Results: Using sequence profile searches and homology-based structure prediction, we have identified a previously uncharacterized family of P-loop NTPases, which includes the neuronal me
Trang 1A novel family of P-loop NTPases with an unusual phyletic
distribution and transmembrane segments inserted within the
NTPase domain
L Aravind, Lakshminarayan M Iyer, Detlef D Leipe and Eugene V Koonin
Address: National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, MD 20894,
USA
Correspondence: L Aravind E-mail: aravind@ncbi.nlm.nih.gov
© 2004 Aravind et al.; licensee BioMed Central Ltd This is an Open Access article: verbatim copying and redistribution of this article are permitted in all
media for any purpose, provided this notice is preserved along with the article's original URL.
A novel family of P-loop NTPases with an unusual phyletic distribution and transmembrane segments inserted within the NTPase domain
tional properties
Abstract
Background: Recent sequence-structure studies on P-loop-fold NTPases have substantially advanced the
existing understanding of their evolution and functional diversity These studies provide a framework for
characterization of novel lineages within this fold and prediction of their functional properties
Results: Using sequence profile searches and homology-based structure prediction, we have identified a
previously uncharacterized family of P-loop NTPases, which includes the neuronal membrane protein and
receptor tyrosine kinase substrate Kidins220/ARMS, which is conserved in animals, the F-plasmid PifA
protein involved in phage T7 exclusion, and several uncharacterized bacterial proteins We refer to these
(predicted) NTPases as the KAP family, after Kidins220/ARMS and PifA The KAP family NTPases are
sporadically distributed across a wide phylogenetic range in bacteria but among the eukaryotes are
represented only in animals Many of the prokaryotic KAP NTPases are encoded in plasmids and tend to
undergo disruption to form pseudogenes A unique feature of all eukaryotic and certain bacterial KAP
NTPases is the presence of two or four transmembrane helices inserted into the P-loop NTPase domain
These transmembrane helices anchor KAP NTPases in the membrane such that the P-loop domain is
located on the intracellular side We show that the KAP family belongs to the same major division of the
P-loop NTPase fold with the AAA+, ABC, RecA-like, VirD4-like, PilT-like, and AP/NACHT-like NTPase
classes In addition to the KAP family, we identified another small family of predicted bacterial NTPases,
with two transmembrane helices inserted into the P-loop domain This family is not specifically related to
the KAP NTPases, suggesting independent acquisition of the transmembrane helices
Conclusions: We predict that KAP family NTPases function principally in the NTP-dependent dynamics
of protein complexes, especially those associated with the intracellular surface of cell membranes Animal
KAP NTPases, including Kidins220/ARMS, are likely to function as NTP-dependent regulators of the
assembly of membrane-associated signaling complexes involved in neurite growth and development One
possible function of the prokaryotic KAP NTPases might be in the exclusion of selfish replicons, such as
viruses, from the host cells Phylogenetic analysis and phyletic patterns suggest that the common ancestor
of the animals acquired a KAP NTPase via lateral transfer from bacteria However, an earlier transfer into
eukaryotes followed by multiple losses in several eukaryotic lineages cannot be ruled out
Published: 16 April 2004
Genome Biology 2004, 5:R30
Received: 19 January 2004 Revised: 8 March 2004 Accepted: 11 March 2004 The electronic version of this article is the complete one and can be
found online at http://genomebiology.com/2004/5/5/R30
Trang 2The P-loop NTPase domains constitute one of the largest
apparently monophyletic groups of globular protein domains
in the proteomes of most cellular organisms [1,2] These
domains are implicated in nearly all biochemical and
mechanical processes in the cell, including translation,
tran-scription, replication and repair, intracellular trafficking,
membrane transport, and activation of various metabolites
[1,3] At the sequence level, most of the P-loop domains are
characterized by two conserved motifs, termed the Walker A
and B motifs [4] Structurally, P-loop domains adopt a
globu-lar fold with at least 5 α/β units (the P-loop NTPase fold), with
the strands typically forming a core parallel sheet [5,6] The
Walker A motif (typically, Gx4GK[T/S], where x is any
resi-due) encompasses the first strand and helix, and is involved
in binding the triphosphate moiety of the substrate NTP The
Walker B motif (typically, hhhhD, where h is a hydrophobic
residue) encompasses the third universally conserved strand
in the P-loop NTPase fold and coordinates a Mg2+ ion which
directs an attack on the bond between the β and γ phosphates
of the NTP [1,3,4]
A series of recent comparative studies on the sequences and
structures of P-loop NTPases defined the probable major
evo-lutionary events in the diversification of these domains
[6-12] In particular, these studies delineated two major
divi-sions of P-loop NTPases, the KG (kinase-GTPase) division
and the ASCE division (for additional strand, catalytic E) The
KG division includes kinases and GTPases that share many
structural similarities, such as the adjacent placement of the
P-loop and Walker B strands [9,10] The ASCE division is
characterized by an additional strand in the core sheet, which
is located between the P-loop strand and the Walker B strand
(Figure 1) [10] As opposed to kinases and GTPases, ATP
hydrolysis by the ASCE proteins typically depends on a
con-served catalytic (proton-abstracting) acidic residue (usually
glutamate) that primes a water molecule for the nucleophilic
attack on the γ-phosphate group of ATP ([10] and references
therein) As a consequence, ASCE division proteins typically
are more active NTPases than those of the KG division and do
not require accessory factors, such as GTPase-activating and
GDP-exchange proteins [9] In addition, most of the ASCE division NTPases possess a conserved polar residue at the carboxy terminus of strand 4, which is inserted between the strands associated with the Walker A and B motifs [10] The ASCE division includes AAA+, ABC, PilT, superfamily 1/2 (SF1/2) helicases, and RecA/F1/F0 classes of ATPases, and a large assemblage of NTPases related to the AP(apoptotic) and NACHT families [6-8,11,13,14]
Recognition of these distinctive sequence and structural fea-tures allows classification of uncharacterized P-loop NTPase families into one of the principal divisions and facilitates pre-dictions of their potential catalytic capacity Systematic anal-ysis of the P-loop NTPases further demonstrated that most of the conserved families of the ASCE division ATPases could be confidently placed within one of the six large classes men-tioned above [11] However, several families of ASCE NTPases remained outside this classification scheme Here, we apply sequence and structural analysis to characterize one such pre-viously unexplored family, which includes animal proteins participating in neural development and receptor tyrosine kinase signaling, and prokaryotic plasmid-encoded proteins that confer resistance to bacteriophages We investigate the evolutionary implications of their unusual phyletic distribu-tion and their unique structural feature, namely the inserdistribu-tion
of multiple transmembrane helices into the P-loop NTPase fold We also present predictions regarding their potential biochemical roles in eukaryotes and bacteria
Results and discussion
Identification and classification of the KAP family of predicted ATPases
During our systematic analysis of the P-loop NTPase fold, we detected the mammalian neuronal membrane protein named kinase D-interacting substance of 220 kDa (Kidins220) or ankyrin repeat-rich membrane spanning protein (ARMS) [15,16] in various searches initiated with position-specific scoring matrices (PSSMs) for different ASCE division ATPases, such as the AAA+ class The alignments produced in these searches indicated that the ARMS protein contained the
Multiple alignment of the KAP family NTPases
Figure 1 (see following page)
Multiple alignment of the KAP family NTPases The secondary structure predicted by the PHD program is displayed above the alignment, where E designates a β-strand and H designates α-helix The helix and strand numbering is given for the secondary structural elements of the conserved P-loop fold The 80% consensus coloring reflects the following amino acid classes: h (hydrophobic residues: ACFILMVWY), a (aromatic residues: FHWY), and l (aliphatic residues: VIL) are shaded yellow; b (big residues: LIYERFQKMW) are shaded gray; p (polar residues: CDEHKNQRST), - (acidic residues: DE), + (basic residues: HKR) and c (charged residues:HRKDE) are colored magenta; o (alcohol-group-containing residues: ST) are colored blue; s (small: GASCVDNPT) and u (tiny: GAS) residues are colored green The protein identifiers in the alignment include the name of the protein/gene, species abbreviation and the GenBank gi separated by underscores The groups discussed in the text are indicated to the right in the last block of the alignment The asterisk next to the rat sequence indicates a Kidins paralog with a potentially inactive NTPase domain Species abbreviations are as follows: Atu:
Agrobacterium tumefaciens, Ana: Anabaena sp pcc 7120, Ce: Caenorhabditis elegans, Cpe: Clostridium perfringens, Cgl: Corynebacterium glutamicum, Ceff: Corynebacterium efficiens, Dr: Deinococcus radiodurans, Dm: Drosophila melanogaster, Ec: Escherichia coli, Plaf: F plasmid, Gsu: Geobacter sulfurreducens, Hs: Homo sapiens, Kpne: Klebsiella pneumoniae, Lme: Leuconostoc mesenteroides, Mcsp: Magnetococcus sp mc-1, Mde: Microbulbifer degradans, Npu: Nostoc punctiforme, Pput: Pseudomonas putida, Pfl: Pseudomonas fluorescens, Psy: Pseudomonas syringae, Rme: Ralstonia metallidurans, Rn: Rattus norvegicus, Step: Staphylococcus epidermidis, Ssp: Synechocystis sp, Tm: Thermotoga maritima, Vpar: Vibrio parahaemolyticus, Vvul: Vibrio vulnificus.
Trang 3Figure 1 (see legend on previous page)
N-term helix Str-1 Helix 1 Transmembane helix-1
Sec Structure .HHHHHHHHHHHHHH EEEEEE HHHHHHHHHHHHHHH HHHHHHHHHHHHHHHHHHHHHH Kidins_Hs_14133247 433 HLSP TE TDG D MLGYDLY S ALA D ILSEPTM QP P V LY A QW G K FLL K KLE D EMK T FAGQQIEPL -FQFSWLIVFLTLLLCGGLGLLFAFTVHP -CG30387_Dm_28573593 428 RLNT NE DSE G MLGYELY S ALA D VLSEPTL TT P V LY A KW G K FLL N KLR D EMN N FARQWAEPPIRTSGLLFIVCLHVALLIGTIVGLSTW -Kidin_Ce_17540190 415 PIDA ED KMD T AMGYDVY S VLA D IVCEPSL SL P I LY A KW G K ALLAKLK E AMH S FSRDWLDGVS LSVSFALFFAIFLFFGMFSLTFTMLIAISNSVT LOC308414_Rn_27676618 172 GSFT S YGADILTEDDVY C CLA K TLCHVP- -V P V FY A PF G RLHLML D KIM T LMQ Q EAAQRESEE
Mdeg2631_Mde_23028847 277 SAEN KE VIK D SLARDRY V ALA K IIKNKRN DW N I LF A RW G K GLL S LLS K NLR N
all7130_Ana_17233146 1443 FRND TD LNE D LLNLKDEI D ALA N MLLMRDL EP P V IL G GW G K YIL H LMQ N RIL E
GSU0709_Gsu_39995815 227 TADDP T SAT D LMDVRQE A AFA R LAAGRAI RP P I VF G EW G K FFM K LMH E HVA R
DRC0009_Dr_10957551 1 MWAD TE TDR D YLNFTSV A TVA E LIVGSA- GN P I VS G AW G K SMI K LIR R NLN E
Reut2660_Rme_22977923 1 -D NE TKV D LLNNEAI A TIIGLLRAKP- DH P I VH G DW G K SVL E MIEAGFA D
Reut1119_Rme_22976310 1 MWHD NE TTV D YVNFKLV A VCA D LIRNSG- GD P I VS G GW G K SLV R MIEAELI S
AGR_pAT_30p_Atu_16119253 1 MWADV E TGR D FLNFNVM A LIS Q MILDAN- GE A I IS G GW G K SMV K LIEADLR T
c4514_Ec_26250336 1 MWSD KE SSE D YLNFGEV S LAV D VLTTKD- ML P I IF G NW G K SLL K LIE Q KLE Q
pifA_Plaf_9507753 14 DAAV ED VPE D RYGFGNI A NIS R SILTLPL EA S V IE G AW G K SLL N LIL R NLALK PifA_Kpne_38639573 14 DAAV EN VPE D RYGFRNI A NIS R SILSLPQ EA S I IE G AW G K SLL N LIL K SLF Q
PSPTO3386_Psy_28870550 70 DRAI T APEF D ALGRAPFI S SLV K TLVHTDY 11 AT G V LT G EW G K SVL N LLE H DLK Q
Lmes0002_Lme_23023289 11 DVPI KS SND D LLDRKQF A QLA R SILDYKQ SD S I LY G KW G K SVL N MTV E YLL D
all7133_Ana_17233149 21 DKPL SD PKD D KLGYAPF A NLA E SICKMSP PD G I VY A PW G K TLL N FII H YLK Q
Npun6978_Npu_23130674 12 DSSLV D PEK D LLGHANF A YLA D SICKMTF PE G I VY G SWN S S TLL N FVV H YLQ Q
TM1189_Tm_15643945 10 DEPL KS PDQ D KLGFAPF A RIA T VIQSVQL RE S F VY G KW G K TFI N FLT S YLN H
Cgl1727_Cgl_21324496 28 DLPI TK ISE D RFERSAY S AQLA N IICDVAP 1 GA S F LT G QW G K SLV N LIR S EESLS CE3P015_Ceff_23578001 6 DDPI KS VEE D EFGRSGY A AHVA K LINNSHS 1 ET S F LT G AW G K SMLAMIE K ELK E
Mmc11613_Mcsp_22999934 4 LNDT ET IDIEQLGAAQF A PIQ S MILEV TP P F IG A RW G K STL R ALWASLT H
VV12408_Vvul_27365727 1 ATRV CE SSEYLFGREAF A SLL N IFSNS ES G L ID A TW G K AFI H QLI H DLKAT VP2903_Vpar_28899677 4 DTQL T FEAR D EFNRKSI A KVI T LLRSD IT V LVID G SW G K EFC Q KLL S LMS T
PP1936_Pput_26988664 155 DDEI HK STE D ALHCDPQ A SFA K TIMASHA HP G F ID G PW G K SFI N LAA R YWE K
Pflu0188_Pfl_23057821 198 DRVI EE SEE D LLNVKEQ A IFA E RVLNGGS SE S F ID A PW G K SFV K LCC N YWE K
CPE1287_Cpe_18310269 169 FLNE EE ESY D LLERNNII E KLY E AIVNCNP KRKFII S LE G NW G K TIL N IVS K KIN D
slr1135_Ssp_16329878 1 -MI E DNQ S HNENIKEYLNYY K KLD SP G ILLK G EW G K HFI K NYF Q LED K
p415_Step_32470570 1 -MDKFKKAI T NYI E KDE N LFID G EW G K HFF E YD -Consensus/80% pp sbh hsp.hhp.h shshuh.u.aGsGKo.hhpbh.p.h.p.
Transmembrane helix-2 Str-2 Transmembrane helix-3
Sec Structure HHHHHHHHHHHHHHHHHHHHHHHHHH EEEEEE HHHHHHHHHHHHHHHHHH HHHHHHHH EEEEEE HHHHHHHHHHHHHHHHHHHHHHHHH Kidins_Hs_14133247 NLGIAVSLSFLALLYIFFIVIYFGGRR 45 V R FLFTDY N RLSSVGG- ET SLAEMIATLSDACEREFGFLATRLFRVFK TEDTQGKKKWK KTCCLPSFVIFLFIIGCIISGITLLAIFRVDPK -CG30387_Dm_28573593 SAVVGVSAAVGFLLLAYLLLAAVRYCN 44 V R FHFAEA NS ASPTG D GAVAHMLAALLDAIESHYGWLATRLYRAFR PKCLKVDVGWRWRRMCCIPIVLIFELALVTVVTGISLTVAYFTFADEKE Kidin_Ce_17540190 AYLISWSVFLLIFIIFCSLIVVVYYGD 43 V S FLFADYHRLSSIGG- EQ ALAKIVATLFEAAETHFGVLPVRLFCCMK PPYPGIHGSLR RHCGVPHVILLIVAVFLLIMAQVFGTVWLLSDR -LOC308414_Rn_27676618 39 V R FLFIRF SA WQYAGT- DK LWAGLV T TLCEGIRHHYGALPFSVYSVLG 4 GPRDGLCQREW-HCRRRVCLALLALLAALCLGVGLLYLSLGGHAPG -Mdeg2631_Mde_23028847 4 N K CYIANF NA WAYQGA- ES VRAAMA H EIVKTLTTKYYREYANEDHAER NWFMISIEK -VFGFVVEIRGVFTNLVCRFILAIKFTRRKS all7130_Ana_17233146 24 G H IYQIKF DA WTYAK SD LWASLM Q TIFFELDRQISLEQQLIKVGIE 203 YQSITLYSVREWAKKNKLLIIIFFVCLLLAILLPAGIQFFNNLGS GSU0709_Gsu_39995815 11 G N IVQIRF NA WHYVES N LWASLV D YIFTELDRWLKERPENPNETVD 104 GRARTLGRSAMATLGRPRWLAALALILVAAPVAVVWFRDILGRTEVLSW DRC0009_Dr_10957551 23 P K MVFVEF NA WLYQGY- DD ARAALM D VIARELTAEAERQKTGMDHVKD FVSRINWMRGARVAAHLGA
Reut2660_Rme_22977923 D D VLCLKF NG WRFQGF- ED AKIALI E GIVTGLIEKRPALKKAAVAIKD VFRRIDWLKVAKRSGGLAL Reut1119_Rme_22976310 11 EPYVVVTF NP WLYQGF- ED ARTALL Q TVGDAVLKQAEGSQTLTDKAKA FVKRINLLRLAQLGGEVAA AGR_pAT_30p_Atu_16119253 11 R S LLFVNF NA WLYQGH- DD AKAALM E EIANALMIRAKQQQTSVQKGMN LLKRIDVFRGIWMLGELAV c4514_Ec_26250336 1 K D WIVINF DS WLYQGY- DD TRAALL E VIATELTKAAEGNSTLISKTKR LLSRVDGFRAMGLLAEGTA pifA_Plaf_9507753 2 A H THVLHI SP WLSGGSPV E ALFLPVATVIQQEMEIRYPPKGFKKLWRK 5 EAQKVIEYAQDTSSRVLPL PifA_Kpne_38639573 2 G H THVLHV SP WLSGSDPV E ALFLPVATVIQQEMEKRYPPKGFKKFWRK 5 EAQKVIEYAQDTSSRVLPL PSPTO3386_Psy_28870550 E H VAVATL NP WLFKGR- DE VVEAYF N ALREALGFSSSEKARKLLVHLA 11 TTAVVIDFVVGTGSATAIW Lmes0002_Lme_23023289 5 N K PEIIRF NP WMFTDE- SQ LINQFF K QLSSNFIGKKDKKKLGDQLQIL GDVLGLTTFVPGVGILGTA all7133_Ana_17233149 3 E Q PIIVPY NP WWFSGQ- ED LTKSFF E QLSGVLYEKWQSLGRKFKNQIE SFAERVSTVPGLWTKGFAA Npun6978_Npu_23130674 3 E Q PIIVPF NP WLLSGH- QN ITRRFF E QLQNVLSQQSSVPKGLKERLAD FAAIISDIPLPYAQTGKAL TM1189_Tm_15643945 S S ITIVKF DP WWFSEK- ED LIRQFL S NLQFTLNKSTKFKDIAKMLKPY IETLGEIPKFGWIFKIASR Cgl1727_Cgl_21324496 1 E K WTIVDF NP WVASDP- QS LIEEFY R VIVGTVPDDKTGQKIKTVLQKT FSTIGSIAGGVGGFGVLEA CE3P015_Ceff_23578001 1 G D WHIAYF TP WATSDV- N GLFADFY S SLEHALSSEGERE-FSTILGEM LTIAAPIAKIIPVVGDATQ Mmc11613_Mcsp_22999934 27 LYVKTVWF NP WQHQHE- QN PLVPLL H EIREQMRHQTLHQGLAGCATVF EAGIHTMGALIDDAQNISY VV12408_Vvul_27365727 E K IIPIYY DA FSN D FSNDTFL S IGATIFHEVEGYFESTGKSVKV KKQLEHLKDLT-KKTAGEL VP2903_Vpar_28899677 E T HHLIYI DA FKA DH ADEPLL T VLAKVLEVLPSQEEQQGLIQKA IPALRYGLKTGGKALVAHI PP1936_Pput_26988664 1 N E IIICRFE P LRFASE-P D LTDRLI K ELSATIQREAYAPEFRPAAS RYSRLIKGKADISFLGFKL Pflu0188_Pfl_23057821 2 Q S IIVHHFE P LRYEDG- TD LTEKFV D DLISTIQQHVFAPSLRPLFKRY ENLVKDKKKTSLLDIKTTF CPE1287_Cpe_18310269 2 DIKIISSF DP WSYNDQ-I S MFRSMF D ILLKETGISYSIGKTKRLVNDI YNILFSTKYTKGIKDLNFF slr1135_Ssp_16329878 1 N E SFNFKKKYFSLK - NN QHKENS K AIYISLYGIKDIESIDILIIQK LIPILADRKIQLTGSVINI p415_Step_32470570 YFFNEI D ENNEDIQ- KN YNKSSY K KEYISVYGKHSLKQIQEIIVTK LLSHVDEDVINQNIKKGLN Consensus/80% p hh.hssh pph hhp.l h
Walker A
Transmembrane helix-4 Helix-2 Str-3 Helix-3 Sec Structure HHHHHHHHHHHHHHHHHHHHHHH.HHHHHHHH HHHHHHHHHH HHHHHHHHHHH EEEEEEE HHHHHHHHHHHHHHH
Kidins_Hs_14133247 HLTVNAVLISIASVVGLAFVLNCRTWWQVLDSLLNSQRKRLHNAASKLHKLKSEGFM KVLKC E VE -LMA R MA K TID S 3 NQT R LVVII D D AC EQ DK VLQML D TV R VLFSK -CG30387_Dm_28573593 KEHILVALYVIAAVMGTLICTHLHVLAKVFVSLFTSHIRVLKRAVRSSESAPL TMLGA E VA -VMT D MV K CLDA 3 QQS R LVGVI D D SC DT ER ILTLL N AV Q TLLSSPN-Kidins_Ce_17540190 DPNNFNLFIAIAFLCGFVMIAIYPLALIIMYSWTNVPRRRVNAAARNAHKLRFEGLM QKLQT E VD -LLA D MI R SLDA 3 SHT R LVVVV D D NC EQ ER MVQTL D AL E LLFSARKH LOC308414_Rn_27676618 HAERGVLKALGGAATTLSGSGLLMAVYSVGKHLFVSQRKKIERLVSREKFGSQLGFM CEVKK E VE -LLT D FL C FLEI 3 RRL R VVLEVT G TC YP ER VVGVL N AI N TLLSDSH-Mdeg2631_Mde_23028847 ILKLMATSIVVVLSAPFVYSGLSDFIASFFKDWRLINPSDVNYLAAVEASIGVLVSV 36 MSQDL K IL -CGI Q LGAGAR E 1 YTR R MVVIV D DR C EP DC IVKVF E AI K LVMDI -all7130_Ana_17233146 -SKVIAQVVGFFTPMLPAIATLQALWTTGKKWYDETQLALNEYKTSYEQALEERVQK 128 PADSK D YA AKIDFLK K AFP R GPA R VILYI D DR C SP DT VVQVL E AV Q LLVKN -GSU0709_Gsu_39995815 LKEVNAAVLGLSSVMASVAGFAGTALKRTATALDTLEGFRANLETAIAERTEEFRKN 117 DVLTD E EV -AAL R AS T TFDA 4 LFE R IILYI D DR C PP EK VVEVL Q AI H LLLCF -DRC0009_Dr_10957551 54 TSPPQ E IQ ALRS S FE T ALE K LDVVLVVLI D DR C LP ET TISTL E AI R LFLFL -Reut2660_Rme_22977923 51 KNVPE E VE AFRKAFD Q LLK D 1 GIK Q LVVLI D DR C LP DT AIETL E AI R LFVFT -Reut1119_Rme_22976310 50 RSLPK E IQ GFRD D LE E LLS E LGV T LVVFV D DR C LP KT AIATL E AI R LLLFL -c4514_Ec_26250336 54 KSPPQ Q ID AFRK E YG E ILE E LGKPLIVVI D DR C LPA N AIHTL E AI R LFLFL -AGR_pAT_30p_Atu_16119253 55 QTPPQMIH AIRQ Q FE E LLE D LNL T LVVFV D DR C LPP T VIGTL E AM R LFLFM -pifA_Plaf_9507753 26 AVDQK T TT KLRA E IAGQLV S LDL K FIVVM D DR L EP SQ VAEVF R LV R AVADL -PifA_Kpne_38639573 26 AVDQK T TT KLRA E IA K QLV T LDL K FIVVM D DR L EP SQ IAEVF R LV R AVADL -PSPTO3386_Psy_28870550 12 KSRGL S AN EERK N LEAKLA E AKIAIVMLI D EL V ED EE VRVVA Q LV K AVGDI -Lmes0002_Lme_23023289 14 SALNK N IQ KIKD D LV S EIK K NNI K FIILI D DR L STI D IQSVF K LV Q SIADF -all7133_Ana_17233149 4 VISPK D IH KLKQ E IE E TLK K QQK R ILVVI D DR L TA EE IRQLF R VI K AVANF -Npun6978_Npu_23130674 4 DEKDK E AA QLKE E VE D TLV Q QQR R IVVTI D DR L PA ED IKQLF R IF K AMRNF -TM1189_Tm_15643945 2 KNLQK S VI ETKE E II N RLK E KDG K IVVII D DR L TA KE IRELF T IV K AIADF -Cgl1727_Cgl_21324496 17 KQEQD S WP TLYT R AA N HFK D LNK R ILIVV D DR L HT DE LALLM K VI R LLGRF -CE3P015_Ceff_23578001 9 LQDQPPWK ETFE K AS S EIK K LNR K ILIIA D DR L QG EE LMALL K VV R LLGRF -Mmc11613_Mcsp_22999934 26 FSGRL E SQ YFRSAFE D AVI K 13 TGV R LVVFI D DR C SD QT VFTLL E SI K LYLSS -VV12408_Vvul_27365727 45 FKAYE N AK SNIQ S YV D ALE S 3 NGE K VIFFI D EL C RP D FAVEVL E KV K HLFAA -VP2903_Vpar_28899677 31 LKDHV E AE SSLQALQ Q ALK S 2 EQKPIVLFI D EL C RP N FSVLML E TI K HTFDV -PP1936_Pput_26988664 2 EPSQE T LD ELLD D ID D VLR R IGR R VIIVI D DR L DS KT ANSVLFAT R RTFKL -Pflu0188_Pfl_23057821 SLNND S ID ATLE E MEYVLN N INT R IIVIV D DR M HW SS AKSILFSI K RSFRL -CPE1287_Cpe_18310269 1 HDKTT E IE KMKKMIN N YLHI SNK R IVFII D DR A EK EN IILLF K LV N NVFNF -slr1135_Ssp_16329878 7 IDLKDLKN TKIL N EF T NLD N KILIL D ER C KI D INDLLGYINFFVEH p415_Step_32470570 5 LDIKYIKN 12 TKAI N KI K KNL N 1 NGA E VVLII D ER LSSSI N LKEFLGFIR N VLLDS -Consensus/80% .p b.ph.p.h.p phlhhhDsl-Rh pph hphhp.hh
Str-4 Helix-4 Str-5 Helix-5 Sec Structure EEEEEEE HHHHHHHHHH HHHH.EEEEEEEEE HHHHHHHHHHH HHHHHHHH HHHHHHHHHHHHHHH
Kidins_Hs_14133247 GPFIAIF A P IIIK A IN Q NLN 7 IN G YM R NIV H VFLNSRGLSNA R KFLVTS -AT N GDVPCSDTTGI 45 FDLTKLLVTED 1 FSDI S PQTM RR N IVSVTGRLL 959\Animal CG30387_Dm_28573593 RPFVLLI S P VIAK A AEANSR 7 IG G FL R NLV H VYLQNSGLRKV Q RAQMTA LLF K RSGGGDYQTDD 62 LDLSRIVLTDD 1 FSDV N SM LM IYITVRLL 972|KIDINS Kidins_Ce_17540190 RPFITII A P VIVS A IN H NMH 7 LT G YL K NII S FYLHNSALRQL Q SKLREK R E SMAEWKERFKR 35 RNMNDGILGED 1 FSNM N AM IV LTLTGRLM 937|
LOC308414_Rn_27676618 APFIFIL V P ILAA C LE S AGN 5 DN G YLFLNRTV T FSVPVMGRRTKLQFLHDA-VRSR D DLLFRELTIKL 41 EALCCLHDEGD 5 VPD- N VVSM RR NT VPITVRLL 641/*
Mdeg2631_Mde_23028847 PNVIVII S H IALS A LS E NYQ 12 SI A YLG K II N ICLPPLSSDNV K AYIAHL -IE E SAAETLNSQSI 51 DIQRSLADWAI 1 LGIN N QI LY YHMMINIY 739\Bacterial all7130_Ana_17233146 RLFIAVV A E YINR A LA K YYQ 8 PS P YL II Q YRVTSIADSAL R QYLKSQ VAIQDSGISGNKF 5 EEFNILVQCCQ EVDL S SL LT YKLFKVLN 2172|KIDINS GSU0709_Gsu_39995815 PLFVVVV A A WVSR S LK E VYP 46 AS S YL IF Q YWVRAMDADAC R NYIKGIVAAES T VQADQAPLSPE 61 PHETAFMAELA 1 HAGG T RGL R FV YRLIRTSL 940/
DRC0009_Dr_10957551 KRTAFVI A D MIKH A VR K HFE 5 AA V YF LI Q VRVPPLSTQDV R AYLLLL-LVED S ELEAEKKDRVV 39 DHLAPLLATAN GIDG N LI FL LSIRRAVA 404\DRC0009 Reut2660_Rme_22977923 AQTAFVV A EAMIEY A VR K HFP 9 DY A YL LI Q FRIPALGRSRDANLRGVV AGR R RSRRGRRGLRE 59 QALSQYAVAAR THCDRA R FR HQ RARKAHAR 399|-like Reut1119_Rme_22976310 KGSAFVV A DVFIRG A VRVHFT 6 DV V YF LI Q LRVPRLGPNET K AYAALL -FL E RAHREKSIDDT 45 ERLSPLLLNAR AVQS N LV FL VFLRQAMA 393|group AGR_pAT_30p_Atu_16119253 KGTAFII A D MIKE A VRVHFP 6 DI V YF LI Q LRVPPLGTNEV K AYLMLL -FV E SSRIPPAEKEI 41 DRLARQMIISP KVNG N LI FM LSIRRSLA 394|
c4514_Ec_26250336 TNTAFII A E MIRS S VA D YFK 4 RHQI D YL LI Q IRVPKAGVREI R SYLFML -YAIEHGLEGEKITM 42 DRIAPILANSP IIHG N IV LL VKMRSQIA 382/
pifA_Plaf_9507753 PRFTHIL C R IITH A VE H ALN 1 ED G YLQ K II Q FKLPRPEAFDL R NEFRQR A E ALYQQINNQPP 5 RDLIAVTDTYG AALS T EI AI LIFLYPGM 334\PifA-PifA_Kpne_38639573 PRFTHIL C R IITH A VEYALN 1 ED G YLQ K II Q FKLPRPEAFDL R NEFRQR A E ALYQQINNQPP 5 GDLAAVTDTYG GALS T EI AI LIFLYPGM 334|like PSPTO3386_Psy_28870550 KGISYLV A P RVAQ A LG K GST 5 KA G YL II Q IPLRPLFMDEA R DLLLQA M R NNDVTMPAESQ SYQTEILNQLL RVIR T EI LI FAVLEEIV 389|group Lmes0002_Lme_23023289 PNTIYLL A Y IVTR A LE E VQK DN G YL II Q FNLPVISEVKI T QIFISE L N KIFKNIPEDKF 3 AWAELLHGSIS YYLQ S DLA R LN IGSGANSV 313|
all7133_Ana_17233149 PNVVYLLLF D VVIK A LE E IQK IN G EVYL EK Q FELPLPDRIQL S RLFDSQ L D KIISGTPEELF 3 YWLEIYWQGIE HFIT T SIL R LA LMVTYPGV 311|
Npun6978_Npu_23130674 TNVVYLL V K VVMK T IA D PKE IS G YL II Q FELPVPDKISL R RLLFEK L D NIFTESPKPEI 3 RWGEIYFQGID RFIN S DIT R FV LTVTYPAV 302|
TM1189_Tm_15643945 PNTVYIL A K IVIR A LE K VQE GK G YL II Q IELPLADKTSI R KMLFEE L D AVLSGTSNELF 3 YWRNVYWDGID PFIN T NV LI IRVTYPSV 295|
Cgl1727_Cgl_21324496 PQVNYLL V YEE E SLLT T LA R STA 5 DD A FM IV Q FDVPPLTSFQI E KELSAL F D KLFQGVSLSGD 5 LVKSRMFDVWE KTLV T LL FA A LLTNWTRIY 337|
CE3P015_Ceff_23578001 PGVDFLL A E TVTQ T LAAMGV 5 SG S FM II Q LAIPPLLPTQLISNLMHK L D PYLEQMEESDT 2 IRLQHLRPVLL AQLS T AIG R YI A QVHHHLATF 303/
Mmc11613_Mcsp_22999934 KYCIFVF G RGHVEN A VA K AAM 3 VE A YV LF Q TRLTLPSPSHDQI K KFVQEM -LK K TEEFKSLEDEK LSRLAELLSV- LSPN N FI LI LILYKKLF 351\Other VV12408_Vvul_27365727 KNVIFVI S YNK S QLSKIIS H VYG 3 KD A YL FI H IEANLPVVDEKSS T SSYEQL F D SFVREFNIELP 8 LKNMFTLLCQP 1 HLNM N EI AF S YVSFCFAAL 335|bacterial VP2903_Vpar_28899677 EGVQFVLITNT N QLKA S IN H CYG 2 ID A YL FI R FTLPHTTNENR H DVTMAS V T HYKNLVAKSER 9 SDFWLVAQVIN TNNI S EV LVRHIEIYQALF 323|KAP PP1936_Pput_26988664 SQATFIL C T ILAGIQE E T SR A FL FV T VKLSLFVDSSSIQ N -FLTRD W Q NEEQKLTSVPS 15 ILEGDNAASYL PYVR N KV FV LLILQMER 448|NTPases Pflu0188_Pfl_23057821 PNISYVI C T KINV T PE N PDS EK T FL FI N IKTSIFLGAQDLTAFVKRYF - D SVLSKTLNISS 15 LFNDKDFPHYT PFIG D KI LI LVLLDIDK 494|
CPE1287_Cpe_18310269 EYVTYIL S D KLKKILE N QL- DI D FIS K IV Q IKIPPLDLEVK N EVISTC F K NIIRLYGEDNL EKYNDLINSLS KLII D DF FI VVSVHYKN 451|
slr1135_Ssp_16329878 QALKVILIA D KIEG N II Q SYE 1 KTFD K IK VIGKRFTVNTSFNKAF E QFLNLV -CK D EQEKTYLSK KRDFIKELFET SDSN N TL IIY D FDRIYSYL 275|
p415_Step_32470570 FNCKVIL V GNK N SINS A HQ E - -GMT E HW VI S LKFPSNLEVAK N ILEDDL -KTIDFEKNEIQEIK 1 FICIYSLSKSE SSVL N TL K LVI AD FKNLYDQL 274/
Consensus/80% hlhshD.p.h sh.p s.phhcKhhphsh.h p h p s.R.hcphhssh h
Walker B
Trang 4characteristic sequence signatures of the Walker A and B
motifs However, examination of these alignments also
showed that ARMS contained one or more long inserts (>100
amino acid residues) within the potential P-loop NTPase
domain
To further investigate the structure and evolutionary
connec-tions of this protein, we performed PSI-BLAST searches
(expectation value of 0.01 for inclusion of sequences into the
PSSM, with the statistical correction for compositional bias)
using as the query the sequence of the putative P-loop NTPase
domain of ARMS (GenBank identifier gi: 14133247, residues
433-959) The first iteration of this search retrieved apparent
orthologs of ARMS from other animals, such as Danio,
Dro-sophila, Anopheles and Caenorhabditis, and a homolog from
the cyanobacterium Anabaena The subsequent iterations
also detected, with significant E-values (e < 10-5) apparent
divergent homologs from bacteria spanning a broad phyletic
range (Figure 1) A possible pseudogene belonging to this
family was also detected in the genomes of the archaea
Meth-anococcus jannaschii and Methanosarcina (see below) The
prokaryotic proteins detected in these searches included the
PifA protein, which is encoded in the enterobacterial F
plas-mid and is required for exclusion of bacteriophage T7 [17,18]
All these proteins contain the typical Walker A and B
signa-tures, suggesting that they are functional P-loop NTPases In
contrast to the animal ARMS orthologs, most of the bacterial
proteins, except for those from Anabaena species, Geobacter
sulfurreducens and Microbulbifer degradans, lacked the
large inserts within the P-loop NTPase domain Reciprocal
PSI-BLAST searches initiated with these bacterial proteins as
queries first retrieved a consistent set of proteins that
included the animal ARMS orthologs before the retrieval of
other ASCE NTPases, such as the AP/NACHT-NTPases,
AAA+ and ABC classes These observations suggested that
ARMS homologs define a novel group of P-loop NTPases that
is distinct from all the previously described classes of P-loop
domains Hereinafter, we refer to them as the KAP family of
(predicted) NTPases (after Kidins220/ARMS and PifA) In
addition, the above searches retrieved a vertebrate paralog of
the ARMS protein (for example, Rattus norvegicus protein
LOC308414), in which Walker A and B motifs are disrupted
(Figure 1), indicating that, unlike other ARMS homologs, it
might lack NTPase activity
To further explore the functional features and evolutionary
relationships of the KAP family, we constructed a multiple
alignment of the KAP proteins and compared its sequence
conservation pattern and predicted secondary structure with
those of other P-loop NTPases (Figure 1) The Walker B motif
in the KAP family sequences typically has the form hhhhD[D/
G]hD (where h is any hydrophobic residue) The second
aspartate (D) immediately after the Walker B aspartate (first
aspartate) is present in most of the bacterial KAP domains but
is replaced by a glycine or an alanine in the animal sequences
(Figure 1) An acidic residue in this position is an ancestral
feature of the ASCE division of ATPases, and the presence of
an aspartate is specifically characteristic of the AP/NACHT-NTPases as opposed to the glutamate, which is most common
in the SFI/II helicase and AAA+ ATPases [7,13,14,19,20] Furthermore, the third aspartate located three positions downstream of the Walker B aspartate is a shared feature of the KAP and NACHT families [13] In the KAP family pro-teins, one of these aspartates might function as the proton-abstracting negative charge in NTP hydrolysis The KAP fam-ily proteins contain another conserved polar residue (typi-cally, D) at the end of strand 4 (Figure 1) This feature is also characteristic of the ASCE NTPases and corresponds to the sensor I motif of the AAA+ domains and its counterparts in other proteins of the ASCE division [7,11,14] These conserved features, together with the consistent detection of various ASCE NTPases in database searches with the profiles of KAP family PSSM, strongly suggest that this family belongs to the ASCE division
The conserved core of the P-loop NTPase domain of the KAP family contains an α-helix amino-terminal of the Walker A strand and an α-helical extension with three to four predicted helical segments occurring carboxy-terminal of strand 5 (Fig-ures 1, 2) Similar structural feat(Fig-ures are also seen in the AAA+ ATPases and the NACHT/AP-NTPases, suggesting that the KAP family might form a higher-order group with these classes of NTPase domains within the ASCE division [11,13] However, the specific extended sequence signatures associ-ated with the Walker B motif, strand 5 of the core P-loop NTPase domain, and the carboxy-terminal helical module (Figure 1) clearly distinguish KAP ATPases from all other ASCE NTPases Although most proteins of the KAP family have a conserved lysine at the beginning of strand 5, this res-idue does not appear to be equivalent to the arginine finger, which is found in ring-forming ASCE NTPases, such as the AAA+ and VirD4-like ATPases [6,7,11,14] This suggests that KAP ATPases do not have an arginine finger and are unlikely
to function as oligomeric rings However, the KAP family pro-teins contain a conserved arginine in the carboxy-terminal helical segment, which could potentially function similarly to the sensor-2 arginine of the AAA+ ATPases (Figure 1) Exam-ination of the multiple alignment suggests that, in addition to the five conserved strands of the core P-loop domain, the KAP family NTPase domain contains an additional strand after the core strand 2 (Figure 1) By analogy with the RecA and VirD4/ PilT classes, this additional extended segment might stack externally on the β-sheet alongside strand 2 (Figure 2) [6,8] Most of the NTPases of the KAP family have a variable α-hel-ical insert amino-terminal to the Walker B motif Remarka-bly, all animal KAP NTPases and three bacterial ones, those
from Anabaena, G sulfurreducens and Microbulbifer,
con-tain two membrane-spanning helices inserted in this region (Figures 1, 2) The animal proteins additionally contain two more transmembrane helices inserted in the region between helix 1 (associated with the Walker A motif) and strand 2 of
Trang 5the core NTPase domain Insertion of membrane-spanning
helices into globular domains is extremely rare in proteins
[21], and, to our knowledge, the KAP family is the first such
instance among P-loop NTPase domains In the NTPase
domains that do not form ring structures, most residues
involved in NTP-binding and hydrolysis are located at the
carboxy termini of the strands forming the core parallel
β-sheet (Figures 1, 2) This causes a polarity in the structure of
the NTPase domain with respect to the location of catalytic
surface, thus allowing it to accrete inserts in regions that are
spatially disjointed from this catalytic surface This might
explain the ability of the KAP NTPase domain to retain its
structural and functional integrity despite the insertion of
transmembrane helices Superposition of the multiple
align-ment of the KAP family onto known structures of the P-loop
NTPase domains suggests that the membrane-spanning
inserts project outward from the conserved intracellular
glob-ular core, probably from the surface opposite to the
NTP-binding surface (Figure 2)
Prediction of functional features of the KAP NTPases
In mammals, Kidins220/ARMS localizes to the tips of neur-ites and is abundantly expressed in the neural tissues in regions that are enriched in receptors for ephrins and ligands
of the neurotrophin family Furthermore, Kidins220/ARMS physically interacts with TrkA and p75 neurotrophin recep-tors and is phosphorylated upon activation of the neutrophin and ephrin receptors [15,16] Kidins220/ARMS also appears
to be a physiological substrate for protein kinase D, suggest-ing that it might be a key target for multiple neuronal signal-ing cascades [15,16] Kidins220/ARMS and all its animal orthologs contain 10 or more amino-terminal ankyrin repeats
[22], while the Anabaena homolog with transmembrane
seg-ments contains approximately 40 TPR repeats amino-termi-nal to the P-loop NTPase domain [23] Similarly, the
membrane-associated KAP proteins from Microbulbifer and
G sulfurreducens contain a large amino-terminal segment
with predicted coiled-coil structure Phosphorylation of Kidins220/ARMS by various kinases suggests that this pro-tein might function as a signaling nexus associated with the cell membrane The α-superhelical structure domains present in animal (and some bacterial) KAP NTPases, such as ankyrin and TPR repeats, could provide extended surfaces to mediate interactions with various protein complexes The likely function for the KAP NTPase domain is the regulation
of assembly/disassembly of these complexes in an NTP-dependent manner In particular, Kidins220/ARMS and the orthologous KAP NTPases in other animals might regulate the assembly of neurite-membrane-associated signaling com-plexes that are positioned downstream of different receptor tyrosine kinases in the respective signaling pathways Con-sistent with this proposal, the high-throughput screens for
protein-protein interactions in Drosophila recovered the
PDZ-domain protein Dlg, which binds the carboxy-terminal tails of neural membrane proteins, as an interacting partner for the Kidins220/ARMS ortholog [24] The vertebrate para-logs of Kidins220/ARMS with apparently inactive NTPase domains lack the ankyrin repeats and might function as dom-inant-negative regulators of active KAP NTPases
The bacterial KAP proteins without the transmembrane regions contain a variable helical insert (Figure 1), which could function as a site for interactions with other proteins
The prokaryotic KAP family members have not been charac-terized biochemically, but potential leads to their functions are suggested by the available data on the PifA protein, which
is encoded in enterobacterial F plasmids and is required for exclusion of bacteriophage T7 from plasmid-containing cells [17,18] The exclusion process involves interactions between PifA and the products of T7 genes 1.2 and 10, which code for the major phage capsid proteins, and is accompanied by an increase in membrane permeability [17,25] These observa-tions imply that PifA might reorganize certain membrane-associated complexes in an ATP-dependent manner and thereby disrupt the T7 life cycle While it is not clear whether the principal function of PifA is in bacteriophage exclusion,
Predicted topology of the KAP P-loop NTPases and comparison with
other P-loop NTPases
Figure 2
Predicted topology of the KAP P-loop NTPases and comparison with
other P-loop NTPases The core conserved strands that are shared by all
ASCE division NTPases are numbered 1-5, and X indicates additional
strands that are observed only in certain NTPases.
KAP ATPase
(hypothetical)
DD
DS
ASCE ATPases
ankyrin repeats
Cell membrane
RuvB - AAA+ (1HQC)
Thermus thermophilus
Arg
Cdc6 - AAA+ (1FNN)
Pyrobaculum aerophilum
RecA (2REB)
2 2 2
5 5
5
5 1
1
1
1 4
4
4
4
3
3
3
3
x
x 1 2
Trang 6some other lines of circumstantial evidence support this
possibility
The sporadic distribution of the KAP family in prokaryotes
and its presence on plasmids (and a filamentous phage in
Vibrio) in various species (Figure 3) suggests that it was
widely disseminated by these laterally mobile replicons
Pro-tection of bacterial cells from phages could be one of the
func-tions of KAP NTPases in prokaryotes, a role that is conducive
to rapid horizontal spread, by analogy with the dissemination
of antibiotic-resistance determinants In at least six
prokary-otes, including both occurrences in archaea, the genes for
KAP NTPases were disrupted by frameshifts Although some
sequencing errors cannot be ruled out, it seems extremely
unlikely that such errors occurred independently in
homologous genes in several species Furthermore, on several occasions, species or strains closely related to those that har-bor a frameshift in the KAP gene have an intact counterpart, suggesting multiple recent pseudogene formation events in the KAP family Inactivation of KAP NTPases might be driven
by phages acquiring resistance to the KAP-mediated path-ways, thereby rendering KAP genes superfluous Coexpres-sion of PifA with plasmids encoding genes 1.2 and 10 of T7
resulted in lethality in Escherichia coli [26] Such deleterious
effects of KAP NTPases under certain circumstances, such as expression of high levels of certain phage proteins, could be
an alternative selective pressure for their inactivation
In prokaryotic genomes, genes coding for functionally inter-acting proteins often co-occur in conserved operons or form
Phylogenetic tree and domain architectures of KAP NTPases
Figure 3
Phylogenetic tree and domain architectures of KAP NTPases Proteins are denoted by their gene names and species abbreviations Plasmid-borne genes are denoted by red asterisks, and phage genes are denoted by a red +; the eukaryotic branches are colored green Species abbreviations are as in Figure 1 Filled yellow circles indicate nodes with bootstrap support of greater than 75% in the full maximum-likelihood analysis The bootstrap values obtained through different methods (Full maximum likelihood, Rell bootstrap with Protml/Rell BP, Puzzle bootstrap/Puzzle-B, Neighbor Joining, Minimum evolution) are specifically shown for the clade that includes animal and bacterial proteins In the schematics of protein and gene structure, conserved operons are shown as boxed arrow, and transmembrane regions inserted into the KAP domain are shown in blue DRC0009-C and PifA-C refer to carboxy-terminal globular regions shared by the DRC0009-C and PifA subfamily KAP ATPases Note that CPE1287 and Lmes0002 do not have the PifA-C domain.
PSPTO3386_Psy
CPE1287_Cpe
pifA_F plasmid*
pifA_Kpne*
Cgl1727_Cgl
CE3P015_Ceff*
Lmes0002 Lme
TM1189 Tma all7133 Npun6978 Npun VV12408_Vvul VP2903_Vpar+
slr1135_Ssp
p415_Step*
PP1936_Ppu Pflu0188_Pfl Mmc11613_Mcsp Mdeg2631_Mde
Full ML:80 Rell BP:98 Puzzle-B:76 NJ:100 ME:100
LOC308414_Rn
Kidin_Ce
CG30387_Dm
KIAA1250_Hs
all7130_Ana*
GSU0709_Gsu
Reut2660_Rme
c4514_Ec Reut1119_Rme AGR_pAT30p_Atu*
DRC0009_Dr*
Kidins220/ARMS_Hs
//
Ankyrin repeats
all7130_Ana
KA P
TPR repeats
40
GSU0709_Gsu
KA P
Helical
pifA_F plasmid
LOC308414_Rn
KAXP
DRC0009_Dr gene neighborhood
DRC0007
c4514_Ec
Ana*
Trang 7gene fusions to give rise to a single gene Consequently,
evo-lutionarily conserved juxtaposition of functionally
uncharac-terized genes with genes whose functions are known has the
potential to throw light on the functions of the former
[27-29] In the case of KAP NTPases, a conserved gene
neighbor-hood was detected in E coli (strain cft073), Deinococcus
radiodurans plasmid CP1, and Agrobacterium tumefaciens
plasmid AT, in which the gene for the KAP NTPase is located
next to genes encoding a TIM barrel DNase of the TatD family
[30] and an ATP pyrophosphohydrolase of the PP-loop fold
[31] Although the exact functional implications of this
link-age are unclear, it seems likely that these enzymes cooperate
with the KAP NTPases in the inhibition of phage
reproduc-tion; the DNase, in particular, is a candidate for a role in
deg-radation of phage DNA
Evolution of the KAP NTPase family
Phylogenetic trees of the conserved NTPase domain of the
KAP family were constructed using the maximum likelihood,
neighbor-joining, and minimum evolution methods (see
Materials and methods for details) The trees constructed
with each of these methods had similar topologies and
sug-gested existence of several subfamilies within the KAP family
One of these, the ARMS subfamily, includes all animal KAP
proteins and three bacterial members, those from M.
degradans, G sulfurreducens and Anabaena (Figure 3) In
this case, phylogenetic analysis strongly supported
mono-phyly of this group, which was independently suggested by
their shared derived character, the insertion of
transmem-brane helices into the P-loop domain A second subfamily
consists of proteins from phylogenetically diverse bacteria,
such as E coli (strain cft073), D radiodurans plasmid CP1, A.
tumefaciens plasmid AT, Ralstonia and Magnetococcus, and
is also supported by an apparent shared derived character, a
carboxy-terminal globular domain that is unique to this
family This bacterial subfamily groups with the ARMS
sub-family, to the exclusion of homologs from all other
prokaryotes (Figure 3) The third major subfamily includes
the F-plasmid-borne PifA and its homologs from plasmids
and chromosomes of Klebsiella, Pseudomonas,
Corynebacte-rium, Nostoc, Thermotoga, Clostridium and Leuconostoc.
The validity of this family is supported by the presence of a
unique carboxy-terminal domain that shows no obvious
rela-tionships with any previously conserved globular domains
Thus, on more than one occasion, the phylogenetic tree of the
KAP family brings together phylogenetically distant bacteria
(for example, Deinococcus, Agrobacterium and E coli) in
well-supported clades, strongly suggesting a major role of
plasmid-mediated horizontal transfer in the evolution of this
family (Figure 3) The most striking feature of the tree is the
nesting of the animal ARMS homologs within a clade
contain-ing bacterial members Among the currently available
mem-bers of the KAP family, the greatest diversity is seen in
bacteria, and almost all subfamilies contain multiple
plas-mid-borne members It seems likely that the original KAP
NTPase evolved on a bacterial plasmid and had a role in the modification of the bacterial membrane that results in exclu-sion of bacteriophages from the plasmid-carrying bacteria
Subsequently, the KAP NTPase in one of the bacterial line-ages acquired the pair of transmembrane helices inserted into the P-loop domain, which made it an integral membrane pro-tein The apparent preponderance of horizontal gene transfer
in the evolution of the KAP family and the phylogenetic affin-ities of the animal KAP NTPases suggest that the gene for a membrane-spanning KAP NTPase was laterally transferred
to eukaryotes before the divergence of the major animal line-ages, probably from a bacterial plasmid or chromosome As
no eukaryotes other than animals are currently known to have a KAP NTPase, it seems likely that this gene transfer occurred relatively late in evolution - that is, after the separa-tion of the lineage leading to the animals from other crown-group eukaryotes However, given the sparse sampling of large eukaryotic genomes from different crown-group line-ages, the possibility remains that the transfer occurred ear-lier, but KAP genes have been lost in the currently sampled taxa
Evidence of independent insertion of transmembrane helices in other P-loop NTPase domains
In search of other possible instances of insertion of trans-membrane segments into P-loop NTPase domains we ana-lyzed all uncharacterized NTPase domains detected in our searches using the TMHMM program for transmembrane helix prediction As a result, we identified another small fam-ily of predicted NTPases containing transmembrane helices inserted into the P-loop domain This family is present in
sev-eral bacteria and includes the yobI gene of Bacillus subtilis and its orthologs from Clostridium perfringens, Bacteroides
thetaiotaomicron and Streptococcus mutans (Figure 4) All
these proteins contain a pair of predicted transmembrane helices inserted after the second conserved strand-helix unit
of the NTPase core The location of this insert thus differs from that seen in the ARMS subfamily of the KAP family, where the transmembrane helices are inserted immediately after the Walker A associated strand-helix unit (Figures 1, 4)
The P-loop domain of these proteins shows the hallmarks of the ASCE division but no specific affinity with the KAP family, suggesting an independent origin of the inserts In addition, these proteins contain a large conserved carboxy-terminal extension that is predicted to adopt an α-superhelical struc-ture The presence of these predicted NTPases in a taxonom-ically disjointed set of bacteria suggest a horizontal mode of dissemination similar to that discussed above for the KAP family
Conclusions
We describe here a previously unnoticed family of P-loop NTPases that displays unusual structural features and phyletic patterns The P-loop NTPase domain of this family, designated the KAP family, belongs to the ASCE division of
Trang 8P-loop NTPases and might be distantly related to the AAA+
and AP/NACHT NTPases [10,11,13] All eukaryotic and
sev-eral bacterial members of the KAP family contain two or four
transmembrane segments inserted into the P-loop NTPase
domain and, accordingly, are predicted to be integral
mem-brane proteins, with the P-loop domain attached to the
intra-cellular side of the membrane In addition, we identified
another small family of predicted bacterial NTPases, which
do not seem to be specifically related to the KAP family, but
also contain two transmembrane helices inserted into the
P-loop domain Insertion of transmembrane helices into
globu-lar domains is generally rare and, to our knowledge, has not
been described in P-loop NTPases so far It is well known,
however, that the P-loop domain tolerates extremely long
inserts of hydrophilic domains, such as the coiled-coil
domains in the SMC family ATPases involved in chromatin
dynamics and repair [32,33] Furthermore, many P-loop
NTPases are involved in membrane transport and secretion
In particular, these are the principal functions of the
ABC-class ATPases, and some of these, such as the CFTR protein in
animals, contain multiple transmembrane helices, which,
however, are located outside the P-loop domain [34] The
dis-covery of two families of predicted P-loop NTPases with
transmembrane helices inserted into the P-loop domain itself
unifies these two structural themes and further expands our
notion of the enormous structural and functional plasticity of
this widespread domain
Among eukaryotes, the KAP family is so far represented only
in animals and is typified by the neuronal membrane protein
Kidins220/ARMS and its paralog, which seems to have a
catalytically inactive NTPase domain In prokaryotes, KAP NTPases are often encoded by plasmids and might function in exclusion of bacteriophages from the plasmid-bearing bacte-rial cells We predict that both eukaryotic and bactebacte-rial KAP NTPases regulate NTP-dependent assembly or disassembly
of membrane-associated protein complexes Phyletic pattern and phylogenetic analysis suggest that lateral transfer from bacteria to the animal lineage (or an earlier ancestral form) before the diversification of the latter gave rise to the ancestor
of the eukaryotic KAP NTPases However, given the evidence
of rampant gene loss in diverse eukaryotes [35,36], it is con-ceivable that the KAP NTPases were acquired early in eukary-otic evolution and subsequently lost in several non-animal lineages Regardless of the exact origin scenario, these NTPases provide a remarkable example of recruitment of a protein originally acquired from bacteria for animal-specific functions, such as receptor tyrosine kinase-mediated signal-ing in neural growth and development
Materials and methods
The non-redundant (NR) database of protein sequences (National Center for Biotechnology Information, NIH, Bethesda) was searched using BLASTP [37] Iterative data-base searches were conducted using PSI-BLAST with either a single sequence or an alignment used as the query, with the PSSM inclusion expectation (E) value threshold of 0.01 (unless specified otherwise); the searches were iterated until convergence [37] For all searches with compositionally biased proteins, the statistical correction for this bias was used [38,39] Multiple alignments were constructed using the
Multiple alignment of the YobI family NTPases
Figure 4
Multiple alignment of the YobI family NTPases The coloring scheme and labeling conventions are as in Figure 1 Species abbreviations are as follows: Bs:
Bacillus subtilis, Bat: Bacteroides thetaiotaomicron, Cpe: Clostridium perfringens, Smu: Streptococcus mutans.
Nter helix Str-1 Helix- 1 Str-2
Secondary structure HHHHHHHHH EEEEEE HHHHHHHHHHHH EEEEEEEE .HHHHHHHHHHHHHHHHHHHHHHH
BT4745_Bat_29350153 18 V SQ FQ L TLLK E SAYESV R R L E VL N IAL T PY G K SS H LMYLK-D EK W YLPI S LA T LD KH Q T KD 38 RI E IL Q QLIY R I DT L S RF I TH I
yobI_Bs_16078957 26 E ES ED L SNDV D GKY S K S L K VK N IAL T PY G K SS IL N FQKQY-S RE Y FLNI S LA T F- DT D EN KL E IL Q QMIY R D RT I S RF I KH I
CPE0369_Cpe_18309351 70 Y KK EY L KDNL E NSY I K K I L RK N IAI S IY G K SS E FKQQY KE Y YLDI S LA T FI EE N L EE EL E IL N QIFY K Y DK M S RF I KN I
SMU.1577c_Smu_24379961 1 M TQ IF L INDA D QAH I D N I K VL N VAV S NY G K SS E YKEKFNN KK K FLHV S LA H F- ED G T ER 15 IL E IV N QLLH Q A DK I T IF K QH P
Consensus/100% .ppbbcpLoP -.p a cslpbulcsbc.bNlAloG.YGSGKSShlpohbbbb ccbpaL.lSLAph.sscp.p.cp blE.pIlpQhhap.p.cphP.obF+pbpp.p
Transmembrane helix-1 Transmembrane helix-2 Helix-2
Secondary structure HHHHHHHHHHHHHHHHHHHHH HHHHHHH HHHHHHHHHHHHHHHHHHHHHHHHHHH HHHHEEEEEEE HHHHHHHHHHHHHHHHHH
BT4745_Bat_29350153 PK H ISKLACGFIGTIL A FAILFEPSWMR -IDSFYRVFS Q GFVFNLIGDI V ALLYL-LFVLYTIAQY -VIRIYG S L KL N FKD G E I - KDEN IF N HL D ILYFF Q D
yobI_Bs_16078957 TK S IIINLIFFFAFII V GIYLFKPDALKGIYAETLVSRSLGT E D -QQQIRLTILL A LFFIV-YPLLAYKRIY HFVRA N L KV T IAN T E KNTG EENS IF D YL D ILYFF E K
CPE0369_Cpe_18309351 FL H IFKVTLIFISLIL S LSLLIKPELIEKFTSNVSKLKELFS T IPILKYNVNLSLII V ICLCV-ITILYTTMIL -IKFILS K I KI Q TKN G Q LAK- REES TF N YL D IMYFF E K
SMU.1577c_Smu_24379961 KR Q ILGWFILLTILIL S MLALW - T FPNLSWDSWIKQVL V ILLIISISVLIYQLMKLQFYRKLFK S F GA S VS- G E IF-G KSDA YF D YL D VLYLF D Q
Consensus/100% bpI h.h Ils hLh p b l lshhhhl.h.lLh bb blb.p.php.hp splpb cppsp.Fs+aLD-lhYhFps.p
Str-3 Helix-3 Str-4 Helix-4 Str-5 Helix-5
EEEEEE HHHHHHHHHHHHH EEEEEEEEEE HHHHHH.EEEEEE HHHHHHHHHH HHHHHHHHHHHHHHHHHHHHHHHHH BT4745_Bat_29350153 Y D VVVI ED L DR F PDIFL K RE L FLL N SAVV - GRK IKFIYAV KD D MF K DSSR T FF D YIT T VI P VI S K KL ELE K H EEI K - D DL I FFI DD M LL K IA N YH yobI_Bs_16078957 Y N VVIF ED L DR F IGIFE R RE L ELI N SEQI - DRR VVFIYAI KD D IF 7 L TRDR T FF D FII P VI P II S G IL KIK H Y DLI N - H FL V IYI DD M VL K IF N FV CPE0369_Cpe_18309351 Y D IVFF ED L DR F LEIFT K RE L TLI N AESI - SRK VTFVYAI KD E IF 19 M NKNR T FF D FII P VI P IV E Y IL KIE Q K YGV Q SIISK E LL L MFI DD M LL T IY N FL SMU.1577c_Smu_24379961 S D IIVF ED I DR F NLIFS K KE I TLV N KRKARGE DNK LLFMYLV KD E MF I SKER T FF D FII P VI P AI S R KF ILA D C EDF E - S FL I IYI DD L LV T IC N YV Consensus/100% sllhhEDlDRFps IF.+L+ElN.LlNp h sp+l.FhYhlKD-hF bsppRTKFFDaI.sVIPhlsspNS.-bhpcbl.p.s.p hps p.LpclshaIDDhRllpNIhNEa.
Secondary structure HHHHH EEEEEEEE HHHHHH EEEEEEEE HHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHH
BT4745_Bat_29350153 QYH KR L 2 N GT E H SK LLAMIVY K YY P F AL H NR R KVY Q CVCH ETKQ ELT K FAL Q IL N K EE KR T KER N RHL K AG E LRMIYV 485
yobI_Bs_16078957 IYQ QK L S AI D P NK MLAMIIY K IY P F KL Q YN K LVY E IF QKKQ LII E EQI K LI N I QQ RK N IEV E SLK S IA E LNFIYL 463
CPE0369_Cpe_18309351 IYY KK L 4 K NK T S DN LLAIIVY K LY P F KL Q NR E MVY N VF SEKN DIA D RAV H KL N I KE TN H LEK E ILE N EE E LYLIYN 531
SMU.1577c_Smu_24379961 LYK NN L 6 N KL K N EK LFAMIVY K VF P F EL Q VG S FIH R FF QEKD KLR E EQL H DI N I SE QK S AGN E ALN N EL E LYSSIL 442
Consensus/100% bY.ppL p pLs.pphhAhIlYKNhaP.DFo.Lp pGblaphh ppKp.l.cb.lp.lN.pbpph.ppb.p p p EL h.
Walker A
Walker B
Trang 9T_Coffee or PCMA programs, followed by manual correction
based on the PSI-BLAST results [40,41] All large-scale
sequence analysis procedures were carried out using the
SEALS package [42] Transmembrane regions were predicted
in individual proteins using the TMPRED [43], TMHMM2.0
[44] and TOPRED1.0 [45] programs with default parameters
For TOPRED1.0, the organism parameter was set to
'prokary-ote' or 'eukary'prokary-ote' depending on the source of the protein
Protein-structure manipulations were performed using the
Swiss-PDB viewer program [46] and the ribbon diagrams
were constructed using the MOLSCRIPT program [47]
Pro-tein secondary structure was predicted using a multiple
align-ment as the input for the PHD program [48] Similarity-based
clustering of proteins was carried out using the BLASTCLUST
program [49]
Phylogenetic analysis was carried out using the
maximum-likelihood, neighbor-joining, and minimum evolution (least
squares) methods Maximum-likelihood distance matrices
were constructed with the TreePuzzle 5 program using 1,000
replicates generated from the input alignment and used as the
input for construction of neighbor-joining trees with the
Weighbor program [50,51] Weighbor uses a weighted
neigh-bor-joining tree construction procedure that has been shown
to correct effectively for long-branch effects [51] The minimal
evolution trees were constructed using the FITCH program of
the Phylip package, [52] followed by local rearrangement
using the Protml program of the Molphy package [53] to
pro-duce the maximum likelihood (ML) tree The statistical
sig-nificance of the internal nodes of the ML tree was assessed
using the relative estimate of logarithmic likelihood bootstrap
(Protml RELL-BP), with 10,000 replicates [53] A full ML tree
was constructed using the Proml program of the Phylip
pack-age [52] This tree was used as the input tree to generate
fur-ther full ML trees using the PhyML program with 100
bootstrap replicates generated from the input alignment [54]
The consensus of these trees was derived using the Consense
program of the Phylip package to obtain the bootstrapped ML
tree [52] A gamma distribution with one invariant and eight
variable sites with different rates was used in the ML analysis
Gene neighborhoods were determined by searching the NCBI
PTT tables with a custom-written script These tables can be
accessed from the genomes division of the Entrez retrieval
system
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