Alpha-keto-aldehyde binding mechanism reveals a novel lead structure motif for proteasome inhibition

Elucidation of the alpha-keto-aldehyde binding mechanism: a lead structure motif for proteasome inhibition

Macropain subunit Y7, Proteinase YSCE subunit 7, Multicatalytic endopeptidase complex subunit Y7

Macropain subunit Y13, Proteinase YSCE subunit 13, Multicatalytic endopeptidase complex subunit Y13

Macropain subunit PRE6, Proteinase YSCE subunit PRE6, Multicatalytic endopeptidase complex subunit PRE6

Macropain subunit PUP2, Proteinase YSCE subunit PUP2, Multicatalytic endopeptidase complex subunit PUP2

Macropain subunit PRE5, Proteinase YSCE subunit PRE5, Multicatalytic endopeptidase complex subunit PRE5

Macropain subunit C1, Proteinase YSCE subunit 1, Multicatalytic endopeptidase complex subunit C1

Macropain subunit C7-alpha, Proteinase YSCE subunit 7, Multicatalytic endopeptidase complex C7, Proteasome component Y8, SCL1 suppressor protein

Macropain subunit PUP1, Proteinase YSCE subunit PUP1, Multicatalytic endopeptidase complex subunit PUP1

Macropain subunit PUP3, Multicatalytic endopeptidase complex subunit PUP3

Macropain subunit C11, Proteinase YSCE subunit 11, Multicatalytic endopeptidase complex subunit C11

Macropain subunit PRE2, Proteinase YSCE subunit PRE2, Multicatalytic endopeptidase complex subunit PRE2

Multicatalytic endopeptidase complex subunit C5

Macropain subunit PRE4, Proteinase YSCE subunit PRE4, Multicatalytic endopeptidase complex subunit PRE4

Macropain subunit PRE3, Proteinase YSCE subunit PRE3, Multicatalytic endopeptidase complex subunit PRE3

MTDRYSFSLTTFSPSGKLGQIDYALTAVKQGVTSLGIKATNGVVIATEKKSSSPLAMSETLSKVSLLTPDIGAVYSGMGP
DYRVLVDKSRKVAHTSYKRIYGEYPPTKLLVSEVAKIMQEATQSGGVRPFGVSLLIAGHDEFNGFSLYQVDPSGSYFPWK
ATAIGKGSVAAKTFLEKRWNDELELEDAIHIALLTLKESVEGEFNGDTIELAIIGDENPDLLGYTGIPTDKGPRFRKLTS
QEINDRLEAL

GSRRYDSRTTIFSPEGRLYQVEYALESISHAGTAIGIMASDGIVLAAERKVTSTLLEQDTSTEKLYKLNDKIAVAVAGLT
ADAEILINTARIHAQNYLKTYNEDIPVEILVRRLSDIKQGYTQHGGLRPFGVSFIYAGYDDRYGYQLYTSNPSGNYTGWK
AISVGANTSAAQTLLQMDYKDDMKVDDAIELALKTLSKTTDSSALTYDRLEFATIRKGANDGEVYQKIFKPQEIKDILVK
TGIT

GYDRALSIFSPDGHIFQVEYALEAVKRGTCAVGVKGKNCVVLGCERRSTLKLQDTRITPSKVSKIDSHVVLSFSGLNADS
RILIEKARVEAQSHRLTLEDPVTVEYLTRYVAGVQQRYTQSGGVRPFGVSTLIAGFDPRDDEPKLYQTEPSGIYSSWSAQ
TIGRNSKTVREFLEKNYDRKEPPATVEECVKLTVRSLLEVVQTGAKNIEITVVKPDSDIVALSSEEINQYVTQIEQEKQE
Q

DRGVSTFSPEGRLFQVEYSLEAIKLGSTAIGIATKEGVVLGVEKRATSPLLESDSIEKIVEIDRHIGCAMSGLTADARSM
IEHARTAAVTHNLYYDEDINVESLTQSVCDLALRFGEGASGEERLMSRPFGVALLIAGHDADDGYQLFHAEPSGTFYRYN
AKAIGSGSEGAQAELLNEWHSSLTLKEAELLVLKILKQVMEEKLDENNAQLSCITKQDGFKIYDNEKTAELIKELKEKEA
AE

FRNNYDGDTVTFSPTGRLFQVEYALEAIKQGSVTVGLRSNTHAVLVALKRNADELSSYQKKIIKCDEHMGLSLAGLAPDA
RVLSNYLRQQCNYSSLVFNRKLAVERAGHLLCDKAQKNTQSYGGRPYGVGLLIIGYDKSGAHLLEFQPSGNVTELYGTAI
GARSQGAKTYLERTLDTFIKIDGNPDELIKAGVEAISQSLRDESLTVDNLSIAIVGKDTPFTIYDGEAVAKYI

GTGYDLSNSVFSPDGRNFQVEYAVKAVENGTTSIGIKCNDGVVFAVEKLITSKLLVPQKNVKIQVVDRHIGCVYSGLIPD
GRHLVNRGREEAASFKKLYKTPIPIPAFADRLGQYVQAHTLYNSVRPFGVSTIFGGVDKNGAHLYMLEPSGSYWGYKGAA
TGKGRQSAKAELEKLVDHHPEGLSAREAVKQAAKIIYLAHEDNKEKDFELEISWCSLSETNGLHKFVKGDLLQEAIDFAQ
KEIN

AGYDRHITIFSPEGRLYQVEYAFKATNQTNINSLAVRGKDCTVVISQKKVPDKLLDPTTVSYIFCISRTIGMVVNGPIPD
ARNAALRAKAEAAEFRYKYGYDMPCDVLAKRMANLSQIYTQRAYMRPLGVILTFVSVDEELGPSIYKTDPAGYYVGYKAT
ATGPKQQEITTNLENHFKKSKIDHINEESWEKVVEFAITHMIDALGTEFSKNDLEVGVATKDKFFTLSAENIEERLVAIA
EQD

TTIVGVKFNNGVVIAADTRSTQGPIVADKNCAKLHRISPKIWCAGAGTAADTEAVTQLIGSNIELHSLYTSREPRVVSAL
QMLKQHLFKYQGHIGAYLIVAGVDPTGSHLFSIHAHGSTDVGYYLSLGSGSLAAMAVLESHWKQDLTKEEAIKLASDAIQ
AGIWNDLGSGSNVDVCVMEIGKDAEYLRNYLTPNVREEKQKSYKFPRGTTAVLKESIVNICD

SDPSSINGGIVVAMTGKDCVAIACDLRLGSQSLGVSNKFEKIFHYGHVFLGITGLATDVTTLNEMFRYKTNLYKLKEERA
IEPETFTQLVSSSLYERRFGPYFVGPVVAGINSKSGKPFIAGFDLIGCIDEAKDFIVSGTASDQLFGMCESLYEPNLEPE
DLFETISQALLNAADRDALSGWGAVVYIIKKDEVVKRYLKMRQD

MDIILGIRVQDSVILASSKAVTRGISVLKDSDDKTRQLSPHTLMSFAGEAGDTVQFAEYIQANIQLYSIREDYELSPQAV
SSFVRQELAKSIRSRRPYQVNVLIGGYDKKKNKPELYQIDYLGTKVELPYGAHGYSGFYTFSLLDHHYRPDMTTEEGLDL
LKLCVQELEKRMPMDFKGVIVKIVDKDGIRQVDDFQAQ

TTTLAFRFQGGIIVAVDSRATAGNWVASQTVKKVIEINPFLLGTMAGGAADCQFWETWLGSQCRLHELREKERISVAAAS
KILSNLVYQYKGAGLSMGTMICGYTRKEGPTIYYVDSDGTRLKGDIFCVGSGQTFAYGVLDSNYKWDLSVEDALYLGKRS
ILAAAHRDAYSGGSVNLYHVTEDGWIYHGNHDVGELFWKVKEEEGSFNNVIG

QFNPYGDNGGTILGIAGEDFAVLAGDTRNITDYSINSRYEPKVFDCGDNIVMSANGFAADGDALVKRFKNSVKWYHFDHN
DKKLSINSAARNIQHLLYGKRFFPYYVHTIIAGLDEDGKGAVYSFDPVGSYEREQCRAGGAAASLIMPFLDNQVNFKNQY
EPGTNGKVKKPLKYLSVEEVIKLVRDSFTSATERHIQVGDGLEILIVTKDGVRKEFYELKRD

TQQPIVTGTSVISMKYDNGVIIAADNLGSYGSLLRFNGVERLIPVGDNTVVGISGDISDMQHIERLLKDLVTENAYDNPL
ADAEEALEPSYIFEYLATVMYQRRSKMNPLWNAIIVAGVQSNGDQFLRYVNLLGVTYSSPTLATGFGAHMANPLLRKVVD
RESDIPKTTVQVAEEAIVNAMRVLYYRDARSSRNFSLAIIDKNTGLTFKKNLQVENMKWDFAKDIKGYGTQKI

TSIMAVTFKDGVILGADSRTTTGAYIANRVTDKLTRVHDKIWCCRSGSAADTQAIADIVQYHLELYTSQYGTPSTETAAS
VFKELCYENKDNLTAGIIVAGYDDKNKGEVYTIPLGGSVHKLPYAIAGSGSTFIYGYCDKNFRENMSKEETVDFIKHSLS
QAIKWDGSSGGVIRMVVLTAAGVERLIFYPDEYEQL

Crystalls appeared during one week using 100mM MES, 20mM MG2Ac, 12% MPD, pH 6.9, VAPOR DIFFUSION, HANGING DROP, temperature 293K

20S prteasome bound to calpain inhibitor I (archaeal CP, however high resolution)

Yeast 20S proteasome in complex with the natural product fellutamide B (aldehyde compound)

Yeast 20S proteasome in complex with the natural product epoxomicin (bivalent binding of the ligand via formation of a morphine-ring)

THE UNBOUND FORM OF THE INHIBITOR IS PEPTIDYL ALPHA-KETO ALDEHYDE Z-LEU-LEU-TYRCOCHO. IN A TWO-STEP BINDING MECHANISM THE INHIBITOR REACTS WITH THR1 OG AND THR1 N FORMING A 5,6-DIHYDRO-2/H/-1,4-OXAZIN RING. THE BOUND FORM OF THE INHIBITOR IS REPRESENTED BY CHEMICAL COMPONENT EP9.

The assymetric unit is composed of the physiological 20S proteasome
ubiquitin, protease, inhibitor, central antiparallel beta-sheet flanked by helices, turnover of ubiquitinated substrates, nucleus, cytosol, HYDROLASE-HYDROLASE INHIBITOR complex