Protein aggregationProtein aggregationvia 3D domain swappingvia 3D domain swapping

Mariusz JaskMariusz Jaskoolskilski

Dept. of Crystallography, A.Mickiewicz Univ.Dept. of Crystallography, A.Mickiewicz Univ.Center for Biocrystallographic ResearchCenter for Biocrystallographic Research

PoznanPoznan, Poland, Poland

3D domain swapping (D.Eisenberg)...3D domain swapping (D.Eisenberg)...

a protein domain breaks its contact with other domains;a protein domain breaks its contact with other domains;its place is taken by the same domain of another protein;its place is taken by the same domain of another protein;

interlaced dimer or higher oligomer is formedinterlaced dimer or higher oligomer is formed

• swapped domain: 2-ry str. element or globular domainswapped domain: 2-ry str. element or globular domain• closed monomer: conformation before transitionclosed monomer: conformation before transition• open monomer: conformation after transitionopen monomer: conformation after transition• hinge loop: links the swapped domain to the resthinge loop: links the swapped domain to the rest• closed interface: preserved in the oligomerclosed interface: preserved in the oligomer• open interface: new, absent in the monomeropen interface: new, absent in the monomer

3D domain swapping3D domain swappingexamplesexamples• diphteria toxindiphteria toxin

• RNase (A, BS)RNase (A, BS)• CksHs (cell cycle regulation)CksHs (cell cycle regulation)• CD2CD2• staphylococcal nucleasestaphylococcal nuclease• cro (DNA represor)cro (DNA represor)• spectrin (cytoskeleton)spectrin (cytoskeleton)• antibody fragmentsantibody fragments• human prion proteinhuman prion protein• human cystatin Chuman cystatin C• crystallinscrystallins• growth factors/cytokinesgrowth factors/cytokines• feromon transport/odorant bindingferomon transport/odorant binding• SH3 (signal transduction)SH3 (signal transduction)

bona fidebona fide

quasiquasi

Human cystatin C (Human cystatin C (HHCC)CC)

• small protein (120 aa)small protein (120 aa)• abundant in body fluidsabundant in body fluids• high concentration in cerebrospinal fluidhigh concentration in cerebrospinal fluid• potent inhibitor opotent inhibitor off cysteine proteases cysteine proteases (monomer) (monomer)• N-term. and 2 N-term. and 2 -hairpins bind the enzyme-hairpins bind the enzyme• degradation degradation N-truncated variant N-truncated variant• two S-S bridgestwo S-S bridges in C-terminal part in C-terminal part• structure of structure of N-N-truncated chicken analog knowntruncated chicken analog known• endemic L68Q mutant causes HCCAAendemic L68Q mutant causes HCCAA

AmyloidogenAmyloidogenicic properties ofproperties of HHCCCC

• L68Q mutant more stable as dimer

• dimeric L68Q mutant found in blood of HCCAA patients

• L68Q mutant forms mssive amyloid deposits

• massive brain hemorrhages and early death in HCCAA

• fevers accelerate progress of HCCAA

• N-truncated protein (THCC) found in amyloid deposits

• normal variant (L68) also participates in fibril formation

• elevated temp. or lower pH lead to oligomerization in vitro

EnergyEnergy

partial unfoldingpartial unfoldingthermalthermalenergyenergy

openopeninterfaceinterface

L68QL68Qdestabilizationdestabilization

DimerDimerDimerDimer

MonomerMonomer

MonomerMonomer

WTWT L68QL68Q

HCC – speculative thermodynamicsHCC – speculative thermodynamicshydrophilic patchhydrophilic patchon exposed surfaceon exposed surface

L68Q HCC dimers are observed in blood plasma of HCCAA patientsL68Q HCC dimers are observed in blood plasma of HCCAA patients

Even with one exchangEven with one exchangeeble ble domaindomainopen-ended 3D domain swapping open-ended 3D domain swapping may lead to infinite polymerizationmay lead to infinite polymerization

Incubation at pH 37° CIncubation at pH 37° C

+0.5 M Gnd.HCl, pH 7.4+0.5 M Gnd.HCl, pH 7.4

HCCHCC L68Q HCCL68Q HCC

L68Q HCCL68Q HCC

HCCHCC

stab1 and stab2 of L68Q / HCCstab1 and stab2 of L68Q / HCC

ConclusionsConclusions

• Full-length and N-truncated HCC are both Full-length and N-truncated HCC are both capable of dimerization via 3D domain swappingcapable of dimerization via 3D domain swapping

• The dimer open interface in the hinge region is The dimer open interface in the hinge region is flexibleflexible

• S-S bridges preventing domain swapping S-S bridges preventing domain swapping inhibit dimerizationinhibit dimerization

CollaborationCollaboration

Robert JanowskiRobert Janowski A.Mickiewicz Univ. PoznanA.Mickiewicz Univ. PoznanMaciej KozakMaciej Kozak

Magnus AbrahamsonMagnus Abrahamson Lund Univ. Lund Univ.Anders GrubbAnders GrubbMaria NilssonMaria NilssonXin WangXin Wang

Zbigniew GrzonkaZbigniew Grzonka Univ. of Gdansk Univ. of Gdansk