Structural analysis of human FANCL, the E3 ligase in the Fanconi anemia pathway.

Abstract

The Fanconi anemia (FA) pathway is essential for the repair of DNA interstrand cross-links. At the heart of this pathway is the monoubiquitination of the FANCI-FANCD2 (ID) complex by the multiprotein "core complex" containing the E3 ubiquitin ligase FANCL. Vertebrate organisms have the eight-protein core complex, whereas invertebrates apparently do not. We report here the structure of the central domain of human FANCL in comparison with the recently solved Drosophila melanogaster FANCL. Our data represent the first structural detail into the catalytic core of the human system and reveal that the central fold of FANCL is conserved between species. However, there are macromolecular differences between the FANCL proteins that may account for the apparent distinctions in core complex requirements between the vertebrate and invertebrate FA pathways. In addition, we characterize the binding of human FANCL with its partners, Ube2t, FANCD2, and FANCI. Mutational analysis reveals which residues are required for substrate binding, and we also show the domain required for E2 binding.

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Structural Analysis of Human FANCL, the E3 Ligase in the
Fanconi Anemia Pathway*
Receivedforpublication,April4,2011,andinrevisedform,June23,2011 Published,JBCPapersinPress,July20,2011,DOI10.1074/jbc.M111.244632
Charlotte Hodson, Ambrose R. Cole1, Laurence P. C. Lewis2, Jennifer A. Miles, Andrew Purkiss, and Helen Walden3
FromtheProteinStructureandFunctionLaboratory,Lincoln’sInnFieldsLaboratoriesoftheLondonResearchInstitute,
CancerResearchUK,44Lincoln’sInnFields,London WC2A 3LY, UnitedKingdom
TheFanconianemia(FA)pathwayisessentialfortherepairof
DNA interstrand cross-links. At the heart of this pathway is the
monoubiquitination of the FANCI-FANCD2 (ID) complex by
the multiprotein “core complex” containing the E3 ubiquitin
ligaseFANCL.Vertebrateorganismshavetheeight-proteincore
complex,whereasinvertebratesapparentlydonot.Wereporthere
thestructureofthecentraldomainofhumanFANCLincompari-
son with the recently solved Drosophila melanogaster FANCL.
Our data represent the first structural detail into the catalytic
core of the human system and reveal that the central fold of
FANCL is conserved between species. However, there are mac-
romolecular differences between the FANCL proteins that may
account for the apparent distinctions in core complex require-
ments between the vertebrate and invertebrate FA pathways. In
addition, we characterize the binding of human FANCL with its
partners, Ube2t, FANCD2, and FANCI. Mutational analysis
reveals which residues are required for substrate binding, and
we also show the domain required for E2 binding.
Fanconi anemia (FA)4is an X-linked or autosomal recessive
disorder with a range of clinical presentations including bone
marrow failure and high incidence of cancer (1). Mutations in
one of at least 14 genes associated with FA lead to dysfunction
in DNA interstrand cross-link repair, rendering patients sus-
ceptibletoDNA-damagingagents(2).OftheFAgenes,eightof
the gene products form the FA core complex (FANCA, -B, -C,
-E, -F, -G, -L, and -M) (3–6). The role of the complex, in con-
junction with the E2 ubiquitin-conjugating (UBC) enzyme
Ube2T (7), is to monoubiquitinate each of the components of
the FANCI-FANCD2 (ID) complex (8–11). Mutation in any of
the genes coding for the core complex results in the loss of this
monoubiquitination event. The FA pathway is at least partially
present in all eukaryal organisms, with yeast having only a
FANCMhomologue(12)butallhigherorganismspossessingat
least FANCD2. However, in the case of the core complex,
vertebrateshavethefullcomplement,conservedfromzebrafishto
humans(13),whereastheinvertebratesvaryintheirFAcomposi-
tion; Caenorhabditis elegans have no obvious core complex (14)
and the slime mold Dictyostelium discoideum has a FANCE
homologue (15), whereas Drosophila melanogaster has FANCM
andFANCLhomologues(16).
FANCL is the E3 ubiquitin ligase of the core complex (17). It
wasoriginallypredictedfromprimarysequencetobeaWD40-
propellerfold,withaRINGdomainattheCterminus.Arecent
study also suggested the presence in human FANCL of an
N-terminal RWD domain (18). The RWD classification is a
submember of the UBC superfamily (19, 20). The UBC super-
family has a core four-stranded ?-meander, flanked by an
N-terminal helix and one or two C-terminal helices. In the cat-
alytic E2s, this meander has a C-terminal ?-flap containing the
catalytic cysteine (Fig. 1A). Most E2 folds contain a YPXXXP
motifbetweenstrands3and4thatformsatripleturnmotifalso
predicted to be characteristic of the RWD-like fold (18, 20). In
the RWD fold, the ?-flap assumes a helical conformation (19,
20) (Fig. 1A).
We recently reported the structure of full-length FANCL
fromD.melanogaster(21).DmFANCLcomprisesthreedomains,
an N-terminal E2-like fold (ELF), a double RWD (DRWD)
domain, and a C-terminal RING domain. The DmFANCL
DRWD domain harbors no YPXXXP motifs, yet each lobe con-
tains a helix in place of the ?-flap, giving rise to the DRWD
terminology.
Conservation in the core between the Drosophila and the
vertebrateFANCLssuggeststhattheywouldbebroadlysimilar
in structure. However, the sequence identity between human
and DmFANCL is low, at 21% (21). Of the three domains, the
DRWD domain, particularly the N-terminal half, has the least
sequenceidentity,at9%.Giventhattherequirementsforacore
complex are apparently different between species and that in
humans, the loss of any member of the complex results in fail-
uretoubiquitinatetheFANCI-FANCD2complex,wesetoutto
solve the structure of the human FANCL protein. We report
here the structure of the central domain of human FANCL,
representing ?50% of the protein. We show that it is globally
similar to the DmDRWD domain but that there are macromo-
lecular differences between the proteins that may account for
the biological differences in the systems. In addition, we dem-
onstrate that the central human FANCL domain is dispensable
* This work was supported by a grant from Cancer Research UK.
Author’s Choice—Final version full access.
Theatomiccoordinatesandstructurefactors(code3ZQS)havebeendepositedin
the Protein Data Bank, Research Collaboratory for Structural Bioinformatics,
RutgersUniversity,NewBrunswick,NJ(http://www.rcsb.org/).
1Present address: Dept. of Biological Sciences, Institute of Structural and
Molecular Biology, Crystallography, Birkbeck College, Malet St., London
WC1E 7HX, United Kingdom.
2Present address: Sensory Neurogenetics Research Group, Max Planck Insti-
tute of Neurobiology, Am Klopferspitz 18, D-82152 Martinsried, Germany.
3To whom correspondence should be addressed. Tel.: 44-20-72693106; Fax:
44-20-72693258; E-mail: Helen.Walden@cancer.org.uk.
4The abbreviations used are: FA, Fanconi anemia; DmFANCL, D. melano-
gaster FANCL; Ub, ubiquitin; UBC, ubiquitin-conjugating; URD, UBC-RWD
domain; DRWD, double RWD; DmDRWD, D. melanogaster DRWD; ELF,
E2-likefold;ITC,isothermaltitrationcalorimetry;RWD,foundinRING,WD-
containing proteins, DEXD helicases.
THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 286, NO. 37, pp. 32628–32637, September 16, 2011
© 2011 by The American Society for Biochemistry and Molecular Biology, Inc.
Author’sChoice
Printed in the U.S.A.
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for interaction with Ube2T but is essential to bind substrates
FANCD2 and FANCI.
EXPERIMENTAL PROCEDURES
Protein Expression and Purification—Previous efforts to
structurally characterize human FANCL had proved unsuc-
cessful, but the understanding of the domain architecture of
DmFANCL allowed us to design new constructs. We synthe-
sized a codon-optimized version of the human FANCL coding
sequence, using GeneArt. We then subcloned the central
domain (residues 109–294) and the RING domain (residues
289–375) into a vector containing an N-terminal His-Smt3 tag
using restriction-free cloning (22). Human Ube2T cDNA was
purchasedasanI.M.A.G.E.clone(Geneservice)andclonedinto
the same vector as described above. Proteins were expressed in
Escherichia coli BL21 cells (Invitrogen) in lysogeny broth
mediumsupplementedwithantibioticsandwith0.5mMZnCl2
in the case of the RING domain. Cells were cultured at 37 °C to
an A600of 0.6 nm, induced with 0.25 mM isopropyl-1-thio-?-D-
galactopyranoside (0.5 mM isopropyl-1-thio-?-D-galactopyra-
noside in the case of Ube2T), and cultured overnight at 16 °C.
Cells were harvested by centrifugation and lysed by sonication
on ice (10-s bursts followed by 30 s on ice, repeated four times)
in100mMTris,pH8.0,500mMNaCl,20mMimidazole,250?M
tris(carboxyethyl)phosphine (10 ?M ZnCl2added when purify-
ingtheRINGdomain).Sonicatedsamplesweresubjectedhigh-
speed centrifugation at 4 °C to remove cell debris. Clarified
supernatantswereappliedtoanNi2?-nitrilotriaceticacidaffin-
ity resin (Invitrogen) equilibrated with sonication buffer.
Fusion proteins were cleaved overnight at 4 °C with Smt3 pro-
tease, Ulp1, at a w/w of 1:30 Ulp1:His-Smt3-protein. Flow-
throughwascollected,concentrated,andappliedtoaSuperdex
200orSuperdex75columnforsize-exclusionchromatography.
Purified proteins were concentrated to 20 mg/ml (central
domain),8mg/ml(RING),and15mg/ml(Ube2T),flash-frozen
in liquid nitrogen, and stored at ?80 °C. Human central
domain mutants were generated using the Stratagene site-di-
rected mutagenesis method with Pfu Turbo DNA polymerase
(Stratagene) and verified by sequencing. The central domain
mutants were prepped as the wild type (WT). Human His-
FANCD2 was a kind gift from M. R. Hodskinson and K. J. Patel
and prepared as described (23). Xenopus laevis FLAG-FANCI
was a kind gift from P. Knipscheer and J. C. Walter and pre-
pared as described (24).
CrystallizationandStructureDetermination—HumanFANCL
central domain crystals were grown by hanging drop vapor diffu-
sionat4°C,inspacegroupC2,withcelldimensionsa?147.6Å,
b?102.5Å,c?65.8Å,??90,??94.09,??90.Solvent-content
estimates suggested two monomers per asymmetric unit, with a
solvent content of 79%. Crystallization conditions were: 0.2 M
L-proline, 0.1 M trisodium citrate, pH 5.5, 2% PEG 3350. Crystals
wereharvestedandcryocooledafterprotectionwith30%glycerol.
Data were collected at the DIAMOND synchrotron light source
beamlineI03,at0.98Åwavelength.Bothalow-resolutionpassand
ahigh-resolutionpasswerecollected(3.5and1.75Å,respectively).
FIGURE1.StructuralevolutionoftheUBCfamilyfolds.A,theUBCfoldofacatalyticE2(Ube2T,PDBcode1YH1)showninblue,ofanon-catalyticE2(MMS2,
PDBcode1J74(39))showninpink,andtheRWDfold(RWDD1a,PDBcode2EBM).Agoldstarshowsthecatalyticcysteine,andahotpinkpatchshowstheRING
domain-binding region. The ?-flap indicative to a UBC fold is labeled, as is the YPXXXP motif. B, a structural alignment of UBC folds for both catalytic E2s
(Ube2D1 and Ube2T) and non-catalytic E2s (Ube2V1 and Ube2V2) and for the RWD folds (RWDD1a and RWDD2a). A scale of red-orange-white shows conser-
vation,withredbeing100%conservedandwhitebeing0%.Agoldcirclelabelsthecatalyticcysteine.Thesecondarystructureisshownaboveinblue,withthe
main difference between the UBC fold (a ?-strand) and the RWD fold (?2) shown in light green. The most conserved region is the YPXXXP motif.
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ThetwodatasetswereprocessedseparatelyusingMosflm(25)and
then merged using XDS and scaled using XSCALE (26, 27). The
last55imagesfromthehigh-resolutiondatasetwereomittedfrom
data processing due to radiation damage to the crystal, which
accounts for 75% completeness in the outer shell (2.05–2.0 Å,
overall completeness 79%). The data were phased using the
DmFANCLDRWDdomain,residues109–292,asasearchmodel
(Protein Data Bank (PDB) code 3K1L (21)), with the program
PHASER (28). The model was rebuilt manually through iterative
cycles of refinement using COOT (29)for building and PHENIX
(30) for refinement. Omit maps were generated across regions of
theproteintoassessmodelbias.Thestructurewasrefinedto2.0Å
resolution.Theasymmetricunitcontainstwomolecules,AandB.
Aiscompleteandinasingleconformationthroughoutandisused
for the analyses reported. Copy B, however, has a region of disor-
derwhereresiduesCys-174–Ser-182inloop6appearintheelec-
tron density maps in multiple conformations. To reflect this
movement/disorder, these atoms have been assigned varying
occupanciesinthePDBfileasjudgedmanuallyinthefinal2Fo?Fc
and Fo? Fcmaps. Along with the high resolution of the data and
thefactthatchainBhascrystalcontacts,thenon-crystallographic
symmetrywasnotusedforsomeoftheloopregions(atotalof80
residues left out of the non-crystallographic symmetry). The final
modelpossessesexcellentstereochemistrywithallresiduesinthe
favoredregionsoftheRamachandrandiagram(98.7%)onthebasis
of an analysis with MolProbity (31). Table 1 summarizes the data
collectionandrefinementstatistics.
StructuralAnalyses—Allstructuralanalyseswerecarriedout
onChainAofthemodel.FigureswereproducedusingPyMOL
(32), and structure-based alignments were produced using
MegaAlign and manual adjustments. To produce the electro-
staticsurfaces,programspdb2pqr(33)andapbs(34)wereused
in conjunction with PyMOL.
Thioester Charge Assay—2 ?M E2, His-UbcH7 (Boston
Biochem),orhumancentraldomainwaschargedwithanexcess
(50 ?M) of HA-Ub (Caltag Medsystems) using 150 nM E1 (Bos-
ton Biochem). A total reaction volume of 10 ?l was made up
with assay buffer containing 50 mM Tris, pH 8, 250 mM NaCl, 2
mM dithiothreitol (DTT), 5 mM MgCl, and 5 mM ATP. Reac-
tions were left for 30 min at room temperature. Reactions were
then terminated by the addition of 40 ?l of 2? non-reducing
SDS buffer. 20 ?l of each reaction then had 1 ?l of 1 M ?-mer-
captoethanol added to reduce the thioester bond. 4 ?l of each
sample was loaded on SDS-PAGE gels and visualized using
Westernblots.Antibodiesusedwereanti-His(GEHealthcare),
anti-HA, and anti-FANCL raised against recombinant human
FANCL central domain (Pettingill Technology Ltd.).
In Vitro Pulldown Assays—In a total reaction volume of 1 ml
containing assay buffer: 0.5 M NaCl, 0.1 M Tris, pH 8, and 250
?M tris(carboxyethyl)phosphine, human His-FANCD2 (225
nM) or X. laevis FLAG-FANCI (225 nM) was added with an
excess of human FANCL central domain or mutants at 454 nM.
Reactions were left to bind for 1 h at room temperature. 100 ?l
ofNi2?-nitrilotriaceticacid-agarose(Qiagen)or100?lofanti-
FLAG affinity gel (Sigma) was equilibrated in assay buffer,
addedtothe1-mlreaction,andleftonarollerat4 °Cforatleast
1 h. Samples were washed with 10 ml of assay buffer. Agarose/
affinitygelwasthenresuspendedin100?lofassaybuffer,50?l
of 2? SDS buffer was added, and the sample was boiled. 9 ?l of
the samples was loaded onto SDS page gel, subjected to West-
ernblotting,andprobedwiththeappropriateantibodies.Anti-
FLAG antibody was purchased from Abcam.
Analytical Gel Filtration—Initial establishment of interac-
tions between the human central domain (109–294), and
human RING domain (289–375) with Ube2T was carried out
byanalyticalgelfiltration.Interactionbuffercontained100mM
Tris, pH 8, 250 ?M tris(carboxyethyl)phosphine, 10 ?M ZnCl2
with100mMNaCl.AnincreasedamountofNaClto500mMwas
used for central domain interactions as the protein is unstable in
loweramountsofNaCl.Sampleswereincubatedonicefor1hwith
atotalvolumeof500?l.SampleswerethenloadedontoaSuper-
dex 75 column (equilibrated with the sample buffer), and 0.5-ml
fractionswerecollectedforanalysison12%SDS-PAGEgels.
Isothermal Titration Calorimetry (ITC)—Kinetic informa-
tion of Ube2T binding interactions with FANCL domains was
established using the iTC200 microcalorimeter (MicroCal,
Northampton, MA). Sample buffers were the same as interac-
tion buffers used for analytical gel filtration. Experiments were
carried out at 8 °C. The syringe contained Ube2T between 650
and 750 ?M that injected a total volume of 40 ?l by 2.5-?l
injections. The cell contained a total volume of 205 ?l of either
RING (70 ?M) or central domain (65 ?M). Cell concentrations
were adjusted for a 1:1 stoichiometric interaction, and Micro-
Cal Origin software version 7.0 was used to determine dissoci-
ation constants (Kd). All measurements were repeated at least
twice.
TABLE1
Data collection and refinement statistics
Highest resolution shell is given in parentheses.
Data collection
Wavelength (Å)
Space group
Cell dimensions (Å)
Cell dimensions (o)
Resolution range (Å)
No. observations
No. unique reflections
Completeness (%)
Multiplicity
I/?
Rmeas
Refinement
Number of reflections used
Number of protein atoms
Number of solvent atoms
Water
Na?ion
PEG
Proline
Mean B values (Å2)
Protein
Water
Na?ion
PEG
Proline
r.m.s.adeviations
Bond lengths (Å)
Bond angles (°)
Final Rwork
Final Rfree
Ramachandran regions
Favored
Outliers
ar.m.s., root mean square.
0.98
C121
a ? 147.6, b ? 102.5, c ? 65.8
? ? 90, ? ? 94.09 ? ? 90
45–2.0 (2.05–2.0)
153,360
103,454
79% (75%)
1.5 (1.5)
8.91 (2.03)
7.2% (59.1%)
98,143
3031
413
2
103
16
39.5
54.5
35.0
62.8
81.3
0.007
1.0
0.183
0.207
98.7%
0.0%
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RESULTS
The Structure of Human FANCL Central Domain—We have
determined the structure of the predicted human DRWD
domain (residues 109–294) to 2.0 Å resolution. Overall, the
human FANCL central domain has a fused bilobal shape with
dimensions: 70 ? 25 ? 20 Å, consisting of four ?-helices and
nine ?-strands (Fig. 2A). Each lobe contains a four-stranded
?-meanderfollowedbya?-elementintheN-terminallobeand
an ?-helix in the C-terminal lobe. The lobes are fused by a
kinked helix that runs from one lobe to the other. In addition,
there is an ?-helix N-terminal to the first ?-meander and a
C-terminal ?-helix (Fig. 2B).
Global Comparison with DmFANCL—A comparison of
known structures using the DALI server unsurprisingly reveals
DmFANCL (PDB code 3K1L) as the closest structural homo-
logue.However,aDALIcomparisonusingonlytheN-terminal
lobe(residues109–194)orC-terminallobe(residues195–294)
ofthehumancentraldomainlistsE2UBCenzymesasthemost
structurally similar for both, with significant RWD fold hits
only for the C-terminal lobe (Fig. 2C). This is due to the single
biggestdifferencebetweentheDmDRWDandthehumancen-
traldomainbeingthechangeofsecondarystructureelementin
the N-terminal lobe (Fig. 2D). In the DmDRWD, both lobes
containahelixafterthefour-stranded?-meander,indicativeof
an RWD fold, even in the absence of the triple turn YPXXXP
motif. The human central domain, however, harbors the
YPXXXPmotifintheN-terminallobeandarelatedHPXXXP
motif in the C-terminal lobe (Fig. 3A). However, the human
N-terminal lobe has a ?-element in the position normally
occupied by a helix in RWD folds. This puts the N-terminal
lobe of the human central domain in an almost intermediary
position between an E2 fold with a ?-flap in the catalytic
position and an ?-helix in the non-catalytic RWD-like folds
(Fig. 2C). Therefore the human central domain is not a
DRWD but in fact a UBC-RWD domain. For the remainder
of this study, the human central FANCL domain will be
referred to as the UBC-RWD domain (URD). Structure-
based sequence alignment with other FANCL proteins
reveals that this element swap is likely to be in all the verte-
brate FANCLs (Fig. 3A). Therefore we refer to this as a ver-
tebrate-specific feature, and it represents a key difference
between the vertebrate and invertebrate FANCLs.
Intriguingly, the human ?-element in the N-terminal lobe
contains a cysteine (Fig. 3A). Superposition of the human
N-terminal lobe with Ube2T, with a root mean square devi-
ation of 2 Å, reveals that the cysteine in the URD ?-element
lies structurally in the same position as the catalytic cysteine
of Ube2T (Fig. 2C). We wondered whether this cysteine was
capable of forming a thioester bond with ubiquitin as in the
caseofthecatalyticE2s.TotestwhetherthecysteineintheURD
is catalytic, we carried out a thioester charge assay. The thioester
assay revealed that the URD is unable to form a thioester linkage
between its cysteine and the activated C terminus of ubiquitin
(Fig.3B).
The interacting residues between the N- and C-terminal
lobes of the URD and DmDRWD are not conserved (Fig. 3, A
and C). However the residues that make up the hydrophobic
core between the two lobes are highly conserved (Fig. 3A). A
FIGURE2.StructureofhumanFANCLcentraldomain.A,surfacerepresentationofthehumancentraldomain,withhotpinkrepresentingtheN-terminallobe
and magenta representing the C-terminal lobe. B, secondary structural representation of the human central domain. C, structural comparison of the human
N-terminallobe,showninhotpinkoverlaidwiththeUBCfoldofUbe2Tshowninbluewithitscatalyticcysteinehighlightedbyagoldstar.Acomparisonofthe
human C-terminal lobe is shown in magenta overlaid with the RWD fold of GCN2, shown in cyan. D, a secondary structure comparison of the human central
domain shown in hot pink and magenta with the DmDRWD domain overlaid shown in yellow and green.
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structural alignment of the human and Drosophila domains
showsanoverallconservationof50%,withonly13%ofresidues
absolutely conserved (Fig. 3A). There are also differences in
conservation between the two lobes, with the C-terminal lobe
being more conserved than the N-terminal lobe (30 and 20%,
respectively).
Surface Composition Reveals Extensive Hydrophobic Expo-
sure—Itislikelythatdifferencesinacorecomplexrequirement
for the vertebrate system arise from the need to stabilize
FANCL. We reasoned that there would likely be differences in
the surface conservation and surface chemical composition
between DmFANCL and human FANCL (HsFANCL). To
FIGURE 3. Structural comparison of human and Drosophila central domains. A, a structural alignment of the central domains across species. A scale of
red-orange-white shows conservation, with red being 100% conserved and white being 0%. Secondary structure is shown above with hot pink for the human
centraldomainandgreenfortheDmDRWDdomain.TheYPXXXPandHPXXXPmotifsarehighlightedinblue.Thecysteinefoundinthediffering?-strandregion
ofthehumancentraldomainishighlightedinlilac.Residuesinvolvedinthelobeinteractionshaveagreencircleabovethem,andthosethatarefoundinthe
hydrophobicgrooveofthelobeinteractingsitehaveredcirclesabovethem.Residuesmutatedtoalaninetodeterminetheregionforsubstratebindinghave
a square above them, with blue squares representing patch 2 and pink squares representing patch 4. mm, Mus musculus; Bt, Bos taurus; gg, Gallus gallus; xl,
X. laevis; dr, Danio rerio. B, thioester charge assay to determine whether the human ?-element cysteine thioester links ubiquitin in a manner analogous to
catalyticE2s.Lanes1and2foreachpanelcorrespondtothesampleinnon-reducingbufferandreducingbuffertodetermineathioesterlinkage,respectively.
ItisclearthatthattheURDcannotformathioesterlinktoubiquitin.C,aclose-upviewoftheinteractionsbetweenthehumanN-terminal(pink)andC-terminal
(mauve) central domain lobes. Hydrogen bonds are depicted as black dashed lines.
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assess this, we generated surface maps of the URD and com-
pared it with the DmDRWD domain (Fig. 4).
The surface conservation between DmDRWD and the URD
is?32%.ThesurfaceismoreconservedontheC-terminallobe
at 20% as compared with the N-terminal lobe at 12% (Figs. 3A
and 4A).
Comparison of solvent-exposed hydrophobic residues
betweentheURDandtheDmDRWDrevealsadditionalsurface
hydrophobic residues on the URD. A small hydrophobic patch
resides on the N-terminal lobe of the URD, between the last
?-strand of the four-stranded ?-meander (?4) and the ?-ele-
ment (residues Leu-149, Tyr-165, Phe-166, and Val-167, Fig.
4B).AdditionallyapatchontheURDliesbetween?-helix1and
the N-terminal region of the large kinked helix, corresponding
to residues Leu-121, Ala-140, Ile-184, Tyr-187, and Leu-191
(Fig. 4B). Interestingly, a reasonably conserved region resides
on the N-terminal region corresponding to residues Tyr-112,
Tyr-113, and Leu-116 in DmDRWD, and more extensively in
humans,Ile-115,Trp-123,Leu-126,Val-127,Tyr-128,Ala-129,
and Phe-133 (Fig. 4B). On the C-terminal domain of the
DmDRWD is a hydrophobic groove between the last ?-strand
of the four-stranded ?-meander and ?-helix 3 that follows
(residues Ile-243, Met-246, Leu-248, Leu-264, Leu-268, and
Trp-271). In the human URD, there is also a hydrophobic
groove formed by residues Phe-253, Val-260, and Leu-268, but
it is extended by comparison with the DmFANCL to include
FIGURE4.SurfacecomparisonsofthehumanURDandtheDrosophilaDRWDdomains.A,asurfaceconservationcomparisonbetweenthehumanURDand
theDrosophilaDRWD.ThesurfaceshownhereisofthehumanURD.B,acomparisonofsurfacehydrophobicelementsshowningreen,forboththehumanand
the Drosophila domains. C, a comparison of surface electrostatics for both the human and Drosophila domains, with blue for basic charge and red for acidic.
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Leu-248,Phe-252,Leu-254,Ile-265,andIle-271(Fig.4B).Inthe
URD, there is an additional small hydrophobic patch that con-
sists of Trp-212, Val-213, and Leu-214. In Drosophila DRWD,
the corresponding residues are His-208, Val-209, and Leu-210.
This patch in the Drosophila DRWD is partly occluded by the
DRWD-RING loop in the full-length FANCL structure.
A comparison of the electrostatic potential of the surfaces
of the URD and the DmDRWD reveals distinct differences
betweenthespecies(Fig.4C).Interestingly,thereisalargepos-
itive patch on the DmDRWD, which is absent in the URD.
Overall, the URD has a greater negative charge then the
DmDRWD,whichwrapsaroundtheURDlikearing.However,
on the backside of the central FANCL domain in both human
and Drosophila, there is a similar overall negative charge.
TheHumanRINGDomainBindsUbe2T—Toassesswhether
the URD plays any role in Ube2T binding, we carried out size-
exclusion chromatography and ITC. Initial results from the
size-exclusionchromatographyshowednochangeinmigration
that would correspond to a complex formation between the
URDandUbe2T(Fig.5A).Tofurtherassesstheinteraction,we
employedITC,whichagainshowednointeractionbetweenthe
URD and Ube2T (Fig. 5B). We then tested the purified RING
domain to see whether it was sufficient for Ube2T binding. We
initially observed a 1:1 stoichiometric interaction between the
RING and Ube2T as judged by the migration of the complex
during size-exclusion chromatography (Fig. 5A). To ascertain
the affinity of RING for Ube2T, we carried out ITC. After titra-
tion of Ube2T into RING, the data were fitted with a stoichi-
ometry of 1:1 as seen in the size-exclusion chromatography,
resulting in an equilibrium binding dissociation constant (Kd)
of 454 nM (Fig. 5B). Based on the structure of UbcH7 bound to
theRINGE3c-cbl(35),wemutatedPhe-63inUbe2T,predicted
to be interacting with the RING, to alanine. No complex shift
wasobservedbetweenUbe2T-F63AandRINGusinganalytical
gel filtration (Fig. 5A). Further analysis using ITC revealed that
mutant Ube2T-F63A was able to bind the RING domain, but
withan11-folddecreaseinbindingaffinity(Kd?5?M,Fig.5B).
The Human Central Domain Binds FANCI and FANCD2—In
the Drosophila system, pulldown studies indicated that the ELF
domainwasnotrequiredforsubstratebinding(21).Giventherole
FIGURE5.RINGdomainisnecessaryandsufficientforE2binding.A,size-exclusionchromatogramsshowingmolecularmassshiftsifcomplexformationhas
occurred. Green lines on the chromatogram represent the proteins allowed to form complexes, and red lines show Ube2T or Ube2T-F63A. From the size-
exclusion chromatography experiments, it is clear that complex formation only occurs between RING and Ube2T. The SDS-PAGE gel also show that both the
RINGandtheUbe2Tproteinsarepresentintheshiftedpeak.mAU,milliabsorbanceunits;MW,molecularmassmarkers.B,ITCexperimentsoftheinteractions
between URD-Ube2T, RING-Ube2T, and RING-Ube2T-F63A.
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oftheRINGdomaininE2binding,wehypothesizedthattheURD
domain would be sufficient for substrate binding. To test this, we
performedinvitropulldownexperimentswithFANCI,FANCD2,
and the URD (Fig. 6). As expected, the URD was pulled down by
bothsubstrates.Ourstructuralanalysisrevealsseveralhydropho-
bicpatchesexposedonthesurfaceoftheURD(Fig.4B).Toestab-
lish which patch(es) may be required for substrate binding, we
generated the following mutants: L149A/F166A; L248A/F252A/
L254A/I265A; V127A/Y128A; and W212A/L214A (Fig. 6A). All
mutantswerepurifiedtohomogeneityaswildtypeURDandsub-
jected to pulldown arrays with FANCI and FANCD2. Both sub-
strates showed reduced binding with mutants L248A/F252A/
L254A/I265A and W212A/L214A as compared with wild type,
indicatingthattheexposedhydrophobicpatchesontheC-termi-
nallobearerequiredforsubstratebinding.Incontrast,mutations
in the patches on the N-terminal lobe behaved as wild type
(Fig. 6B).
DISCUSSION
The structure of the human central FANCL domain (URD)
adopts the same overall topology as the Drosophila DRWD
domain, confirming that the FANCL proteins belong to the
UBC and RING superfamilies and are not WD40 proteins as
predicted(17).Thehumancentraldomaindoesdifferfromthe
DmFANCL as it does not have a DRWD but contains a UBC
foldfusedtoanRWDfold(URD).Geneduplicationresultingin
fused domains is not a novel occurrence in protein topology.
However,thefusedconsecutiveUBCsuperfamilyfolds,seenin
both the URD and the DmDRWD, are unique and appear to be
specific to FANCL.
In the context of the full-length human FANCL, there may
be subtle differences in domain associations. Structure-based
sequence alignments of vertebrate FANCL proteins to Dro-
sophila FANCL reveal that the loops connecting ELF-URD (or
ELF-DRWD)andURD-RING(orDRWD-RING)aredivergent
between species (21). The ELF-URD loop is conserved in
length, but not composition, and in the Drosophila structure,
therearenointeractionsbetweentheELFandanyotherpartof
the protein. One possible explanation for the existence of a
?-element in place of the helix in human FANCL URD may be
toallowamoreintimateassociationbetweentheURDandELF
domains. The URD-RING loop in human FANCL is 7 residues
shorter than in DmFANCL. We show a high affinity of human
RINGfortheE2,Ube2T,whichmayreflectadifferentarrange-
ment of the URD and RING domains in the human protein.
Further structural analyses of the relationship between the
human URD and RING domains and other members of the
FANCL family will be required to tease out these differences.
Although we have shown that the URD domain is not
required for Ube2T binding, the URD domain appears to be
essentialforthefunctionofFANCL(36).Ithasbeenpreviously
shown in the Drosophila system that the ELF domain is not
required to bind substrates FANCD2 and FANCI (21). In this
analysis, we show that the human URD domain is sufficient for
binding substrates. Additionally, we also reveal the molecular
determinants of this binding (Fig. 6). In particular, the residues
Trp-212, Leu-214, Leu-248, and Leu-254 are conserved across
species (Fig. 3A). Previous mutational work based on the pre-
dicted structure of FANCL indicated that the then uncharac-
FIGURE 6. URD domain binds substrates FANCD2 and FANCI. A, structural representation of the surface hydrophobic patch mutants of the URD domain.
B,pulldownofsubstratesHis-FANCD2andFLAG-FANCIwithWTURDandthepatchmutantsoftheURD,withthemutationsindicatedinA.URDinputandURD
incubatedwithbeadsalonecontrolareshownusinganti-FANCLantibody.ThepulldownshowseitherHis-FANCD2withanti-HisorFLAG-FANCIwithanti-FLAG
and the URD species that have been pulled down with anti-FANCL antibody.
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terized URD domain was essential for core complex assembly
(36), with point mutants R226E, W201A, and W274A (human
numbering, numbered as 275 in Ref. 36) abolishing core com-
plexassembly.OurstructureofhumanFANCLURDprovidesa
molecular rationale for these observations and indicates that
loss of core complex assembly is in fact due to structural insta-
bility (Fig. 7). As predicted from the Drosophila FANCL struc-
ture (21), Arg-226 forms buried salt bridges with Asp-208 and
the main-chain oxygen of Ala-223 (Fig. 7). Mutation of this
residue to the oppositely charged glutamate therefore destabi-
lizes these salt bridges and consequently the surrounding fold.
Trp-201isrightinthemiddleoftheinterfaceconnectingtheN-
and C-lobes of the URD domain (Fig. 7). A W201A mutation
wouldthereforeimpactthefoldingofthelobesasthisresidueis
clearly seen in the structure to be stacking against and making
multiplecontactswithresiduesHis-148,Arg-146,andArg-221.
Therefore, although the hydrophobic nature of the residue is
maintained, the contacts in the core are not (Fig. 7). Finally,
Trp-274 is completely conserved between all the FANCL spe-
cies, including Drosophila (21). In our URD structure, Trp-274
forms a hydrogen bond between the side-chain nitrogen of the
tryptophanandthemain-chainoxygenofThr-246.Mutationof
this Trp-274 to alanine removes this hydrogen bond as well as
multiple van der Waals contacts with residues Met-247 and
Asn-283 (Fig. 7). We can conclude that these particular muta-
tions destabilize the URD domain, and we can speculate, based
on the extent of surface-exposed hydrophobic patches on the
URD domain, that this domain is very likely to be involved in
binding one or more of the core complex proteins. The pres-
ence of such patches certainly suggests a number of protein-
protein interaction sites. Interestingly, a recent study impli-
cates the URD domain in (PCNA) proliferating cell nuclear
antigenbinding,providingapotentiallinkbetweenthetransle-
sion synthesis and FA pathways (37). Coupled with our obser-
vationthattheURDbindsFANCIandFANCD2,itappearsthat
the URD domain of FANCL is responsible for several interac-
tions. Finally, there has been speculation that phosphorylation
of FANCI, shown to trigger the FA pathway in the chicken
system,mayenhancetheabilityofFANCLtobindFANCI(38).
For this to be the case, we would anticipate the existence of a
basic patch on FANCL to mediate interaction with a cluster of
negatively charged phosphate ions. From our surface electro-
static analysis of the URD domain, there is no such patch, yet
intriguingly, there is in the Drosophila protein. Further struc-
tural studies of FANCL in complex with substrates and other
binding partners will be required to determine the functions of
these surfaces.
Acknowledgments—We thank Michael Way for discussions and crit-
icisms on the manuscript and members of the Protein Structure and
Function laboratory for suggestions and support. We also thank K. J.
Patel for the plasmid for HIS-FANCD2 and P. Knipscheer and J. C.
Walter for the FLAG-FANCI plasmid. We thank the protein produc-
tion facility for generating the viruses for the insect cell protein
expression.
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