggregates within biological cells,including amyloid aggregates

The Amyloid State of ProteinsUCLA: Rebecca Nelson, Michael Sawaya, Marcin Apostol, Melinda BalbirnieMagdalena Ivanova, Stuart Sievers, Jed Wiltzius, Minglei Zhao, Cong LiuLuki Goldschmidt, Heather Mcfarlane, Howard Chang, Anni ZhaoLin Jiang, Jiyong Park, Jacques Colletier, Poh Teng, Boris BhrumsteinUniv. of Washington: John Karanicolis, David BakerESRF: Christain Riekel ETH: Roland Riek, Alice SoragniStuartSieversMike SawayaMelindaJedBalbirnieWiltziusRebecca NelsonMarcinApostolMarcinApostolJacques ColletierArtLaganowskyDuilio Meytal LandauCascioLukiGoldschmidtPoh Teng

CollaboratorsChristian RiekelESRFDavid BakerUniversity ofWashingtonJamesNowickUCIRoland Riek (ETH Zurich), John Karanicolas (U. Kansas),Jan Münch (Ulm)