Cell, Vol. 100, 447±456, February 18, 2000, Copyright 2000 by Cell Press
Crystal Structure of the VHS and FYVE Tandem
Domains of Hrs, a Protein Involved in Membrane
Trafficking and Signal Transduction
ceptors are primarily trafficked to lysosomes whereas
others suchas thetransferrinreceptorarerecycled back
to the plasma membrane. Regulatory molecules that
mediate this trafficking decision might be expected to
interact with cargo proteins, membrane phospholipids,
and members of the trafficking machinery.
One potential candidate that satisfies these criteria is
a newlyidentified proteinnamed Hrs (hepatocytegrowth
factor regulated tyrosine kinase substrate). As its name
implies, Hrs is tyrosine phosphorylated in response to
a variety of growth factors including HGF, EGF, and
PDGF (Komada and Kitamura, 1995). Hrs is expressed
in the cytoplasm of all cells and is predominantly local-
ized to endosomes (Komada et al., 1997). This localiza-
tion is consistent with the finding that Vps27p, the yeast
homolog of Hrs, is also expressed on endosomes, and
null mutants are unable to traffic most proteins to the
vacuole (yeast equivalent of the lysosome), leading to
an exaggerated ªclass Eº compartment (Piper et al.,
1995). Similarly, mice lacking Hrs die early in embryonic
developmentand haveabnormally enlarged endosomes
(Komada and Soriano, 1999). Thus, Hrs has been pro-
posed to play a role in trafficking cargo from the endo-
some to the lysosome. Interestingly, Hrs has also been
implicated in exocytosis of synaptic vesicles. A splice
variant of rat Hrs named Hrs-2 is a calcium-dependent
ATPase, tightly binds the t-SNARE SNAP-25, and inhib-
its neurotransmitter release when injected into perme-
abilized PC12 cells (Bean et al., 1997). This suggests
that Hrs may regulate synaptic vesicle exocytosis by
directly interacting with the fusion machinery.
Hrs contains several conserved domains that are
present in proteins implicated in signal transduction
and/or membrane trafficking. The VHS (Vps27p, Hrs,
STAM) domain is present at the amino terminus of sev-
eral proteins believed to play a role in tyrosine kinase
receptor signaling. STAM (signal-transducing adaptor
molecule) is tyrosine phosphorylated by J ak3 and J ak2
tyrosine kinases in response to cytokines IL-2 and gran-
ulocyte-macrophage colony-stimulating factor (GM-CSF)
(Takeshita et al., 1996, 1997). Interestingly, Hrs is also
phosphorylated in response to these cytokines and
binds to STAM via a coiled-coil domain (Asao et al.,
1997).AnotherVHS-containing proteincalled EAST (epi-
dermal growth factor receptor-associated protein with
SH3 and TAM domains) is tyrosine phosphorylated by
the activated EGF receptor, colocalizes with clathrin,
and coimmunoprecipitates eps15, an essential compo-
nentofthe ligand-dependentendocytic machinery (Car-
bone etal., 1997;Lohietal., 1998). Althoughthe function
of the VHS domain is not yet known, its presence in
STAM, EAST, and Hrs strongly suggests a role for this
domain in tyrosine kinase receptor-mediated endocyto-
sis (Lohi and Lehto, 1998).
Anotherinteresting domain ofHrs is the FYVE (Fab1p,
YOTB, Vac1p, and EEA1) zinc finger domain present in
over 40 proteins. This domain has been shown by sev-
eral groups to specifically bind phosphatidylinositol
3-phosphate (PI(3)P) via a conserved (R/K)(R/K)HHCR
Yuxin Mao,*Alexei Nickitenko,²
Xiaoqun Duan,³Thomas E. Lloyd,§
Mark N. Wu,§Hugo Bellen,³ §?
and Florante A. Quiocho*² ³#
*Structural and Computational Biology
and Molecular Biophysics Graduate Program
²Department of Biochemistry and Molecular Biology
³Howard Hughes Medical Institute
§Department of Molecular and Cellular Biology
?Department of Molecular and Human Genetics
Baylor College of Medicine
Houston, Texas 77030
We have determined the 2 AÊX-ray structure of the
219-residue N-terminalVHS and FYVE tandem domain
unit of Drosophila Hrs. The unit assumes a pyramidal
structure in which the much larger VHS domain (resi-
dues 1±153) forms a rectangular base and the FYVE
domain occupies the apical end. The VHS domain is
comprised of an unusual ªsuperhelixº of eight ? heli-
ces, and the FYVE domain is mainly built of loops,
two double-stranded antiparallel sheets, and a helix
stabilized by two tetrahedrally coordinated zinc atoms.
The two-domain structure forms an exact 2-fold-
related homodimer through antiparallel association of
mainly FYVE domains. Dimerization creates two iden-
tical pockets designed for binding ligands with multi-
ple negative charges such as citrate or phosphatidyl-
Intracellularmembrane trafficking events are tightly reg-
ulated to ensure proper spatial and temporal delivery of
is the ligand-dependent endocytosis of growth factor
receptors. After ligand binding and receptor activation,
a series of phosphorylation events leads to the recruit-
ment of clathrin at the site of the activated receptor
complex. The plasma membrane containing the recep-
tor pinches off from the membrane to form a clathrin-
coated vesicle, the clathrin cage is actively disassem-
bled, and endocytic vesicles fuse with one another to
formearly endosomes. Next, endosomalcargo is sorted
either to recycling endosomes, where it is returned to
the plasma membrane, or to the lysosome, where it is
degraded. Inthis way, signaltransductionpathways can
be regulated by controlling the level of activated recep-
tor present on the surface of the cell. Little is known
about how the balance between surface recycling and
lysosomal degradationof endosomalcargo is achieved.
Some endosomal proteins such as tyrosine kinase re-
#To whom correspondence should be addressed (e-mail: faq@
Table 1. Crystallographic Analysis Statistics
A. Data Collection
Crystal Wavelength (AÊ)dmin(AÊ) Completeness (%)
MAD phasing dataSeMet?1(0.9793)
7.0 (29.1) Refinement data
Observed Diffraction Ratiosc
Figure of merit (FOM)0.51
Bonded Main Chain
Atom B Factor Rmsd (AÊ2)
Bonded Side Chain Atom
B Factor Rmsd (AÊ2)
Resolution Range (AÊ)Rvalued(%)Rfreee(%)
50-2.021.5 25.4 0.0161.6 0.530.45
aValues in parentheses are for the outer resolution shell.
bRsym? ?h?i|II(h) ? ?I(h)|/?h?iII(h).
cValues are ?(?|F|)2?1/2/?|F|2?1/2.
eRfreeis obtained for a test set of reflections, consisting of a randomly selected 10% of the data and not used during refinement.
et al., 1998; Patki et al., 1998). The rab5 effector EEA-1
(early endosomal antigen 1), one of the best character-
ized FYVE-containing proteins, is essential for the teth-
ering and fusion of early endosomes (Christoforidis et
al., 1999). The endosomal localization of EEA-1 is dis-
rupted by deletion of the FYVE domain or by treatment
with the PI3-kinase inhibitor wortmannin, suggesting
that EEA-1 binding to endosomes is mediated by the
FYVE-PI(3)P interaction (Stenmark et al., 1996; Simonsen
et al., 1998). In other FYVE domain±containing proteins
such as Hrs and Vac1p, on the other hand, proteins
deleted of the FYVE domain still localize to endosomes
(Komada et al., 1997; Tall et al., 1999). Furthermore,
Vac1p membrane localization is not disturbed in the
Vps34 (PI3-kinase) null mutant, suggesting that PI(3)P
is not essential for Vac1p binding to membranes (Tall
etal., 1999).Althoughwortmannindoes inhibitHrs mem-
the FYVE domain suggests that interactions with other
PI(3)P-binding proteins mediateits localization(Komada
and Soriano, 1999). Thus, the FYVE domain is required
for membrane localization in some but not all FYVE-
containing proteins, making it likely that this domain
plays other roles as well.
As a first step in understanding the function of Hrs at
the atomic level, we have determined the crystal struc-
ture of the N-terminal 219 amino acids of Drosophila
melanogaster Hrs containing the VHS and FYVE do-
mains. This report describes a prototypic structure for
the VHS domain that is conserved in over 20 proteins.
Moreover, although the structure of the Hrs FYVE do-
main is very similar to that recently reported for Vps27p
(Misra and Hurley, 1999), ourdata suggesta significantly
different model forbinding of the FYVE domain to PI(3)P
Results and Discussion
Structure Determination and Overall Structure
The tandem domain segment (residues 1 to 219) of Dro-
sophila Hrs was obtained by subcloning and overex-
pression in a protein-splicing system and yielded excel-
lent crystals (see the Experimental Procedures). The
presence of citrate and maintaining the pH at 7.2 were
crucial in obtaining the crystals. The crystals belong to
the C2 space group with unit cell dimensions of a ?
116.71 AÊ, b ? 69.67 AÊ, c ? 41.80 AÊ, and ? ? 94.77? and
one molecule in the asymmetric unit. The structure was
determined by selenomethionine MAD phasing (Table
1). With the exception of residues 146±148, which were
assumed to be very flexible, allresidues were positioned
in well-defined density. The current model refined at 2 AÊ
resolutionhas anR value of21.5% and anRfreeof25.4%.
The model has good geometry (Table 1), and 99.5% of
the backbone dihedral angles are in the most favored
or allowed regions.
The three-dimensional structure of the VHS and FYVE
tandem domains is illustrated in Figure 1A. Both do-
mains are aligned to form a pyramid-like structure with
the much largerVHS domain forming a rectangularbase
(45AÊ?35AÊ)witha thickness ofabout1/3ofthe pyramid
altitude (?60 AÊ) and the FYVE domain occupying the
apical end. Following termination of the VHS domain at
Asp-153, which is salt linked to Lys-71, the polypeptide
chain turns sharply into an N-terminal segment of the
FYVE domain. The VHS domain has a long C-terminal
segment which, together with the N-terminal segment
of the FYVE domain, could serve as a flexible tether
between the two domains. As will be discussed below,
the tandem domain unit associates into a homodimer,
and this association may provide high specificity and
affinity of ligand binding.
Structure of VHS and FYVE Tandem Domains
Figure 1. Structure of the Hrs Tandem Domains of VHS (Residues 1±153) and FYVE (Residues 154±219)
(A) Ribbon representation of the overall fold of the tandem domains of VHS (red) and FYVE (green) with the secondary structure labeled and
bound Zn2?atoms represented by gold spheres. ? helices 1, 3, and 6 constitute the A helices of the three repeats of the VHS domain, whereas
2, 4, and 7 ? helices make up the B helices. The segment of the FYVE domain highlighted in blue, which includes ?1 strand, contains
176RKHHCR, a sequence motif conserved among FYVE domains.
(B) Stereoview of the tandem domain homodimer down the ªfrontº or crystallographic 2-fold rotation axis. Every 20 residues are numbered
and identified with small filled circles. The two large filled circles represent Zn2?. Figures 1, 5A, 5C, and 6 were drawn using Molscript (Kraulis,
1991), Raster3D (Merritt and Bacon, 1997), and/or Bobscript (Esnouf, 1997).
VHS Domain: A Motif of Superhelix of Helices
The structure of the VHS domain (residues 1±153) is
comprised of eight ? helices and a long C-terminal ex-
tension (Figures 1A and 1B). The first four helices (?1 to
?4)formtwo repeats, each consisting of two antiparallel
helices (A and B). The succeeding three helices (?5 to
?7) fold into a three-helix hairpin (repeat 3) with ?6 and
?7 analogous to the A and B helices, respectively, of
the first two repeats. The ?8 helix is packed against ?6
and ?7. Repeat 1 helices (?1 and ?2) are related to their
counterparts of repeat 2 (?3 and ?4, respectively) by
a left-handed rotation of ?20? about an axis roughly
perpendicularto the helices. Helices ?3 and ?4ofrepeat
2 are, in turn, stacked essentially in parallel with ?6 and
?7 of repeat 3. These interrepeat geometries give rise
to a left-handed shallow groove, in which the A helices
are located at the outer (convex) face and form the
bottom of the pyramid-like structure and the B helices
are located at the inner (concave) face at approximately
1/3 of the height of the pyramidal structure.
The parallel stacking of the A and B helices of the
three repeats, combined with?8, creates a hydrophobic
channel between the double layer of helices that nearly
spans the entire length of the VHS domain. The channel
contains mostly hydrophobic residues (?30), of which
80% are conserved among known VHS domains (Figure
2). The slightly wider space in the channel between the
two-helix repeat 2 and the three-helix repeat 3 (Figure
1A) has the highest concentration of hydrophobic resi-
dues, mostly with aromatic and large aliphatic side-
chains. The distributionpatternofhydrophobic residues
in the channel is a major determinant of the packing
geometry of the superhelical structure of the VHS
Comparison of the VHS Domain with Other
Superhelices of ? helices have been observed pre-
viously in domains (Das et al., 1998) and subunits of a
variety of enzymes and signaling proteins, one of which
is clathrin, a key playerinendocytosis (Raag etal., 1988;
Thunnissen et al., 1994; Huber et al., 1997; Strickland
et al., 1998; Groves et al., 1999; Vetter et al., 1999). The
first two repeats of the VHS domain are very similar
to the HEAT repeats, whereas the third repeat closely
resembles an ARM repeat (Figure 3A). The combination
of two different repeats makes the VHS domain an un-
usual member of domains or proteins with superhelical
The closest geometrical match to the three repeats
plus the eighth helix of the VHS domain occurs in the
phosphatase 2 PR65/A subunit (Groves et al., 1999), in
which the HEAT repeats 3 to 5 plus helix A of repeat 6
superimpose with an rms deviation of 3.6 AÊover 128
residues (DALI [Holm and Sander, 1995]) ªZº score of
9.1) (Figure 3B). The closer match with the PR65/A sub-
unit is attributable to the similarity of the left-handed
packing geometry between repeats 1 and 2 to that be-
tween repeats 3 and 4 of the PR65/A subunit. Moreover,
the VHS domain and PR65/A subunit are the only ones,
thus far, that have a left-handed superhelical fold of
? helices. Although the amino acid sequences of the
overlapped VHS and PR65/A repeats differ consider-
ably, the hydrophobic nature of the channels is con-
served (Figure 2).
Multipurpose Docking Sites of the VHS Domain
The superhelical structure found in the phosphatase 2
PR65/A subunit and othersubunits and proteindomains
Figure 2. Multiple Sequence Alignment of VHS Domains Constructed Using the Clustal W Program and Colored by the Boxshade Program
Search of sequences similar to the Drosophila VHS sequence was performed using BLAST2 (Altschul et al., 1997). The alignment with the
HEAT repeats 3 to 5 and the A helix of repeat 6 of the X-ray structure of the PR65/A subunit of protein phosphatase 2 is based on DALI (Holm
and Sander, 1995). The ? helices of the Hrs VHS structure are shown above the alignment. The first column gives the name of the protein or
the accession number in the Entrez database for those proteins that have not been characterized. The following capital letters designate the
species (A, Arabidopsis thaliana; C, Caenorphabditis elegans; D, Drosophila melanogaster; G, Gallus gallus; H, Homo sapiens; M, Mus musculus;
Sa, Saccharomyces cerevisiae; and Sc, Schizosaccharomyces pombe). Identical and similar residues have yellow and blue backgrounds,
respectively. The conserved hydrophobic residues in the VHS channel of Hrs are identified by asterisks.
serves as a scaffold forprotein-proteininteractions, par-
ticularly in the vicinity of the inner groove and its ridge
(Raag et al., 1988; Thunnissen et al., 1994; Huber et al.,
1997; Das et al., 1998; Strickland et al., 1998; Groves et
al., 1999; Vetter et al., 1999). Interestingly, the VHS do-
main is engaged in both interdomain and dimeric inter-
actions. The concave surface docks with the proximal
end of the FYVE domain within the monomer and the
apical end of the FYVE domain from the symmetry re-
lated molecule (Figures 1A and 1B). The ?2 helix of
the VHS domain contains three extremely conserved
residues (Trp-23, Asp-31, and Leu-27) that make both
intra- and intermolecular interactions with FYVE do-
mains. Trp-23 is engaged in contacts with hydrophobic
moieties atthe tip oftheloop between?3and ?4strands
of the FYVE domain within the monomer, whereas Asp-
31 is involved in hydrogen-bonding interactions be-
tween monomers. The O?1 and O?2 of the carboxylate
sidechain of Asp-31 form strong hydrogen bonds with
the backbone peptide NHs ofresidues Phe-173 and Thr-
174, respectively, at the apical loop preceeding the ?1
strand of the FYVE domain from the symmetry-related
Structure of VHS and FYVE Tandem Domains
Figure 3. Overlap of the VHS Domain Helix
HairpinRepeats withSimilarRepeats ofOther
(A)The firsttwo repeats ofVHS (red)superim-
posed with HEAT repeats 3 and 4 (cyan) of
protein phosphate 2 PR65/A subunit (Groves
et al., 1999) and the third repeat superim-
posed with the ARM repeat 4 (blue) of ?
catenin (Huber et al., 1997).
(B) Overlap, based on DALI (Holm and
Sander, 1995), of the entire eight helices of
the VHS domain with repeats 3 to 5 plus the
first helix of repeat 6 of protein phosphate 2
bond from the hydroxyl sidechain of Thr-174. The third
conservedVHS residue,Leu-27, is involvedinhydropho-
bic interactions with the symmetry-related Phe-173 that
is also a highly conserved residue among FYVE do-
mains. The VHS domain is a prime example of this type
of structure whose concave surface acts as a platform
for both interdomain and dimeric interactions.
The VHS domain may also interact with membranes
and/or proteins of the endocytotic machinery. The
N-terminal portion of EAST containing the VHS domain
is capable of interacting with membranes and partially
colocalizes with clathrin (Lohi et al., 1998). This N-termi-
nal VHS portion of EAST also forms a complex with
eps15, an EGF receptor substrate associated with
clathrin-coated pits and vesicles (Lohi and Lehto, 1998).
Moreover, the presence of the VHS domain in Hrs may
explainwhy theFYVE deletionmutantis notmislocalized
(Komada et al., 1997). Thus, the VHS domain may local-
ize proteins to the membrane through interactions with
the membrane and/or the endocytic machinery. Taken
together, the VHS domainis therefore viewed as a multi-
purpose docking adapter.
array of interactions, involving the invariant Trp-158 and
His-179 residues, not only contributes to the dimer sta-
bility but also creates an environment essential to the
functional role of His-179 in ligand binding (discussed
Dimer formation of the FYVE domain has also been
observed by otherrecentstudies. NMR analysis demon-
strated that the isolated FYVE domain of EEA1 forms
functional homodimers that are in fast equilibrium with
monomers (Kutateladze et al., 1999). More importantly,
the analysis further indicated that the homodimers bind
PI(3)P-bearing membranes much more tightly than the
corresponding monomers. Binding appears to be more
specific forPI(3)P since PI(5)P, a naturally occurring lipid
headgroup, is bound more weakly. The suggestion that
the long N-terminal segment of the EEA1 FYVE domain
contributes to dimerization (Kutateladze et al., 1999) is
consistentwiththe Hrs structure thatshows the involve-
ment of this segment in homodimer formation (see
above; Figure 1B). Interestingly, the N-terminal segment
of the monomeric recombinant Vps27p FYVE domain is
considerably shorter as it is missing several residues
(Kutateladze et al., 1999; Misra and Hurley, 1999). Using
thedynamic lightscattering technique, we havesimilarly
observed a dimer-monomer equilibrium of the Hrs tan-
dem domains in solution. Therefore, the FYVE domain
of Hrs is capable of forming a homodimer. Finally, as
further exemplified by the pleckstrin homology (PH) do-
main(Kleinetal., 1998), domainoligomerizationappears
to be a common feature for high-affinity phosphoinosi-
FYVE Zinc Finger Domain and Its Involvement
in Dimer Formation
The structure of the FYVE domain (residues 154±219)
consists of a nonstandard N-terminal strand, loops, two
double-stranded antiparallel ? sheets, and a C-terminal
helix (Figures 1A and 1B). The fold is stabilized by a pair
of bound Zn2?atoms, each tetrahedrally coordinated
with four Cys sidechains. The X-ray structure of the
FYVE zinc finger domain of Hrs is very similar to that of
the isolated monomeric FYVE domain of Vps27p (Misra
and Hurley, 1999), with an rms deviation of 0.93 AÊ. How-
ever, our crystal structure has revealed the formation of
a homodimer mainly by way of the antiparallel associa-
tion between two FYVE domains (Figures 1B, 4A, and
4B). This homodimer is related by a crystallographic
2-fold axis, and this association buries a total of 1920
AÊ2accessible surface area. The largest area of contact
between the monomers involves mainly residues from
a region of the FYVE domain that includes the long
N-terminalsegmentand thestrands ofthe twoantiparal-
lel ? sheets. An important dimeric interaction is the hy-
drophobic edge-on contact between two Trp-158 resi-
dues flanked by two His-179 residues (Figure 5A). This
Binding Sites in the FYVE Dimer for Ligands
with Multiple Negative Charges
Our structural data have further uncovered two neigh-
boring identical pockets formed between two symme-
try-related FYVE domains (Figures 1B, 4A, and 4B). Each
pocket is lined mainly by residues from the ?1 strand
of one FYVE domain, which contains several basic resi-
dues of the conserved 176RKHHCR motif and the ?4
strand of the symmetry-related FYVE domain, which
contains hydrophobic residues (Figures 1 and 5A). This
pocket is ideally suited for binding ligands with multiple
negativecharges suchas citrate(Figure5A),anessential
component in crystallization of the tandem domains.
The area encompassing the two neighboring pockets
Figure 4. Electrostatic Surface Potential (?10
kT) of the Homodimer of the VHS-FYVE Tan-
(A) Electrostatic surface potential was calcu-
lated and displayed within GRASP (Nicholls
et al., 1993). ªFront viewº of the homodimer
that is identical to that shown in Figure 1B.
As also shown in Figure 1B, the middle of the
dimer contains two symmetry-related multi-
anion-binding pockets with bound citrates
whose C1carboxylates are partially exposed.
The bound citrates are excluded from the
(B) ªBack viewº of the homodimer obtained
by a 180? rotation of the structure shown in
(A) about a vertical axis.
(named ªmultianion-binding sitesº)shows a very intense
positive electrostatic surface potential (Figure 4A),
largely due to the presence of two symmetry-related
conserved basic sequence motifs.
Four key features are associated with molecular rec-
ognitionand binding ofcitrate by the multianion-binding
site of the FYVE dimer, and these are likely to have an
importantbearing onthe binding ofthephosphatidylino-
sitol 3-phosphate head group. First, the citrate is bound
snugly ineachpocketwithabout90% ofits freeaccessi-
ble surface buried (Figure 4A). The C1 carboxylate,
which is partially exposed and hydrated by three or-
dered surface water molecules, occupies the pocket
opening, and the C5 carboxylate occupies the pocket
bottom (Figures 1B, 4A, 5A, and 5B).
of charge-coupling and hydrogen-bonding interactions
mostly with the positively charged residues of the con-
served motif (Figure 5B). Although Lys-177 of the motif
is near the ligand-binding pocket (Figure 5A), it does
not interact with the bound citrate. However, Lys-177
may stabilize the multianion-binding site by forming a
salt link with Asp-160.
Third, the citrate makes a specific hydrogenbond with
the invariant His-179. The hydrophobic environment of
the His-179 sidechain in the pocket bottom, being
flanked on both sides by Val-186 and two symmetry-
related Trp-158 (Figure 5A), would seem to preclude a
protonated positively charged imidazole group. As a
consequence of this environment and the involvement
oftheHis Nd2H as a hydrogenbond donortothe peptide
carbonyl oxygen of Ala-159, it is indicated that the His
Nd1 group accepts a hydrogen bond from one of the
citrate carboxylates that would normally have the high-
est pKaof 6.4 (Figure 5B).
Fourth, the cluster of nonpolar C2 and C4 groups of
citrate interface with the nonpolar groups of C? and C?
of Arg-181 and the sidechains of Ile-203, Val-207 of
the b4 strand from the symmetry-related FYVE domain
(Figure 5A). Ile-203 is weakly conserved, whereas Val-
207 is strongly conserved among FYVE domains. As
thesenonpolarinteractions canonly occurinthedimeric
form of the tandem domains, they provide a specific
evidence for a functional homodimer structure. Given
its ideal binding mode, citrate may prove to be a potent
inhibitor of the functions of FYVE domain±containing
Models for Binding of Phosphatidylinositol
3-Phosphate to the FYVE Dimer
and Membrane Interaction
The bound citrate has seriously hampered ourinvestiga-
tion of the binding of PI(3)P analogs to tandem domain
crystals. Nonetheless, the mode of binding of citrate
to the FYVE dimer provides an excellent paradigm for
modeling a bound PI(3)P headgroup (Figure 5C). The
citrate's C1 and C5 carboxylate oxygens (separated by
?7 AÊ) are homologous to the 1- and 3-phosphates, re-
spectively, of PI(3)P. The orientation of the modeled
PI(3)P head group is closely related to that of citrate;
the 1- and 3-phosphates occupy the opening and the
bottom, respectively, ofthe pocket. The modeling incor-
porates allfourkey features of citrate binding described
above. A large portion of the PI(3)P is buried. The mode
of PI(3)P binding places the 3-phosphate close to His-
179 and the 1-phosphate close to His-178 and Arg-208.
The sidechain of Arg-176 is juxtaposed between the 1-
and 3-phosphates. Arg-181 and its symmetry-related
counterpartmay beinvolved ininteracting withtheinosi-
tol hydroxyl groups. Thus, the phosphate and hydroxyl
groups of PI(3)P are assumed to be extensively involved
in charge-coupling and hydrogen-bonding interactions
with the conserved basic residues.
As in the bound citrate (Figure 5B), a protonated oxy-
gen of the 3-phosphate (normal pKaof 7.2) donates a
hydrogen bond to the neutral His-179 sidechain. This
hydrogen bond, combined with a salt link with Arg-176,
fully satisfies the requirement of the 3-phosphate for
Structure of VHS and FYVE Tandem Domains
Figure 5. Multianion-Binding Site
(A) Stereo view of one of the two sites with the 2 AÊresolution citrate omit (Fo-Fc), ?calc electron density map at ?3?. Residues from the
symmetry-related FYVE domain are distinguished by asterisks.
(B) Schematic diagram of the interactions with the citrate, involving hydrogen bonds (?3.2 AÊ) and/orsalt links (?4 AÊ), along with theirobserved
distances in angstroms.
(C) A model of the binding of PI(3)P headgroup with a glycerol group linked to the 1-phosphate oriented toward the reader. The PI(3)P was
modeled in the binding site with no steric clashes and then energy minimized using CNS (Bru Ènger et al., 1998). Only residues within close
proximity to the model are included. The 3-phosphate group, oriented away from the reader, is within hydrogen-bonding distance to His-179.
The 1-phosphate moiety is close to His-178 and Arg-208. Arg-176 is within salt-linking distance to both 1- and 3 phosphates. The 2- and
3-hydroxyl groups are near Arg-181, and the 4-OH is close to Arg-181 of the symmetry-related FYVE.
ligand specificity. Furthermore, some of the nonpolar
CH groups protruding from the inositol ring interface
with the hydrophobic sidechains from the symmetry-
related molecule identified in citrate binding (Figures 5A
and 5C). The residues of the FYVE domain of EEA1
equivalent to these hydrophobic residues of Hrs, as well
as the polar residues in the binding site (Figures 5A, 5B,
and 5C), have been shown by NMR analysis to undergo
chemical shifts upon binding of PI(3)P analogs (Kutatel-
adze et al., 1999). Several of these shifts would not be
expected if the FYVE binds PI(3)P as a monomer. The
modelofthe Hrs FYVE-PI(3)P complex is consistentwith
the reasonably tight affinity (?M Kd) determined for the
complex of PI(3)P analogs with the EEA1 FYVE domain
(Kutateladze et al., 1999).
ThemodelofPI(3)P binding toHrs differs considerably
from that previously proposed for the Vps27p FYVE do-
main (Misra and Hurley, 1999). The PI(3)P binding to a
small pocket of conserved basic residues was modeled
on the basis of a monomeric Vps27p FYVE structure.
The modeling further relied on the observation that the
pocket is occupied by two carboxylate sidechains of
Asp and Glu residues from a lattice-related molecule.
The locations of the Asp and Glu carboxylates, sepa-
rated by about 9 AÊ, were assumed to be the sites for
binding of the 3- and 1-phosphate groups, respectively,
of the PI(3)P headgroup. The 3-phosphate lies in an
approximately similarlocationas thatofthe modelinHrs
FYVE dimer. However, the locations of the 1-phosphate
group in the two models are completely different. As
a result, the orientations of the PI(3)P headgroup with
respect to the binding site differ greatly between the
two models. The model with Vps27p (Misra and Hurley,
1999) places the phosphatidylinositol in an area of the
monomer which, in the Hrs FYVE domain, is heavily
engaged in dimer formation. The PI(3)P modeled in the
Figure 6. Model of the Interaction of the VHS
and FYVE Tandem Domains with the Mem-
The flat ªfrontº surface of the tandem do-
mains shown in Figures 1B and 4A interfaces
with the membrane. The region of the VHS
domain protruding into the membrane inter-
face contains predominantly basic residues
(Arg 3, Lys-8, and Arg-18) and hydrophobic
residues (Phe-2, Leu-17, and Leu-19) that
Vps27p lies sideways or parallel with respect to the ?1
strand with its 1-phosphate group directed toward the
apical end of the FYVE domain and engaged in an inter-
action with Lys-189 (the equivalent of Lys-177 of Hrs).
In contrast, in our model with the Hrs homodimer, the
PI(3)P is normal to the ?1 strand, its 1-phosphate pro-
truding fromthe opening ofthe pocket and farfromLys-
177 (Figure 5C). Moreover, of the four key features of
ligand recognition and binding to the Hrs dimer de-
scribed above, only some of the electrostatic interac-
tions may have semblance with the proposed model
of PI(3)P binding to the Vps27p FYVE monomer. The
monomeric binding mode, with the ligand also signifi-
cantly exposed to the bulk solvent, may not be sufficient
for high ligand specificity and affinity.
The difference between the two proposed PI(3)P-
binding modes leads to two completely differentmodels
for membrane-targeting of the FYVE domain. In our
model shown in Figure 6, the extended tandem domain
dimeric structure lies in a horizontal direction with re-
spect to the membrane surface. In addition to the asso-
ciation between FYVE and the phosphoinositol 3-phos-
phate headgroup of the membranes (Figures 5C and 6),
the model would place the VHS domain in a position to
interact with membranes (Figure 6), a feature consistent
with the demonstration that the VHS domain interacts
with the membrane (discussed above). In contrast, in
the modelbased onthe ªsidewaysº binding ofthe PI(3)P
proposed by Misra and Hurley (1999), the FYVE mono-
merlies ina verticalorientationwithrespectto the mem-
brane with the tip of the apical end inserted in the mem-
brane's interface. This orientation would place the VHS
domain of Vps27p, assuming similarpyramidal arrange-
ment with the FYVE domain as seen in the Hrs tandem
domain structure, very far from the membrane surface.
Finally, in contrast with the Vps27p model, the finding
that the apical end of the Hrs FYVE domain is buried
and extensively involved in polar and nonpolar interac-
tions withthe symmetry-related VHS domain(discussed
above; Figure 1B) would completely preclude the inter-
action of this end with the membrane's interface.
Since the citrate or the modeled PI(3)P interacts pre-
dominantly with the basic residues of the conserved
motif, ligand binding solely to one FYVE domain is not
precluded. However, binding to the monomer would, as
inthe case also ofthe modelwiththe Vps27p FYVE, lead
to a more solvent exposed ligand [e.g., ?50% exposed
accessible surfacearea ofcitrate orthemodeled PI(3)P)]
and the elimination of the hydrophobic interactions de-
scribed above, resulting in weaker ligand affinities.
Therefore, thefunctionalformoftheFYVE domainfavors
In conclusion, the structure reported here reveals the
unique superhelical conformation of the VHS domain
and its relationship with the FYVE domain. It further
shows the formation of a homodimer mainly through
the antiparallel association of FYVE domains. The VHS
domain's presence in proteins implicated in membrane
trafficking, superhelical folding character, and involve-
ment in interdomain and dimeric interactions strongly
suggest that the VHS domain functions as a multipur-
pose docking adapter. Furthermore, the finding of the
dimeric form of the FYVE domain and its complex with
citrate provide anexcellent paradigmforPI(3)P recogni-
tion. Based onthese results, we have proposed a model
for PI(3)P binding and membrane interaction of the tan-
dem domains. Finally, the structure of the tandem do-
mains reported here serves as a framework for further
studies of Hrs and other related proteins, especially the
distinct role of each domain in membrane trafficking
and signal transduction events.
Expression, Purification, and Crystallization
The DNA sequences corresponding to the tandem domains (resi-
dues 1±219) of Drosophila Hrs were amplified by PCR and cloned
into the pTYB1 expression vector of the IMPACT T7 System (New
England BioLabs) as the N-terminal segment fused to the intein and
chitin-binding domain unit. To facilitate protein splicing, a glycine
residue was introduced between the Hrs protein and the intein.
Proteins were expressed in Escherichia coli ER2566 cells at room
temperature, purified to near homogeneity according to the proce-
dure provided with the IMPACT T7 System, and dialyzed against 50
mM NaCl and 100 mM Tris (pH 8.0), at 4?C. To remove minorimpuri-
ties, the protein (5 mg/ml) was further purified on an HQ column,
and the stock protein solution was concentrated to 10 mg/ml in 1
mM DTT and 50 mM citrate (pH 5.5). The protein was crystallized
at 4?C using the vapor diffusion method with the drop consisting of
a 1:1 mixture of the stock protein solution and the reservoirsolution
of 15% PEG 10000, 5 mM DTT, and 100 mM HEPES (pH 7.4). Prior
to data collection, the crystalwas flash-frozenin liquid nitrogenwith
25% glycerol in the crystallization solution.
Structure of VHS and FYVE Tandem Domains
The structure was determined by multiwavelength anomalous dis-
persion(MAD). A 3-wavelengthdata set was collected froma crystal
of selenomethionine-substituted protein on beamline X4A at NSLS
of the Brookhaven National Laboratory and processed and merged
with DENZO and SCALEPACK (Otwinowski and Minor, 1997), re-
spectively. The positions of four of the six SeMet sites and 2 Zn2?
were determined, heavy atom parameters refined, and MAD phases
calculated at 2.4 AÊresolution using the suite of programs in SOLVE
(http://www.hwi.buffalo.edu/SnB). The calculated electron density
map, which was solvent flattened in DM (Cowton, 1994), was used
to build an initial model of 180 of the total 220 residues by means
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density of the bound citrate was well defined in initial maps, its
model was fitted only in the final rounds of refinement. The final
model was refined against the 2 AÊresolution data (92.4% complete
with 2? cutoff) from the higher-energy remote ?3wavelength (Table 1).
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Protein Data Bank ID Code
Atomic coordinates have been deposited with the ID code 1DVP.