Ci a ion: Es e an-Mo ió, E.;
Ramí ez-La o a, J.S.; Bushi, E.;
Eckha d, U. Dissec ing
Cy ophagalysin: S uc u al and
Biochemical S udies o a Bac e ial
Pappalysin-Family Me allopep idase.
Biomolecules 2024,14, 1604. h ps://
doi.o g/10.3390/biom14121604
Academic Edi o : Pie e La i e
Recei ed: 24 No embe 2024
Re ised: 7 Decembe 2024
Accep ed: 12 Decembe 2024
Published: 16 Decembe 2024
Copy igh : © 2024 by he au ho s.
Licensee MDPI, Basel, Swi ze land.
This a icle is an open access a icle
dis ibu ed unde he e ms and
condi ions o he C ea i e Commons
A ibu ion (CC BY) license (h ps://
c ea i ecommons.o g/licenses/by/
4.0/).
A icle
Dissec ing Cy ophagalysin: S uc u al and Biochemical S udies
o a Bac e ial Pappalysin-Family Me allopep idase
E a Es e an-Mo ió1,2 , Juan Sebas ián Ramí ez-La o a 1,2 , Enkela Bushi 1and Ul ich Eckha d 1,∗
1Syn he ic S uc u al Biology G oup, Molecula Biology Ins i u e o Ba celona (IBMB), Spanish Na ional
Resea ch Council (CSIC), 08028 Ba celona, Spain
2Doc o a e in Bio echnology, Facul y o Pha macy and Food Sciences, Uni e si y o Ba celona,
08028 Ba celona, Spain
*Co espondence: ul ich.eckha [email p o ec ed]
Abs ac : Cy ophaga is a genus o G am-nega i e bac e ia occu ing in soil and he gu mic obiome.
I is closely ela ed o pa hogenic Fla obac e ium spp. ha cause se e e diseases in ish. Cy ophaga
s ain L43-1 sec e es cy ophagalysin (CPL1), a 137 kDa pep idase wi h epo ed collagenoly ic and
gela inoly ic ac i i y. We pe o med highly-con iden s uc u e p edic ion calcula ions o CPL1,
which iden i ied 11 segmen s and domains, including a signal pep ide o sec e ion, a p osegmen
(PS) o la ency, a me allopep idase (MP)-like ca aly ic domain (CD), and eigh immunoglobulin
(Ig)-like domains (D3–D10). In addi ion, wo sho linke s we e ound a he D8–D9 and D9–D10
junc ions, and he s uc u e would be c osslinked by ou disul ide bonds. The CPL1 CD was ound
closes o ulilysin om Me hanosa cina ace i o ans, which assigns CPL1 o he lowe -pappalysin amily
wi hin he me zincin clan o MPs. Based on he s uc u e p edic ions, we aimed o p oduce cons uc s
spanning he ull-leng h enzyme, as well as PS+CD, PS+CD+D3, and PS+CD+D3+D4. Howe e , we
we e success ul only wi h he la e h ee cons uc s. We could ac i a e ecombinan CPL1 by PS
emo al employing ypsin, and ound ha bo h zymogen and ma u e CPL1 we e ac i e in gela in
zymog aphy and agains a luo ogenic gela in a ian . This ac i i y was abla ed in a mu an , in which
he ca aly ic glu ama e desc ibed o lowe pappalyins and o he me zincins was eplaced by alanine,
and by a b oad-spec um me al chela o . O e all, hese esul s p o ed ha ou ecombinan CPL1 is
a unc ional ac i e MP, hus suppo ing he conclusions de i ed om he s uc u e p edic ions.
Keywo ds: me allopep idase; pappalysin amily; ecombinan p o ein exp ession; p o ease ac i a ion;
unc ional cha ac e iza ion
1. In oduc ion
Zinc-dependen me allopep idases (MP) a e ubiqui ous pep ido- and p o eoly ic ca al-
yse s engaged in a ple ho a o biochemical pa hways [
1
], and hey a e subdi ided in o nu-
me ous amilies and clans. These include he aspzincin [
2
], gluzincin [
3
], and me zincin [
4
]
clans, which oge he wi h S2P-zincins [
5
,
6
], F sH-like AAA MPs [
7
], and i on-dependen
pep ide de o mylase [
8
] o m he zincin ibe o MPs [
6
,
9
]. Cha ac e is ic o zincins is a
sho mo i (H–E–x–x–H; amino-acid one-le e -code, x o any esidue), which includes
wo zinc-binding his idines and a ca aly ic glu ama e. The la e ac s i s as a gene al
base and hen as a gene al acid du ing he ca aly ic mechanism, which pi o s a ound
he nucleophilic a ack o a zinc-bound ca aly ic sol en molecule on o he scissile bond
o a pep idic subs a e bound o he ac i e-si e cle [
1
,
6
,
9
]. Glu ama e eplacemen wi h
alanine hampe s ca alysis bu main ains he ac i e-si e geome y [
9
–
12
], so such mu an s
a e o en used o c ea e inac i e ca aly ic domains o zymogens o s uc u al and binding
s udies [
9
]. The me zincin clan, in u n, is hallma ked by a C- e minal ex ension o he
mo i (H–E–x–x–H–x–x–G/N–x–x–H/D), which includes a u he glycine o aspa agine
essen ial o a conse ed u n o he polypep ide chain ha leads o a hi d zinc ligand h ee
posi ions downs eam in sequence, ei he a his idine o an aspa a e [
6
,
9
,
13
,
14
]. A he
Biomolecules 2024,14, 1604. h ps://doi.o g/10.3390/biom14121604 h ps://www.mdpi.com/jou nal/biomolecules
Biomolecules 2024,14, 1604 2 o 20
s uc u al le el, me zincins sha e a compac ellipsoidal ca aly ic domain (CD) o ~130–410
esidues di ided by he ac i e-si e cle in o an uppe N- e minal subdomain (NSD) and a
lowe C- e minal subdomain (CSD) when pic u ed in he consensus s anda d o ien a ion
o MPs [
15
]. Me zincin NSDs sha e an “ac i e-si e helix”, which encompasses he i s hal
o he ex ended zinc-binding mo i , a “backing helix”, and a ou / i e-s anded
β
-shee ,
whose lowe mos
β
-s and o ms he “uppe im” o he ac i e-si e cle . CSDs sha e a
“C- e minal helix” and a “Me - u n” cen ed on a conse ed me hionine esidue ha c ea es
a hyd ophobic base o he ac i e-si e zinc.
Wi hin me zincins, he pappalysin amily [
16
] is named a e i s ounding membe ,
human pappalysin-1 o p egnancy-associa ed plasma p o ein-A, which is a ~180 kDa
mul idomain p o ein i s epo ed om he bloods eam o p egnan women [
17
,
18
] whose
c yo-elec on mic oscopy s uc u e has been ecen ly elucida ed [
12
,
19
]. Pappalysins a e
also g ouped wi hin amily M43 in he MEROPS pep idase da abase (www.ebi.ac.uk/
me ops; accessed on 20 No embe 2024) [
20
], and one sub amily ea u es he “lowe
pappalysins” (LOPAPs) [
21
], p e iously e e ed o as “unicellula pappalysins” [
22
,
23
].
LOPAPs encompass simila sequences om a chaea, bac e ia, and, mo e ecen ly, also
unicellula and mul icellula lowe euka yo es like algae and ungi [
21
]. LOPAPs sha e
a leas a sho N- e minal p osegmen (PS) o zymogenici y, which is a common ea u e
o mos MPs [
9
], and he zinc-dependen me zincin- ype CD. O e he yea s, sub amily
membe s ha e been desc ibed a he ansc ip ional, unc ional, and/o biochemical le els
(see Table 1 in [
21
]), among which wo ha e also been analysed o hei c ys al s uc u es:
mi olysin om he pe iodon opa hogenic G am-nega i e bac e ium Tanne ella o sy hia and
ulilysin om he en i onmen al me hanogenic a chaeon Me hanosa cina ace i o ans [
21
–
27
].
The la e , also known as lysa giNase [
21
,
28
], is equen ly used in bo om-up p o eomics
due o i s ypsin-mi o ing clea age si e speci ici y.
Cy ophaga is a bac e ial genus ha belongs o he Bac e oide es phylum, o me ly e-
e ed o as he CFB (Cy ophaga,Bac e oides,Fla obac e ium) phylum [
29
], which is dominan
in gu mic obial communi ies and inhabi s he o al ca i y, gas oin es inal ac , and u o-
geni al ac o humans [
30
]. Cy ophaga sp. s ain L43-1, which is closely ela ed o he
Fla obac e ium genus [
31
], sec e es cy ophagalysin (CPL1), a 1282- esidue mul i-modula
LOPAP o iginally pu i ied om cul u e supe na an and cha ac e ized [
32
]. Mo eo e , i
was cloned and sequenced (see [
33
,
34
]) and is lis ed wi hin he UniP o da abase (UP) [
35
]
unde en y Q46348. The pu i ied enzyme was shown o clea e
β
-casein, bo h insoluble and
acid-soluble collagens and gela in, op imally a pH 7.5 and 30
◦
C, bu no small luo ogenic
pep ides ha a e clea ed by o he bac e ial collagenases [34].
Closely ela ed o CPL1, an o hologue (UP A0A2H1E968) is encoded by Tenacibac-
ulum ma i imum NCIMB 2154 [
36
], a de as a ing pa hogenic la obac e ium ha causes
enacibaculosis in wild and a med ma ine ish. Ano he pa hogen, Fla obac e ium psy-
ch ophylum, causes cold-wa e disease in Salmonidae and Osme idae ish and encodes he
960- esidue MPs WA-1 and WA-2, which a e ~50% iden ical in sequence o CPL1 [
31
].
O he po en ial o hologues a e encoded by he en i onmen al bac e ia Sedimini omix la a
(UP A0A315ZEZ8) om ma ine sedimen and Chi inophaga solisil ae (UP A0A3S1B1Z0)
om o es soil. Impo an ly, any ype o gela inase o collagenase, including cy opha-
galysin, ep esen s highly sough -a e enzymes o bio echnology. I s abili y o deg ade
collagen makes i aluable in medical issue enginee ing h ough collagen emodelling.
Collagenases play a c ucial ole in en i onmen al bio emedia ion by deg ading collagen-
and gela in-based ma e ials, he eby acili a ing hei u iliza ion in he lea he p ocessing
and ood p oduc ion indus ies.
He e, we aimed o shed ligh on he s uc u e and unc ion o CPL1 and pe o med
s uc u e-p edic ion calcula ions. We analysed hese esul s in he ligh o he accumula ed
knowledge on MPs in gene al and LOPAPs in pa icula . We u he designed CPL1
cons uc s spanning dis inc domains and achie ed hei ecombinan o e exp ession and
pu i ica ion. Finally, we unc ionally assessed hem in pep ide- and p o eoly ic assays
in i o, and pe o med mu an s o alida e hei ac i i y.
Biomolecules 2024,14, 1604 3 o 20
2. Ma e ials and Me hods
2.1. Biocompu a ional S udies
Sequence simila i y sea ches and alignmen s we e pe o med wi h he Blas algo-
i hm [
37
] wi hin UP and Mul Alin [
38
], espec i ely, using s anda d pa ame e s. The
h ee-dimensional s uc u e o ull-leng h CPL1 was p edic ed wi h AlphaFold [
39
] using
pai ed mul iple sequence alignmen s, which enable he ex ac ion o coe olu iona y in-
o ma ion and enhance he p edic ion accu acy [
40
]. The con idence o he i e dis inc
p edic ions ob ained was assessed by means o he p edic ed local-dis ance di e ence es
(pLDDT [
41
]), which eliably es ima es he accu acy o he C
α
local-dis ance di e ence
es [
39
]. In his espec , pLDDT alues >90% accoun o high accu acy o he o e all
p edic ion, and alues >70% quali y as gene ally co ec p edic ions o he backbone [
42
].
The models we e isually inspec ed o chemical sense and cohe ence wi h Coo [
43
]. The
op p edic ion (model_1) was subjec ed o manual model building o co ec clashes and
chemical inconsis encies, ollowed by geome ical egula isa ion wi h Coo . The ea e , he
Geome y_minimiza ion ou ine o he Phenix sui e [
44
] was applied, and he esul ing inal
model, which can be downloaded as pa o he Supplemen a y Ma e ials, was alida ed
wi h Molp obi y [
45
] (Table S1). In e -domain in e aces we e analysed wi h Pisa [
46
] and
s uc u al supe posi ions we e calcula ed using he Ssm ou ine [
47
] in Coo . S uc u al
ela i es p esen in he P o ein Da a Bank (PDB) we e e ie ed h ough Dali [
48
] and
s uc u al igu es we e p epa ed wi h Chime a [
49
] o an open-sou ce build o PyMOL
(Ve sion 2.5 Sch ödinge , LLC, New Yo k, NY, USA) [50].
2.2. Molecula Cloning o CPL1 Cons uc s
The sequence o ull-leng h CPL1 (Q
20
–K
1282
) om Cy ophaga sp. L43-1
31
was codon-
op imized o exp ession in E. coli (Supplemen a y Figu es S4 and S5), and a syn he ic gene
was pu chased om GenSc ip Bio ech o in oduc ion in o a modi ied pC i7a
plasmid 66,
he ea e pC i7a*, using he NdeI and BamHI es ic ion si es. This plasmid added an N-
e minal S ep- ag wi h a ecogni ion sequence o obacco-e ch i us pep idase (TEV) and
a C- e minal His
8
- ag. Th ee C- e minally unca ed a ian s o CPL1, designa ed CPL1_1-2
(Q
20
–N
328
), CPL1_1-3 (Q
20
–V
444
), and CPL1_1-4 (Q
20
–T
591
), we e subsequen ly cloned by
in e se PCR [
51
] using 0.5
µ
M o o wa d and e e se p ime s, 200
µ
M o each o he ou
dNTPs, 0.02 U/
µ
L Q5 High-Fideli y DNA Polyme ase (New England Biolabs), bu no High
GC Enhance . Cons uc s we e p oduced as wild- ype species and as mu an s, in which he
ca aly ic glu ama e om he zinc-binding mo i had been eplaced wi h alanine (E
232
A).
The wo a ian s o CPL1_1-3 and CPL1_1-4 we e also in oduced in o plasmid pC i7b [
52
],
which only adds he C- e minal His
8
- ag, using he same es ic ion si es. Cloning and
mu agenesis p ime s a e lis ed unde Supplemen a y Figu e S6A,B, and he map o pC i7a*
is shown unde Supplemen a y Figu e S6C.
2.3. P o ein Exp ession and Pu i ica ion
Va ious chemically compe en E. coli s ains, including BL21(DE3), O igami 2(DE3),
Lemo21(DE3), and Rose a(DE3), we e p epa ed inhouse ollowing he Inoue me hod [
53
],
ans o med wi h he di e en p o ein-encoding plasmids, and pla ed on aga pla es
con aining Be ani’s lysogeny b o h [
54
] supplemen ed wi h kanamycin a 50
µ
g/mL
(he ea e medium). A e o e nigh incuba ion, a single colony was picked o each s ain
and plasmid, and cul u ed in 5 mL medium a 37
◦
C unde gen le shaking (a 200 pm).
Cul u es we e hen dilu ed 1:1000 in 500 mL esh medium using ou 2 L E lenmeye
lasks, g own a 37
◦
C o OD
600 ≈
1.2, cooled o 15–30 min in a cold oom, induced
wi h 1 mM isop opyl-
β
-D-1- hiogalac opy anoside (IPTG), and placed a 20
◦
C unde
gen le shaking o o e nigh p o ein exp ession. Cells we e subsequen ly ha es ed by
cen i uga ion, esuspended in 30 mL bu e A (50 mM T is
·
HCl pH 8.0, 300 mM sodium
chlo ide,
10 mM
imidazole, 10 mM calcium chlo ide, 50
µ
M zinc chlo ide), and sonica ed
on ice o 1 min a 20% ampli ude and 0.5 s in e als using a B anson 450 Digi al Soni ie
(Ma shall Scien i ic, Hamp on, NH, USA) wi h a 6 mm ape ed mic o ip. The esul ing
Biomolecules 2024,14, 1604 4 o 20
solu ion was incuba ed on ice wi h magnesium chlo ide a 10 mM and 10
µ
g/mL DNase I
o 15 min, and hen lysed using an E1061 con inuous low cell dis up o (Cons an Sys ems,
Da en y, UK) ope a ed a 1.36 kba . Lysa es we e incuba ed o 30 min on ice wi h 1 M
u ea be o e being cen i uged wice a 20,000 pm using a JA-25.50 o o in a Beckman
A an i J-25 cen i uge (~48,000
×
g). The cla i ied supe na an s we e subjec ed o a ini y
ch oma og aphy employing HisPu Ni-NTA esin (The mo Scien i ic, Wal ham, MA, USA)
and bu e A plus 20 mM o 250 mM imidazole o he wash o elu ion s eps, espec i ely.
Elu ed samples we e hen concen a ed using a Vi aspin-20 polye he sul one cen i ugal
de ice wi h 10 kDa molecula -weigh cu o (Sa o ius, Gö ingen, Ge many), and polished
by SEC in Supe dex 200 10/300 GL o Supe ose 6 Inc ease 10/300 GL columns (Cy i a,
Ma lbo ough, MA, USA), p e iously equilib a ed wi h bu e B (50 mM T is
·
HCl pH 8.0,
100 mM sodium chlo ide) and a ached o an ÄKTApu i ie 10 p o ein pu i ica ion sys em
(GE Heal hca e, Milwaukee, WI, USA). Peak ac ions we e pooled, e-concen a ed by
cen i uga ion, and lash- ozen in liquid ni ogen as 50
µ
L and 10
µ
L aliquo s o 10 mg/mL
and 1 mg/mL concen a ion, espec i ely, and s o ed a
−
80
◦
C un il usage. P o ein
concen a ions we e de e mined using a BioD op Duo+ mic o olume spec opho ome e
(BioCh om, Camb idge, UK) based on he abso bance measu ed a
λ
= 280 nm and he
espec i e heo e ical ex inc ion coe icien calcula ed by P o Pa am [55,56]. P o ein pu i y
was assessed by SDS-PAGE analysis on cus om-made 12% and 14% T is/Glycine gels
ollowed by Coomassie B illian Blue (Sigma-Ald ich, Sain Louis, MO, USA) s aining. The
BlueS a Plus P es ained p o ein ma ke (Nippon Gene ics EUROPE, Dü en, Ge many)
was used as molecula -mass e e ence.
2.4. Ac i a ion o CPL1
Fo ac i a ion, he wild- ype CPL1 cons uc s, which encompass he p edic ed PS
o la ency (Q
20
–A
66
; see Sec ion 3.2), we e incuba ed wi h bo ine ypsin, ea ed wi h
N- osyl-L-phenylalanyl chlo ome hyl ke one (TPCK) o p e en ex aneous chymo yp ic
ac i i y (Sigma-Ald ich, Sain Louis, MO, USA), a a weigh a io o 1000:1 o 10–15 min
a oom empe a u e, and ac i a ion h ough PS emo al was moni o ed by SDS-PAGE.
Simila ly, bo ine
α
-chymo ypsin, ea ed wi h N- osyl-L-lysinyl chlo ome hyl ke one
(TLCK) o abla e undesi ed ypsin ac i i y (Sigma-Ald ich, Sain Louis, MO, USA), was
likewise used o assess ac i a ion. In ei he case, eac ions we e s opped by adding he
EDTA- ee HALT inhibi o cock ail (The mo Fishe Scien i ic, Wal ham, MA, USA) a a inal
concen a ion o 2
×
, which is equi alen o 2 mM o he b oad-spec um se ine pep idase
inhibi o 4-(2-aminoe hyl)benzenesul onyl luo ide (AEBSF).
2.5. Ac i i y Assessmen by Zymog aphy
Ac i a ed CPL1 a ian s we e loaded on o 12% o 14% SDS-PAGE gels con aining
0.1% gela in om cold wa e ish skin (Sigma-Ald ich) and subjec ed o elec opho esis
a 160 V o ~1 h on ice. The ea e , gels we e ex ensi ely insed wi h dis illed wa e
and washed h ee imes o 30 min in bu e C (50 mM T is
·
HCl pH 8.0, 150 mM sodium
chlo ide, 2.5% T i on X-100) unde gen le shaking. A e wo u he washes wi h bu e D
(50 mM T is
·
HCl pH 8.0, 150 mM sodium chlo ide, 10 mM calcium chlo ide, 50
µ
M zinc
chlo ide, 0.02% B ij-35) o 20 min, gels we e incuba ed in he same bu e o e nigh a
oom empe a u e, and s ained wi h Coomassie B illian Blue. A e a b ie des aining s ep,
p o eoly ic ac i i y was e ealed as ligh bands agains o e a da k blue backg ound. All
samples o zymog aphy we e p epa ed wi h non- educing sample bu e wi hou hea ing
i no s a ed o he wise.
2.6. Fluo ome ic Gela inoly ic Ac i i y and Inhibi ion
Ac i i y o CPL1 a ian s agains gela in in solu ion was de e mined using he sub-
s a e DQ Gela in om pig skin (The mo Fishe Scien i ic, Wal ham, MA, USA), which
consis s o highly quenched luo escein-labelled gela in ha yields b igh g een luo es-
cence upon p o eolysis o he subs a e. Subs a e a 20
µ
g/mL in 100
µ
L bu e E (50 mM
Biomolecules 2024,14, 1604 5 o 20
T is
·
HCl pH 8, 150 mM sodium chlo ide, 10 mM calcium chlo ide, 50
µ
M zinc chlo ide,
1
×
Hal
TM
P o ease Inhibi o Cock ail; The mo Fishe Scien i ic, Wal ham, MA, USA) was
eac ed wi h CPL1 a 37
◦
C, and he luo escence esponse was moni o ed in a BioTek
Syne gy H1 mul imode mic opla e eade (Agilen Technologies, San a Cla a, CA, USA)
wi h
λexc
= 480 nm and
λem
= 520 nm. Ac i i y inhibi ion was assessed by p eincuba ing
he CPL1 a ian s wi h 20 mM EDTA o 30 min and p oceeding as men ioned.
2.7. Collagen Deg ada ion Assay
Ac i i y o CPL1 a ian s agains he Vi oCol ype-I human a elocollagen (Ad anced
Bioma ix, Ca lsbad, CA, USA) and agains a - ail ype-I collagen (Co ning, Co ning, NY,
USA) was es ed in 40
µ
L eac ion olumes by adding 2
µ
g o enzyme o 10
µ
g o collagen in
bu e E. A e 16 h o incuba ion a 20
◦
C o ensu e na i e collagen condi ions, clea age was
assessed by 12% SDS-PAGE gels. Collagenase ype IA om Clos idium his oly icum, bo ine
TPCK- ea ed ypsin, and bo ine TLCK- ea ed
α
-chymo ypsin (all om Sigma-Ald ich,
S . Louis, MO, USA) we e used as con ols.
2.8. P o ein C ys alliza ion Assays
C ys alliza ion assays we e pe o med by he si ing-d op apo di usion me hod.
Rese oi solu ions we e p epa ed by a Tecan obo and 100 nL c ys alliza ion d ops we e
dispensed on 96-well 2-d op Swissci PS MRC pla es (Molecula Dimensions, She ield,
UK) by a Phoenix nanod op obo (A Robbins Ins umen s, Sunny ale, CA, USA) o a
Ca esian Mic osys 4000 XL obo (Genomic Solu ions, Ann A bo , MI, USA) a he join
IBMB/IRB Au oma ed C ys allog aphy Pla o m (h ps://ibmb.csic.es/en/pla o ms/
au oma ed-c ys allog aphic-pla o m; accessed on 20 No embe 2024). Pla es we e s o ed
in s eady- empe a u e c ys al a ms (B uke , Bille ica, MA, USA) a 4
◦
C o 20
◦
C and
egula ly inspec ed.
3. Resul s and Discussion
3.1. Compu a ional P edic ion o he CPL1 S uc u e
Simila i y sea ches iden i ied mi olysin (UP A0A0F7IPS1 and G8ULV1, w ongly en-
i led “ka ilysin”) and ulilysin (UP Q8TL28) as he closes sequences o CPL1 among
s uc u ally analysed p o eins (p= 1.8
×
10
−23
, 269 common esidues, 30% sequence iden-
i y; and p= 1.5
×
10
−20
, 237 common esidues, 31% sequence iden i y, espec i ely). The
aligned sequence s e ches encompassed in bo h cases he espec i e PSs and CDs, which
suppo s he asc ip ion o CPL1 CD o he LOPAPs (Figu e 1A).
Nex , we pe o med compu a ional s uc u e p edic ions o esidues 1–1282 wi h
he AlphaFold p og am [
39
], which we e highly con iden acco ding o he p edic ed local-
dis ance di e ence es (pLDDT) and sequence co e age (Figu e 1C,D). The highes - anking
model o i e p edic ions was subjec ed o manual ebuilding and geome ic minimiza ion.
Supplemen a y Table S1 depic s he s a is ics o he alida ion o he esul ing wo king
model, which can be downloaded as pa o he Supplemen a y In o ma ion. This model
p oposes he p esence o 11 segmen s and domains plus wo linke s, which include a
19- esidue signal pep ide o sec e ion (SP) ollowed by a 47- esidue PS (Q
20
–A
66
; sequence
numbe ing o CPL1 in supe sc ip , see UP Q46348) (Figu e 1B) ha is absen om he en-
zyme pu i ied om cul u e supe na an [
32
–
34
]. Nex , he me al-dependen CD
(E67–S329)
would con ain wo in adomain disul ides (C
247
–C
273
and C
267
–C
292
) plus a hi d one (C
140
–
C
434
) ha links his domain o downs eam domain D3 (T
330
–P
446
). The la e p ecedes
se en u he domains (D4, Y
447
–M
593
; D5, T
594
–S
713
; D6, P
714
–G
864
; D7, I
865
–A
959
; D8,
S
960
–G
1099
; D9, T
1107
–P
1194
; and D10, K
1207
–K
1282
), wi h wo sho linke s inse ed a he
D8–D9 and D9–D10 junc ions (Figu e 1B).
Biomolecules 2024,14, 1604 6 o 20
Biomolecules 2024, 14, x FOR PEER REVIEW 6 o 20
S960–G1099; D9, T1107–P1194; and D10, K1207–K1282), wi h wo sho linke s inse ed a he D8–
D9 and D9–D10 junc ions (Figu e 1B).
Wi h espec o he ela i e o ien a ions o he domains, only he i s i e domains
consis en ly appea ed in a compa able a angemen in he i e p edic ions (see Figu e 1E).
This is consis en wi h he ac ha ela i e o ien a ions o domains canno usually be p e-
dic ed accu a ely [57]. Indeed, analysis o he p edic ed aligned e o , which accoun s o
he esidue– esidue alignmen con idence and, hus, i domains a e eliably posi ioned
[58], sugges s ha PS–CD, CD–D3, as well as D3–D4 and D4–D5 migh be co ec ly placed
ela i e o one ano he in he p edic ions (Figu e 1F). Visual inspec ion and compu a ional
quan i ica ion o he in e -domain in e aces wi h Pisa [46] sugges ed ha he ela i e a -
angemen o PS–CD and CD–D3 migh ac ually be signi ican . I ea u es in e aces span-
ning 1329 Å2 and 518 Å2, espec i ely, which a e in he ange o alues epo ed o expe -
imen al p o ein–p o ein complexes (955 ± 380 Å2) [59], and he calcula ed ΔiG alues a e -
12.2 kcal/mol and -8.9 kcal/mol, espec i ely. In con as , he D3–D4 and D4–D5 in e aces
ea u e only ew con ac s and may a he ep esen hinge poin s o s uc u e ea ange-
men . Fu u e s udies may include mul i-s a e small-angle X- ay sca e ing (SAXS) p o ile
analysis o nea - o- ull-leng h p o ein p epa a ions o expe imen ally cap u e he s uc-
u al in e play o accesso y domains.
Figu e 1. Biocompu a ional s udies. (A) Sequence alignmen o he p osegmen s (PSs) (g een
backg ound) and ca aly ic domains (CDs) o CPL1 (UP Q46348), mi olysin (UP G8ULV1), and ulilysin
(UP Q8TL28). Iden ical o equi alen esidues a e in ed, and hose sha ed by wo sequences a e
in blue. The PS cys eine engaged in zinc-binding in he zymogen—pu a i ely in CPL1—is amed.
The ex ended zinc-binding mo i , he esidues shaping he common calcium si e, and he Me - u n
mo i a e shown o e ligh -blue, o ange, and cyan backg ound, espec i ely. (B) Domain dis ibu ion
along he chemical sequence p edic ed by AlphaFold, which o esees a signal pep ide o sec e ion
(SP), he PS, he CD, and immunoglobulin-like domains D3 h ough D10. Each domain is labelled,
he espec i e limi ing esidues a e indica ed, and he a e age p edic ed local-dis ance di e ence es
(pLDDT) is shown in pa en hesis. In all cases, hese alues a e close o o exceed he high-accu acy
cu -o o ~90% [
42
], and a e hus classed as high con idence. The only excep ion is he PS, whose
p edic ion e inces an a e age pLDDT ha is sligh ly lowe , bu s ill highly eliable o he main
chain. Two sho linke s (LNKs) would be in e cala ed be ween D8 and D9, and be ween D9 and D10.
P edic ed disul ide bonds a e shown in o ange. The cys eine pu a i ely engaged in la ency in he
zymogen (C
24
) and he ex ended zinc-binding mo i (H
231
–H
241
), as well as he Me - u n me hionine
(M
284
) and he ma u a ion clea age poin (A
66
–E
67
; scisso s) a e u he pinpoin ed. (C) pLDDT
o each esidue o he p edic ion (posi ions 1–1282) o each o he i e dis inc models ob ained.
(D) Sequence
co e age o each esidue o he p edic ion (posi ions 1–1282) s. numbe o sequences.
Biomolecules 2024,14, 1604 7 o 20
(E) Supe posi ion o he i e p edic ed models wi hou u he elaxa ion/minimiza ion wi h each
domain/segmen in he colou o (B). Only PS, CD, D3, D4, and, oughly, D5 appea wi h simila
ela i e o ien a ions in all models. (F) Analysis o he p edic ed aligned e o , which es ima es i
domains a e co ec ly posi ioned ela i e o one ano he , o each esidue o he p edic ion (posi ions
1–1282; model_1). Each segmen /domain o (B) gi es ise o a ma ine blue squa e along he diagonal.
O -diagonal blue alues sugges well-p edic ed in e ac ions be ween domains. (G) Supe posi ion o
he C
α
- aces o he expe imen al s uc u es o p omi olysin (PS in sienna, CD in gold) and p oulilysin
(cyan/dodge blue) in s anda d o ien a ion [
15
] on o he p edic ion o CPL1 (pu ple/pink). The
CPL1 p edic ion ma ches p oulilysin signi ican ly be e . The ca aly ic zinc (magen a sphe e) and he
common calcium ( ed sphe e) o p oulilysin a e u he displayed.
Wi h espec o he ela i e o ien a ions o he domains, only he i s i e domains
consis en ly appea ed in a compa able a angemen in he i e p edic ions (see Figu e 1E).
This is consis en wi h he ac ha ela i e o ien a ions o domains canno usually be
p edic ed accu a ely [
57
]. Indeed, analysis o he p edic ed aligned e o , which accoun s o
he esidue– esidue alignmen con idence and, hus, i domains a e eliably posi ioned [
58
],
sugges s ha PS–CD, CD–D3, as well as D3–D4 and D4–D5 migh be co ec ly placed
ela i e o one ano he in he p edic ions (Figu e 1F). Visual inspec ion and compu a ional
quan i ica ion o he in e -domain in e aces wi h Pisa [
46
] sugges ed ha he ela i e
a angemen o PS–CD and CD–D3 migh ac ually be signi ican . I ea u es in e aces
spanning 1329 Å
2
and 518 Å
2
, espec i ely, which a e in he ange o alues epo ed o
expe imen al p o ein–p o ein complexes (955
±
380 Å
2
) [
59
], and he calcula ed
∆i
G alues
a e
−
12.2 kcal/mol and
−
8.9 kcal/mol, espec i ely. In con as , he D3–D4 and D4–D5
in e aces ea u e only ew con ac s and may a he ep esen hinge poin s o s uc u e
ea angemen . Fu u e s udies may include mul i-s a e small-angle X- ay sca e ing (SAXS)
p o ile analysis o nea - o- ull-leng h p o ein p epa a ions o expe imen ally cap u e he
s uc u al in e play o accesso y domains.
3.2. Compa ison wi h P oulilysin and P omi olysin
Supe posi ion o he expe imen al LOPAP s uc u e o Tanne ella p omi olysin (P o ein
Da a Bank [PDB] access code 6R7V [
23
]) on o he CPL1 wo king model e ealed 213 aligned
esidues (ou o 307 o p omi olysin), wi h a co e msd o 2.7 Å and a sequence iden i y o
30%. The same calcula ions employing Me hanosa cina p oulilysin (PDB 8CD8 [
21
]) e ealed
248 aligned esidues (ou o 306 p oulilysin esidues) de ia ing 1.7 Å (29% iden i y). O e all,
he h ee s uc u es accu a ely ma ch and sha e he common s uc u al elemen s o LOPAPS
(Figu es 1A,G and 2A). Howe e , he di e ences in he msd alues poin o CPL1 being
close o ulilysin han o mi olysin.
In all s uc u es, he PS uns ac oss he cle o he CD in he opposi e di ec ion o a
subs a e, which p e en s au oly ic clea age, and encompasses wo la ge, cha ac e is ic
helices (
α
1p and
α
2p). This segmen blocks access o subs a es o he ac i e-si e cle ,
hus keeping he zymogens inac i e. The helices de ia e in he Tanne ella zymogen when
compa ed wi h he o he wo s uc u es, which accu a ely ma ch (Figu e 1F). Mo eo e ,
C
24
o CPL1’s PS would be opologically equi alen o C
23
o bo h mi olysin and ulilysin
(o he p o eins’ posi ions a e numbe ed in subsc ip ), which ha e been shown o play a
ole in la ency in hese enzymes by ac ing as a “cys eine swi ch” [
21
,
23
]. The cys eine
S
γ
a om blocks he ca aly ic zinc ion as also desc ibed o o he me zincins, like ma ix
me allopep idases, bac e ial as acins, adamalysins, and a-disin eg in-and-me allopep idase
enzymes (ADAMs) [
60
–
63
]. Fu he mo e, he expe imen al ac i a ion clea age si e o CPL1
(A
66
–E
67
) [
32
,
33
], which emo es he PS and yields he ma u e, compe en CD (Figu e 1A,G),
would be opologically equi alen o hose o he a chaeal (S
60
–R
61
) and bac e ial (S
54
–R
55
)
ela i es [21,23].
Biomolecules 2024,14, 1604 8 o 20
Biomolecules 2024, 14, x FOR PEER REVIEW 9 o 20
Figu e 2. S uc u al analysis o he p edic ed CPL1 domains. (A) Ribbon- ype plo o he CPL1 PS
and CD in c oss-eye s e eo. The seconda y s uc u e elemen s a e labelled (α1p, α2p, α1–α9, and
β1–β6). The pu a i e cys eine-swi ch cys eine (C24), zinc-binding esidues (H231, H235 and H241), gen-
e al base/acid glu ama e (E232), Me - u n me hionine (M284) and y osine-swi ch y osine (Y286), cal-
cium-binding esidues (D251 and T256), as well as he pu a i e disul ide-bonded cys eines (C247–C273;
and C267–C292; ) a e shown o hei side chains as s icks and numbe ed. The zinc and calcium
ca ions we e modelled based on he p oulilysin (PDB 8CDB) and ma u e ulilysin (PDB 2CKI) s uc-
u es. The pu a i e ma u a ion si e (A66–E67) and he LNR-loop a e highligh ed by g een and o ange
a ows, espec i ely. Depic ion o he Ig-like domains (D3–D10) showing as ibbon- o Cα-plo s (B)
D3; (C) D5 (cyan Cα-plo ) on o D3 (plum Cα-plo ) in he same o ien a ion as in (B); (D) D4; (E) D6
(b own Cα-plo ) on o D4 (yellow Cα-plo ) in he same o ien a ion as (D); (F) D7; (G) D9 (o ange Cα-
plo ) on o D7 (g een Cα-plo ) in he same o ien a ion as (F); (H) D8 (disul ide bond C963–C1083; )
and (I) D10. The β-s ands and he N- and C- e minal esidues a e numbe ed in all cases.
3.3. Eigh C-Te minal Immunoglobulin-Like Domains (D3–D10)
Downs eam o he CD, domain D3 would adop a compac immunoglobulin-like
(Ig-like) old [67–69], which consis s o 117 esidues o ming an an ipa allel β-sandwich
wi h ou -s anded on and back shee s (β1–β3–β6–β5 and β2–β8–β7–β4, espec i ely,
om le o igh in Figu e 2B), wi h an in e shee angle o ~35° and G eek-key opology.
Figu e 2. S uc u al analysis o he p edic ed CPL1 domains. (A) Ribbon- ype plo o he CPL1
PS and CD in c oss-eye s e eo. The seconda y s uc u e elemen s a e labelled (
α
1p,
α
2p,
α
1–
α
9,
and
β
1–
β
6). The pu a i e cys eine-swi ch cys eine (C
24
), zinc-binding esidues (H
231
, H
235
and
H
241
), gene al base/acid glu ama e (E
232
), Me - u n me hionine (M
284
) and y osine-swi ch y osine
(Y
286
), calcium-binding esidues (D
251
and T
256
), as well as he pu a i e disul ide-bonded cys eines
(C
247
–C
273
;
1
and C
267
–C
292
;
2
) a e shown o hei side chains as s icks and numbe ed. The zinc
and calcium ca ions we e modelled based on he p oulilysin (PDB 8CDB) and ma u e ulilysin (PDB
2CKI) s uc u es. The pu a i e ma u a ion si e (A
66
–E
67
) and he LNR-loop a e highligh ed by g een
and o ange a ows, espec i ely. Depic ion o he Ig-like domains (D3–D10) showing as ibbon- o
C
α
-plo s (B) D3; (C) D5 (cyan C
α
-plo ) on o D3 (plum C
α
-plo ) in he same o ien a ion as in (B);
(D) D4; (E) D6 (b own C
α
-plo ) on o D4 (yellow C
α
-plo ) in he same o ien a ion as (D); (F) D7;
(G) D9
(o ange C
α
-plo ) on o D7 (g een C
α
-plo ) in he same o ien a ion as (F); (H) D8 (disul ide
bond C
963
–C
1083
;
1
) and (I) D10. The
β
-s ands and he N- and C- e minal esidues a e numbe ed in
all cases.
As o he CD (Figu es 1G and 2A), CPL1 would sha e wi h he wo LOPAPs he
i e-s anded
β
-shee ( op o bo om,
β
2+
β
3–
β
4–
β
6–
β
5 in CPL1) including he “LNR-like
loop” (L
131
–G
143
), which p o udes om he molecula su ace and di ides he second
Biomolecules 2024,14, 1604 9 o 20
s and o he shee in wo (
β
2+
β
3). Nex , he ac i e-si e helix (
α
6) and he backing helix
(
α
1), as well as he wo sho helices (
α
2 and
α
3) a e he la e , which o m a cape in he
back o he molecule, would be sha ed. Fu he common elemen s a e he Me - u n, he
C- e minal helix (
α
8), and wo addi ional
α
-helices (
α
8 and
α
9) a e he C- e minal helix,
which a e cha ac e is ic o LOPAPs bu no o me zincins in gene al. Mo eo e , he ac i e
si e cen ed on he ca aly ic zinc ion and he a angemen o he esidues encompassing he
ex ended zinc-binding mo i would be e y simila , which u he suppo s ha CPL1 is
ac ually an MP (Figu e 2A) (see also Sec ion 3.5). In addi ion, he p edic ed CD disul ides
C
247
–C
273
and C
267
–C
292
would be opologically equi alen o C
250
–C
277
and C
269
–C
297
o
ulilysin and C
243
–C
271
and C
262
–C
291
o mi olysin. Fu he mo e, Y
286
(p omi olysin) and
Y
292
(p oulilysin), which a e ound wo posi ions downs eam o he Me - u n me hionine
(Figu e 1A), ha e been epo ed o play a ole in subs a e binding du ing ca alysis, ac ing
as a “ y osine swi ch” [
10
]. This esidue would also be p esen in CPL1 as Y
286
(Figu e 2A),
likely exe ing simila unc ions. Finally, simila ly o p oulilysin, he CPL1 zymogen
would lack one o wo calcium-binding si es, which exe essen ial s uc u al unc ions
in he ma u e s uc u es [
21
–
25
]. In con as , his calcium si e is al eady p esen in he
p omi olysin zymogen [
23
]. As o he second si e, he model adop s a e y simila ajec o y
o he expe imen al s uc u es o he p o ein chain in ol ed (D
251
–T
256
; Figu e 1G), hus
suppo ing his si e would also be p esen in CPL1.
Unique o CPL1, an “adamalysin helix” (
α
4) would be inse ed be ween
β
3 and
β
4,
nes ling on he con ex ace o he
β
-shee (Figu e 2A). Such a helix was i s epo ed o
adamalysins/ADAMs [
64
] and hen o agilysin-3 [
65
,
66
], and i is eplaced in ulilysin
and mi olysin by an i egula loop [
22
,
23
]. Mo eo e , an addi ional helix (
α
5), which is
missing in he o he LOPAPs, would be inse ed be ween shee s ands β5 and β6.
3.3. Eigh C-Te minal Immunoglobulin-like Domains (D3–D10)
Downs eam o he CD, domain D3 would adop a compac immunoglobulin-like (Ig-
like) old [67–69], which consis s o 117 esidues o ming an an ipa allel β-sandwich wi h
ou -s anded on and back shee s (
β
1–
β
3–
β
6–
β
5 and
β
2–
β
8–
β
7–
β
4, espec i ely, om
le o igh in Figu e 2B), wi h an in e shee angle o ~35
◦
and G eek-key opology. The
p edic ed model o he domain a e nex (D5) encompasses 120 esidues and supe poses
e y accu a ely on o D3, wi h 114 aligned esidues o e lapping wi h a co e msd o 1.2 Å
(Figu e 2C). The wo domains ha e 37% sequence iden i y, so hey migh be unc ionally
equi alen . No ably, Ig-like domains also ep esen an in iguing sca old o he de no o
design o an ibody-like s uc u es wi h supe io biophysical p ope ies [70,71].
Wi h espec o unc ion, D3 and D5 bes ma ch domain Ig-2 o he high-molecula -
mass chi inase ChiW om Paenobacillus sp. ( msd 3.0 Å; Z-sco e 9.1 acco ding o [
48
]; PDB
5GZT [
72
]). Like D3, his domain is also immedia ely downs eam o and a ached o a CD,
in his case a ca bohyd a e hyd olase moie y. Bo h models sha e he o e all a chi ec u e,
opology, and connec i i y, bu no he de ailed chain ace, as e ealed by he a he high
msd alue. Domain Ig-2 and o he s alike ha e been p oposed o be linke s ha connec
CDs—o hese wi h subs a e-binding domains—and s abilize hem wi hin la ge mul i-
modula enzymes [
72
]. The unc ion o D3, and by ex ension D5, in CPL1 could be simila .
Indeed, in he p obably mos eliably p edic ed ela i e a angemen be ween domains o
he wo king model (see Sec ion 3.1), D3 would in e ac h ough i s la e al sandwich su ace
amed by s ands
β
2 and
β
8 wi h he LNR-like loop o he p eceding CD. This in e ac ion
would be cemen ed h ough a disul ide bond (C140–C434) ha is unique o LOPAPs.
Linked o D3, he 147- esidue D4 domain would likewise be an an ipa allel 4+4
β
-
sandwich (Figu e 2D) wi h a on shee , in which he i s s and is hal ed by a bulge
( om le o igh ,
β
6–
β
7–
β
8–
β
1a+
β
1b), and a back shee (
β
5–
β
4–
β
3–
β
2). This domain
di e s om D3/D5 in he opology o he
β
-shee s. In addi ion, he di e ence in size is
accoun ed o by an 11- esidue C- e minal ex ension and a wide 41- esidue spi al segmen ,
which connec s s ands β4 and β5 and la e ally con ac s he le sandwich su ace amed
by s ands
β
5 and
β
6 (Figu e 2D). This con e s on D4 a much mo e globula and bulky
Biomolecules 2024,14, 1604 16 o 20
Ig immunoglobulin
Ig-like immunoglobulin-like
IPTG isop opyl-β-D-1- hiogalac opy anoside
LNKs linke s
LOPAPs lowe pappalysins
MP me allopep idase
NSD N- e minal subdomain
PDB P o ein Da a Bank
pLDDT p edic ed local-dis ance di e ence es
PS p osegmen
SDS-PAGE sodium dodecylsul a e polyac ylamide gel elec opho esis
SEC size-exclusion ch oma og aphy
SP signal pep ide
T9SS ype-IX sec e ion sys em
TEV obacco-e ch i us pep idase
TLCK N- osyl-L-lysinyl chlo ome hyl ke one
TPCK N- osyl-L-phenylalanyl chlo ome hyl ke one
UP UniP o da abase
Re e ences
1.
Klein, T.; Eckha d, U.; Du ou , A.; Solis, N.; O e all, C.M. P o eoly ic Clea age-Mechanisms, Func ion, and “Omic” App oaches
o a Nea -Ubiqui ous Pos ansla ional Modi ica ion. Chem. Re . 2018,118, 1137–1168. [C ossRe ]
2.
Fushimi, N.; Ee, C.E.; Nakajima, T.; Ichishima, E. Aspzincin, a Family o Me alloendopep idases wi h a New Zinc-Binding
Mo i . Iden i ica ion o New Zinc-Binding Si es (His
128
, His
132
, and Asp
164
) and Th ee Ca aly ically C ucial Residues (Glu
129
,
Asp
143
, and Ty
106
) o Deu e olysin om Aspe gillus O yzae by Si e-Di ec ed Mu agenesis. J. Biol. Chem. 1999,274, 24195–24201.
[C ossRe ] [PubMed]
3. Hoope , N.M. Families o Zinc Me allop o eases. FEBS Le . 1994,354, 1–6. [C ossRe ]
4.
Bode, W.; Gomis-Rü h, F.X.; S öcke , W. As acins, Se alysins, Snake Venom and Ma ix Me allop o einases Exhibi Iden ical
Zinc-Binding En i onmen s (HEXXHXXGXXH and Me -Tu n) and Topologies and Should Be G ouped in o a Common Family,
he ‘me zincins’. FEBS Le . 1993,331, 134–140. [C ossRe ]
5.
Schneide , J.S.; Glickman, M.S. Func ion o Si e-2 P o eases in Bac e ia and Bac e ial Pa hogens. Biochim. Biophys. Ac a 2013,1828,
2808–2814. [C ossRe ]
6.
Ce dà-Cos a, N.; Gomis-Rü h, F.X. A chi ec u e and Func ion o Me allopep idase Ca aly ic Domains. P o ein Sci. Publ. P o ein
Soc. 2014,23, 123–144. [C ossRe ]
7.
Langklo z, S.; Baumann, U.; Na be haus, F. S uc u e and Func ion o he Bac e ial AAA P o ease F sH. Biochim. Biophys. Ac a
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