Supplemental Information
Structures of apo and product-bound human L-asparaginase:
Insights into the mechanism of autoproteolysis
and substrate hydrolysis
Julian Nomme, Ying Su, Manfred Konrad, and Arnon Lavie
Page 1 of 9
Figure S1. hASNase3 uncleaved structures. The uncleaved state of hASNase3 was
observed in both protomers of the sulfate-complex structure (only protA shown, in cyan), and in
protAs of the malonate-complex (green) and partially-cleaved ASP-complex (magenta)
structures. (a) Overlay of the uncleaved protomers shows an overall identical structure. We
could not model beyond the C-terminal helix of the a-chain until 2-3 residues prior to the scissile
residue T168. While the secondary structural elements that span this region are identical, the
residues preceding V169 occupy slightly differing conformations. This demonstrates the
inherent flexibility of this region. (b) Detailed overlay around T168. Note that the carbonyl of G9
points towards the side chain of H8 in these uncleaved structures. G9 flips in the cleaved state.
Page 2 of 9
Figure S2. Glycine 9 flips conformation between the cleaved and uncleaved states.
Stereo view of the ASP-complex hASNase3 structure, where the uncleaved protA (magenta) is
overlaid on the cleaved protB (pink). The Gly9 carbonyl interacts with the His8 side chain in the
uncleaved state but is flipped in the cleaved state (black arrow).
Page 3 of 9
Figure S3.
Q7L266-ASGL1_HUMAN
Q4R7U8-ASGL1_MACFA
Q32LE5-ASGL1_BOVIN
Q8C0M9-ASGL1_MOUSE
Q8VI04-ASGL1_RAT
Q6GM78-ASGL1_XENLA
Q5BKW9-ASGL1_DANRE
P37595-IAAA_ECOLI
Q7CQV5-IAAA_SALTY
Q9ZSD6-ASPG_LUPLU
P30364-ASPG_LUPAN
P50288-ASPG_LUPAL
Q54WW4-ASGX_DICDI
P50287-ASPGA_ARATH
P20933-ASPG_HUMAN
B4NWI1-ASPG1_DROYA
Q8MR45-ASPG1_DROME
B4QHB1-ASPG1_DROSI
Q9H6P5-TASP1_HUMAN
Q8R1G1-TASP1_MOUSE
-------------------------------------MN--PIVVVHGGGAGPIS-----------------------------------------MN--PIVVVHGGGAGPIS-----------------------------------------MN--PVVVVHGGGASNIS------------------------MACARGTVAPPVRASIDVS--LVVVVHGGGASNIS------------------MATARPSSCGRDSVPATPRASIDVS--LVVVVHGGGASNIS-----------------------------------------MK--PVIVVHGGAGK-IV-----------------------------------------ML--PVVVVHGGAGH-IP-----------------------------------------MG-KAVIAIHGGAGA-ISR----------------------------------------MN-KAVIAIHGGAGA-IAR----------------------------------------MG-GWSIALHGGAGD-IP-----------------------------------------MG-GWSIALHGGAGD-IP-----------------------------------------MG-GWSIALHGGAGD-IP-----------------------------------------MNKKSVLVIHGGAGV-ISK----------------------------------------MV-GWAIALHGGAGD-IP---------------MAR—KSNLPVLLVPFLLCQALVRC--SSPLPLVVNTW----------------------MKRHLKACLWVLCFASTALSSLAD-TTSP-------------KPTLASA
-----------MRRHLRASLWILCLATMAFSILAAVNTSP-------------KPTLTSA
-----------MRTHLRASLWVLCLASMAFSILAAVNTSP-------------KPTLASA
MTMEKGMSSGEGLPSRSSQVSAGKITAKELETKQSYKEKRGGFVLVHAG-AGYHS----MIMEKGMNSGEGLPSRSSQASAAKVTVKELETQQPCKEKRGGFVLVHAG-AGYHS----:
:.
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33
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54
54
Q7L266-ASGL1_HUMAN
Q4R7U8-ASGL1_MACFA
Q32LE5-ASGL1_BOVIN
Q8C0M9-ASGL1_MOUSE
Q8VI04-ASGL1_RAT
Q6GM78-ASGL1_XENLA
Q5BKW9-ASGL1_DANRE
P37595-IAAA_ECOLI
Q7CQV5-IAAA_SALTY
Q9ZSD6-ASPG_LUPLU
P30364-ASPG_LUPAN
P50288-ASPG_LUPAL
Q54WW4-ASGX_DICDI
P50287-ASPGA_ARATH
P20933-ASPG_HUMAN
B4NWI1-ASPG1_DROYA
Q8MR45-ASPG1_DROME
B4QHB1-ASPG1_DROSI
Q9H6P5-TASP1_HUMAN
Q8R1G1-TASP1_MOUSE
----------------------KDRKERVHQGMVRAATVGYGILRE--GGS--AVDAVEG
----------------------KDRKERMHQGIVRAATVGYGILRE--GGS--AVDAVEG
----------------------KDRKERVRQGILRAATVGYNILKQ--GGS--AVDAVEG
----------------------ANRKELVREGIARAATEGYKILKA--GGS--AVDAVEG
----------------------PGRKELVSEGIAKAATEGYNILKA--GGS--AVDAVEG
----------------------EELDATYRAGVKRAVLKGYDVLSQ--GGS--ALTAVEE
----------------------KERTEESTIGVKEAARTGYAILQR--GGS--AVDAVVE
------------------AQMSLQQELRYIEALSAIVETGQKMLEA--GES--ALDVVTE
------------------AQMSHEQELRYIQALSEIVESGQKMLEA--GDS--ALDVVTE
------------------FSLPPERRKPREEGLRHCLQIGVEALKA--QKP--PLDVVEL
------------------FSLPPERRQPREEGLRHCLQIGVEALKS--QKP--PLDVVEL
------------------FSLPPERRKPREEGLRHCLQIGIEALKA--QNP--PLDVVEL
------------------STISKEREEIFLNSLKNILLAGKIILKQ--GGT--SLDVVQE
------------------IDLPDERRIPRESALRHCLDLGISALKS--GKP--PLDVAEL
-------------------------------PFKNATEAAWRALAS--GGS--ALDAVES
FSGKSKTTAVSTALKANKTASELLPMVINTWNFTAANVLAWRILKQSKGGLRQTRNAVVE
FSGKAGTTAV----KANKTTGELLPMVINTWNFTAANVLAWRILKQSKGGLRQTRNAVVE
FSGKAGTPAV----KANKTTGELLPMVINTWNFTAANVLAWRILKQSKGGLRQTRNAVVE
----------------------ESKAKEYKHVCKRACQKAIEKLQA--GAL--ATDAVTA
----------------------ESKAKEYKHVCKRACQKAIEKLQA--GAL--ATDAVAA
.
*
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50
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67
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Q7L266-ASGL1_HUMAN
Q4R7U8-ASGL1_MACFA
Q32LE5-ASGL1_BOVIN
Q8C0M9-ASGL1_MOUSE
Q8VI04-ASGL1_RAT
Q6GM78-ASGL1_XENLA
Q5BKW9-ASGL1_DANRE
P37595-IAAA_ECOLI
Q7CQV5-IAAA_SALTY
Q9ZSD6-ASPG_LUPLU
P30364-ASPG_LUPAN
P50288-ASPG_LUPAL
Q54WW4-ASGX_DICDI
P50287-ASPGA_ARATH
P20933-ASPG_HUMAN
B4NWI1-ASPG1_DROYA
Q8MR45-ASPG1_DROME
B4QHB1-ASPG1_DROSI
Q9H6P5-TASP1_HUMAN
Q8R1G1-TASP1_MOUSE
AVVALED-DPEFNAGCGSVLNTNGEVEMDASIMDGKDLSAGAVSAVQCIANPIKLAR--AVVALED-DPEFNAGCGSVLNTDGEVEMDASIMDGKDLSVGAVSAVRCIANPIKLAR--AVTVLED-DPDFNAGCGSVLNENGEVEMDASIMNGKDLSAGAVSAVRCIANPIKLAR--AVTVLEN-DPEFNAGYGSVLNVNGDIEMDASIMDGKDLSAGAVSAVRCIANPVKLAR--AVTMLEN-DPEFNAGYGSVLNADGDIEMDASIMDGKDLSAGAVSAVRCIANPVKLAR--AVIVLED-EQIFNAGHGSVLNEKGDIEMDAIIMDGKNLDSGAVSAIRNIANPIKLAR--AVALMET-NPRFNAGRGSVLNIKGEVEMDALVMDGRTLDSGAVSAVRRIANPVQLAR--AVRLLEE-CPLFNAGIGAVFTRDETHELDACVMDGNTLKAGAVAGVSHLRNPVLAAR--AVRLLEA-CPLFNAGIGAVYTRDGTHELDACVMDGNTLKAGAVAGVSHVRHPVLAAR--VVRELEN-IEHFNAGIGSVLTNSGTVEMEASIMDGNTMKCGAVSGLSTVLNPISLAR--VVRELEN-IQHFNAGIGSVLTNSGTVEMEASIMDGKTMKCGAVSGLSTVLNPISLAR--VVRELEN-IQHFNAGIGSVLTNSGTVEMEASIMDGNTMKCGAVSGLNTVMNPISLAR--AVRLLEE-DPIYNAGKGSVFTELGTNEMDAAIMDGTNLKAGAVGGVSIIRNPIIAAR--VVRELEN-HPDFNAGKGSVLTAQGTVEMEASIMDGKTKRCGAVSGLTTVVNPISLAR--GCAMCEREQCDGSVGFGGSPDELGETTLDAMIMDGTTMDVGAVGDLRRIKNAIGVAR--GCSKCEKLQCDRTVGYGGSPDELGETTLDAMVMDGATMEVGAVAGLRRIKDAIKVAR--GCSKCEKLQCDRTVGYGGSPDELGETTLDAMVMDGATMDVGAVAGLRRIKDAIKVAR--GCSKCEKLQCDRTVGYGGSPDELGETTLDAMVMDGATMDVGAVAGLRRIKDAIKVAR--ALVELED-SPFTNAGMGSNLNLLGEIECDASIMDGKSLNFGAVGALSGIKNPVSVANRLL
ALVELED-SPFTNAGIGSNLNLLGEIECDASIMDGKSLNFGAVGALSGIKNPVSVAHRLL
*
..* *.
:* :*:*
***. : : ..: *.
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Page 4 of 9
Q7L266-ASGL1_HUMAN
Q4R7U8-ASGL1_MACFA
Q32LE5-ASGL1_BOVIN
Q8C0M9-ASGL1_MOUSE
Q8VI04-ASGL1_RAT
Q6GM78-ASGL1_XENLA
Q5BKW9-ASGL1_DANRE
P37595-IAAA_ECOLI
Q7CQV5-IAAA_SALTY
Q9ZSD6-ASPG_LUPLU
P30364-ASPG_LUPAN
P50288-ASPG_LUPAL
Q54WW4-ASGX_DICDI
P50287-ASPGA_ARATH
P20933-ASPG_HUMAN
B4NWI1-ASPG1_DROYA
Q8MR45-ASPG1_DROME
B4QHB1-ASPG1_DROSI
Q9H6P5-TASP1_HUMAN
Q8R1G1-TASP1_MOUSE
Q7L266-ASGL1_HUMAN
Q4R7U8-ASGL1_MACFA
Q32LE5-ASGL1_BOVIN
Q8C0M9-ASGL1_MOUSE
Q8VI04-ASGL1_RAT
Q6GM78-ASGL1_XENLA
Q5BKW9-ASGL1_DANRE
P37595-IAAA_ECOLI
Q7CQV5-IAAA_SALTY
Q9ZSD6-ASPG_LUPLU
P30364-ASPG_LUPAN
P50288-ASPG_LUPAL
Q54WW4-ASGX_DICDI
P50287-ASPGA_ARATH
P20933-ASPG_HUMAN
B4NWI1-ASPG1_DROYA
Q8MR45-ASPG1_DROME
B4QHB1-ASPG1_DROSI
Q9H6P5-TASP1_HUMAN
Q8R1G1-TASP1_MOUSE
Q7L266-ASGL1_HUMAN
Q4R7U8-ASGL1_MACFA
Q32LE5-ASGL1_BOVIN
Q8C0M9-ASGL1_MOUSE
Q8VI04-ASGL1_RAT
Q6GM78-ASGL1_XENLA
Q5BKW9-ASGL1_DANRE
P37595-IAAA_ECOLI
Q7CQV5-IAAA_SALTY
Q9ZSD6-ASPG_LUPLU
P30364-ASPG_LUPAN
P50288-ASPG_LUPAL
Q54WW4-ASGX_DICDI
P50287-ASPGA_ARATH
P20933-ASPG_HUMAN
B4NWI1-ASPG1_DROYA
Q8MR45-ASPG1_DROME
B4QHB1-ASPG1_DROSI
Q9H6P5-TASP1_HUMAN
Q8R1G1-TASP1_MOUSE
-------LVMEKTPHCFLTDQGAAQFAAAMGVPEIPGEKLVTERNKKRLEKEKHEKGAQ-------LVMEKTPHCFLTDQGAAQFAAAMGVPEIPGEKLVTEKNKKRLEKEKHEKGAQ-------LVMDKTPHCFLTDQGAARFAAANGIPTIPGQQLVTERSRKRLEKEKLEKDAQ-------LVMEKTPHCFLTGHGAEKFAEDMGIPQVPVEKLITERTKKHLEKEKLEKGAQ-------LVMEKTPHCFLTGRGAEKFAADMGIPQTPAEKLITERTKKHLEKEKLEKGAQ-------LVMEKTDHMLLTCEGATLFAKAQGIPEVPNESLVTERSRKRWMKN-LKENSN-------LVMEKTKHLCLTAEGASKFARSMGVPEVPEESLITDYAKMRWKKN-LEPDAN-------LVMEQSPHVMMIGEGAENFAFARGMERVSPEIFSTSLRYEQLLAARKEGATVL
-------LVMERSPHVLMVGEGAENFAFSQGMARVSPDIFSTPARYEQLLAARAAGEMAL
-------LVMDKTPHIYLAFQGAQDFAKQQGVETVDSSHLITAENVERLKLAIEANRVQV
-------LVMDKTPHIYLAFQGAQDFAKQQGVETVDSSHFITAENVERLKLAIEANRVQV
-------QVMDKTPHIFLAFQGAQDLGKQQGVETVDSSHFITEENVERLKLAIEANRVQV
-------AVMEHTNHCLLVGKGAEEFAKSKNLEIVEPSFFFTQNRYDQLLRAKDEKKLIL
-------LVMEKTPHIYLAFDAAEAFARAHGVETVDSSHFITPENIARLKQAKEFNRVQL
-------KVLEHTTHTLLVGESATTFAQSMGFINEDLSTTASQALHSDWLARNCQPNYWR
-------HVLEHTQHTMLVGDAASAFANAMGFESESLVTPESKDMWLQWTAENCQPNFWK
-------HVLEHTQHTMLVGDAASAFANAMGFESESLVTPESKDMWLQWTAENCQPNFWK
-------HVLEHTQHTMLVGDAASAFANAMGFESESLVTPESKDMWLQWTAENCQPNFWK
CEGQKGKLSAGRIPPCFLVGEGAYRWAVDHGIPSCPPNIMTTRFSLAAFKRNKRKLELAE
CEGQKGKLSAGRIPPCFLVGEGAYRWAVDHGIPSCPPSTMTTRFSLAAFKRNKRKLELAE
:
:
.*
.
..
:
▼
-------------------------------KTDCQKN-LGTVGAVALDCKGNVAYATST
-------------------------------KTDCEKN-LGTVGAVALDFKGNVAYATST
-------------------------------KPDCQKN-LGTVGAVALDCQGNLAYATST
-------------------------------NADCPKN-SGTVGAVALDCRGNLAYATST
-------------------------------KADCPKN-SGTVGAVALDCKGNLAYATST
-------------------------------PVADQIG-LGTVGAVAIDCEGNVACATST
-------------------------------PVECQMGKMGTVGAVAVDMDGNIACATST
DHSG---------------------------APLDEKQKMGTVGAVALDLDGNLAAATST
DHSG---------------------------APLDETKKMGTVGAVARDKFGNLAAATST
DYSQYNYPEPVKDDA------------EKELPLTNGDSQIGTVGCVAVDSHGNLASATST
DYSQYNYPQPAQDDA------------EKELPLANGDSQIGTVGCVAVDSHGNLASATST
DYSQYNYTQPVQDDA------------EKELPLANGDSQIGTVGCVAVDSHGNLASATST
DHDGENLLEKEKEKEKNNETSTTTTTISVGVDPIDPKYKMGTVGAVCLDSFGNLAAATST
DYTVP----------------------SPKVPDNCGDSQIGTVGCVAVDSAGNLASATST
NVIPDPSKYCGPYKPP----GILKQD-IPIHKETEDDRGHDTIGMVVIHKTGHIAAGTST
NVHPDPKVSCGPYKPRPTPLTRWKED-RARNEYEIGRKNHDTIGMIAIDVESNIHAGTST
NVHPDPKVSCGPYKPRPTPLTRWKED-RARNEYEIGRKNHDTIGMIAIDVESNIHAGTST
NVHPDPKVSCGPYKPRPTPLTRWKED-RARNEYEIGRKNHDTIGMIAIDVENNIHAGTST
RVDTDFMQLKKRRQS---------------SEKENDSGTLDTVGAVVVDHEGNVAAAVSS
RVETDFIQLKRRRQS---------------SAKENDSGTLDTVGAVVVDHEGNVAAAVSS
.*:* : . .:: ..*:
GGIVNKMVGRVGDSPCLGAGGYADN------DIGAVSTTGHGESILKVNLARLTLFHIEQ
GGIVNKMVGRVGDTPCVGAGGYADN------DIGAISTTGHGESILKVNLARLTLFHIEQ
GGIVNKMPGRVGDTPCVGSGGYADN------DIGAVSTTGHGESILKVNLARLALFHVEQ
GGIVNKMVGRVGDSPCIGAGGYADN------NLGAVSTTGHGESILKVNLARLALFHVEQ
GGIVNKMVGRVGDSPCIGAGGYADN------NLGAVSTTGHGESILKVNLARLALFHVEQ
GGLTNKMVGRVGDTACIGSGGYADN------NVGAVSTTGHGESIMKVILARLILHHMEQ
GGMINKMEGRVGDTPCVGCGGYADN------KIGAVSPTGHGEAIMKVTLSRLVLFHMEQ
GGMTNKLPGRVGDSPLVGAGCYANN------ASVAVSCTGTGEVFIRALAAYDIAALMDY
GGMTNKLPGRVGDSPLVGAGCYANN------ASVAVSCTGTGEVFIRTLAAYDIAALMEY
GGLVNKMVGRIGDTPLIGAGTYAN-------ELCAVSATGKGEEIIRATVARDVAALMEF
GGLVNKMVGRIGDTPLIGAGTYAN-------ELCAVSATGKGEAIISATVARDVAALMEF
GGLVNKMVGRIGDTPLIGAGTYAN-------ELCAVSATGKGEAIIRATVARDVAALMEF
GGMTNKMHGRVGDTPIIGAGVYAN-------KNVAVSSTGTGEAFMRTVAAFDIAAMMEY
GGYVNKMVGRIGDTPVIGAGTYAN-------HLCAISATGKGEDIIRGTVARDVAALMEY
NGIKFKIHGRVGDSPIPGAGAYADD------TAGAAAATGNGDILMRFLPSYQAVEYMRR
NGARHKIPGRVGDSPIPGAGAYADN------EVGAAVATGDGDVMMRFLPSLLAVEAMRA
NGARHKIPGRVGDSPIPGAGAYADN------EVGAAVATGDGDVMMRFLPSLLAVETMRA
NGARHKIPGRVGDSPIPGAGAYADN------EVGAAVATGDGDVMMRFLPSLLAVEAMRA
GGLALKHPGRVGQAALYGCGCWAENTGAHNPYSTAVSTSGCGEHLVRTILARECSHALQA
GGLALKHPGRVGQAALYGCGCWAENTGAQNPYSTAVSTSGCGEHLVRTILARECSHALQA
.*
* **:*::. *.* :*:
*
:* *: ::
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Page 5 of 9
Q7L266-ASGL1_HUMAN
Q4R7U8-ASGL1_MACFA
Q32LE5-ASGL1_BOVIN
Q8C0M9-ASGL1_MOUSE
Q8VI04-ASGL1_RAT
Q6GM78-ASGL1_XENLA
Q5BKW9-ASGL1_DANRE
P37595-IAAA_ECOLI
Q7CQV5-IAAA_SALTY
Q9ZSD6-ASPG_LUPLU
P30364-ASPG_LUPAN
P50288-ASPG_LUPAL
Q54WW4-ASGX_DICDI
P50287-ASPGA_ARATH
P20933-ASPG_HUMAN
B4NWI1-ASPG1_DROYA
Q8MR45-ASPG1_DROME
B4QHB1-ASPG1_DROSI
Q9H6P5-TASP1_HUMAN
Q8R1G1-TASP1_MOUSE
-GKTVEEAADLSLGYMKS-----RVKGLGGLIVVSKT----------------GDWVAKW
-GKTVEEAADLSLGYMKS-----RVKGLGGLIVVSKT----------------GDWVAKW
-GKSLEEAANASLGHMKS-----KVKGVGGIIMVNKA----------------GEWAVKW
-GKTVEEAAQLALDYMKS-----KLKGLGGLILVNKT----------------GDWVAKW
-GKTVDEAATLALDYMKS-----KLKGLGGLILINKT----------------GDWVAKW
-GKSPEEAADAGLNYMKS-----RVGGIGGVIIVNSS----------------GDWTAKF
-GKTPEEASDLALAYMKE-----RVDGLGGVVVVDHN----------------GTWAARF
GGLSLAEACERVVMEKLP-----ALGGSGGLIAIDHE----------------GNVALPF
GGLSLADACERVVMEKLP-----ALGGSGGLIAVDHE----------------GNVALPF
KGLSLKEAADFVIHER-------TPKGTVGLIAVSAA----------------GEIAMPF
KGLSLKEAADYVVHER-------TPKGTVGLIAVSAA----------------GEIAMPF
KGLSLKEAADCVVHER-------TPKGTVGLIAVSAA----------------GEIAMPF
GSLSLKDASNKVVMEKLI-----TVG-DGGVICVDKY----------------GNVEMPF
KGLSLTEAAAYVVDQS-------VPRGSCGLVAVSAN----------------GEVTMPF
-GEDPTIACQKVISRIQK----HFPEFFGAVICANVTG-------------SYGAACNKL
-GKPPADAAEESLRRIIR----HHKDFMGALIAVDRLG-------------RYGAACYGL
-GKPPAEAAQEGLRRILK----HHKDFMGALIAVDRLG-------------NYGAACYGL
-GKPPAEAAQGGLRRILK----HHKDFMGALIAVDRLG-------------NYGAACYGL
EDAHQALLETMQNKFISSPFLASEDGVLGGVIVLRSCRCSAEPDSSQNKQTLLVEFLWSH
EDAHQALLETMQNKFISSPFLACEDGVLGGVIVLRSCRCSSESDSSQDKQTLLVEFLWSH
.
.::
Q7L266-ASGL1_HUMAN
Q4R7U8-ASGL1_MACFA
Q32LE5-ASGL1_BOVIN
Q8C0M9-ASGL1_MOUSE
Q8VI04-ASGL1_RAT
Q6GM78-ASGL1_XENLA
Q5BKW9-ASGL1_DANRE
P37595-IAAA_ECOLI
Q7CQV5-IAAA_SALTY
Q9ZSD6-ASPG_LUPLU
P30364-ASPG_LUPAN
P50288-ASPG_LUPAL
Q54WW4-ASGX_DICDI
P50287-ASPGA_ARATH
P20933-ASPG_HUMAN
B4NWI1-ASPG1_DROYA
Q8MR45-ASPG1_DROME
B4QHB1-ASPG1_DROSI
Q9H6P5-TASP1_HUMAN
Q8R1G1-TASP1_MOUSE
TSTSMPWAAAK--DGKLHFGIDPDDTTITDLP---------------TSTSMPWAAAK--DGKLHFGIDPDDTAITDLP---------------TSTSMPWAAAK--DGKLHSGIDFGDTSIIDLS---------------TSASMPWAAVK--NGKLQAGIDLCETRTRDLPC--------------TSASMPWAAVK--NGKLQAGIDLCETKTRNLPTC-------------STNQMSWAAVK--DDQLHIGIYHGENNVTPLEKAL------------SSLQMSWAAAQ--QGKLHFGLFHGDHFTEPVEEHT------------NTEGMYRAWGYA-GDTPTTGIYREKGDTVATQ---------------NSEGMYRAWGYA-GDTPTTGIYRE-----------------------NTTGMFRACATE-DGYSEIAIWPTT----------------------NTTGMFRASATE-DGYSEIAIWPTT----------------------NTTGMFRACATE-DGYSEIAIWPTA----------------------NTEGMYRGYVII-DNNCENDQNDIINVSIYK----------------NTTGMFRACASE-DGYSEIAIWPNN----------------------STFTQFSFMVYNSE-KNQPT-EEKVDCI-------------------D---EFPFMVSSPAGRDGPTRLETVKCIAGQDKVNIVSF--------A---EFPFMVSSPAGADGPTRLETVKCIGGQDKVNIVAL--------E---EFPFMVSSPAGADRPTRLETVKCIAGQDKVNIVAL--------TTESMCVGYMSAQDGKAKTHISRLPPGAVAGQSVAIEGGVCRLESPVN
TTESMCVGYMSAQDGKAKTHISRLPPGAVAGQSVAIEGGVCRLESPVN
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Figure S3. Comprehensive sequence alignment of Ntn-enzymes most similar to
hASNase3. Asparaginases from different organisms are aligned using the Clustal W program.
N-terminal residues in a black bold font correspond to signal peptides. Strictly conserved
residues as well as H8-G10 (hASNase3 numbering) are shaded in gray. Cyan color indicates
residues that bind the substrate. The cleavage site between G167 (yellow) and T168
(highlighted in red) is indicated by an arrowhead. In green are the glycines orienting the N62
side chain. See text for details.
Page 6 of 9
Figure S4. Structure alignment of hASNase3 with hAGA and E. coli Type III asparaginase.
E. coli Type III (pdb codes: 2ZAK, 2ZAL, panel (a)) and hAGA (pdb code: 1APZ, panel (b)) are
superimposed on the hASNase3 substrate-free structure. (a) Left panel: C-trace
representation of hASNase3 uncleaved protA (green) / cleaved protB (blue) and E. coli Type III
Page 7 of 9
uncleaved (dark gray) / cleaved (light gray). Right zoom panels focus on the active site. Top
right zoom: hASNase3 and E. coli Type III cleaved structures. Bottom right zoom: hASNase3
and E. coli Type III uncleaved structures. Conserved active side residues are shown as sticks,
with hASNase3 numbering. A black arrow indicates the scissile residue. The E. coli Type III
T179A mutation, required for obtaining the uncleaved structure, is labeled. (b) Left panel: Ctrace representation of hASNase3 cleaved in protB (blue), and hAGA cleaved (orange). Right
zoom panel highlights the active site region.
Page 8 of 9
Figure S5. Rationalizing the different substrate specificities of hASNase3 and hAGA.
hAGA surface (pdb code: 1APZ; shown is a single protomer comprising the - and -chains) is
represented in transparency and its bound ASP in sticks. hASNase3 and hAGA were overlaid,
and the region encompassing -strand A to -helix 1 (see labels) from both structures are
shown as a ribbon representation (hAGA in orange, hASNase3 in tan). The linker between
these secondary structural elements is 16 residues longer in hASNase3 compared to hAGA.
The consequence of the longer linker in hASNase3 is a block in accessibility to substrate-linked
oligosaccharides. See also Fig. 2 for the sequence alignment that shows the specific residues
of the linkers.
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