(Received for publication, June 12, 1995; and in revised form, August 2, 1995)
From the
The cytosolic tail of the major histocompatibility
complex-associated invariant chain protein contains two Leu-based
motifs that both mediate efficient sorting to the endocytic pathway.
Nuclear magnetic resonance data on a peptide of 27 residues
corresponding to the cytosolic tail of human invariant chain indicate
that in water at pH 7.4 the membrane distal motif
Leu-Ile
lies within a nascent helix, while the
membrane proximal motif Met
-Leu
is part of a
turn. The presence of a small amount of methanol stabilizes an
helix from Gln
to Leu
with a kink on
Pro
. Point mutations of the cytosolic tail of the protein
suggest that amino-terminal residues located in spatial proximity to
the Leu motifs contribute to efficient internalization and targeting to
endosomes in transfected COS cells. Residues on the spatially opposite
side of the Leu motifs were, on the other hand, mutated with no
measurable effect on targeting. Structural and biological data thus
suggest that the signals are not continuous but consist of
``signal patches'' formed by the three-dimensional structure
of the cytosolic tail of invariant chain.
In transmembrane proteins, specific signals for endosomal or
lysosomal sorting have been identified within the cytosolic tails. In a
number of proteins sorted to the endosomal pathway, a Tyr-containing
motif seems to mediate internalization from the plasma membrane (for
review, see (1) and (2) )) and form a tight turn
functionally comparable to the internalization signal found in cell
surface receptors(3, 4, 5, 6) .
However, recent NMR analysis of a synthetic peptide corresponding to
the extreme 21 carboxyl-terminal amino acid residues of the cytosolic
domain of trans-Golgi network (TGN) (
)protein TGN38/41
(which is routed from the plasma membrane and back to TGN) shows that
this Tyr-containing internalization signal lies within a nascent helix (7) .
Tyrosine signals are not universal for sorting of
membrane proteins to the endosomal/lysosomal pathway. In fact, the
cytosolic tail of the lysosomal membrane protein LIMP II contains no
Tyr but a Leu-Ile signal, located two residues from the carboxylic end,
which mediates efficient sorting(8, 9) . Letourneur
and Klausner (10) reported that a Leu-Leu motif could mediate
lysosomal targeting of the CD3- and -
chains of the T-cell
receptor complex. This signal was functional, even if located at the
carboxyl-terminal end, and together with a Tyr-based motif they were
individually sufficient to induce endocytosis and delivery to
lysosomes. Two similar signals were also reported in the cytosolic tail
of the mannose 6-phosphate-receptor by Johnson and
Kornfeld(11) . The above studies may suggest that the Tyr
signal is primarily mediating sorting to the endocytic pathway via
internalization from the plasma membrane, whereas the Leu signal alone,
or in combination with the Tyr signal, mediates direct sorting from the
TGN to the endocytic pathway. However, both signals internalize
efficiently plasma membrane proteins, and further information is needed
to clarify the requirements for selecting the pathway from the TGN to
endosomes (for further discussion see(2) ).
The invariant chain (Ii), which efficiently targets the associated major histocompatibility complex class II molecules to the endosomal/lysosomal pathway, is another example of a protein with cytosolic Leu-sorting motifs(12, 13, 14) . Invariant chains from different species contain in their cytosolic tails pairs of Leu-Ile and Met-Leu (Ile-Leu), and mutational analysis of the cytosolic Ii tail fused to reporter molecules have shown that these signals can independently mediate endosomal targeting(15, 16) . Distribution and internalization studies also show that a significant fraction of the Ii molecules, alone or in complex with major histocompatibility complex class II, were transported to endosomes via the plasma membrane(17) . Furthermore, the Leu-Ile and Met-Leu motifs individually mediate rapid internalization of a chimeric protein (INA) obtained by fusing neuraminidase (NA) to the cytosolic tail of Ii(15) . In another study the Ii tail was fused to the transferrin receptor, but the measured sorting via the plasma membrane was not sufficient to account for the amount of protein synthesized, indicating sorting also directly from the TGN(16) . The accumulated data thus suggest that Leu-based motifs are actively engaged in the sorting of membrane proteins to the endosomal/lysosomal pathway, both directly from the TGN and via the plasma membrane.
Here we report structural and mutational
studies on the 27-amino acid synthetic peptide Ii-(1-27) (Fig. SI), corresponding to the amino-terminal region of human
Ii. NMR results demonstrate, for the first time, that in water
Leu-Ile
and Met
-Leu
internalization motifs lie within a nascent helix and a turn,
respectively, but can take up a kinked helix in the presence of small
amount of methanol. Site-directed mutagenesis has been performed to
determine in which context the two internalization signals are
functional. By comparing the biological data with the three-dimensional
structure we conclude that the spatial arrangement of the leucine motif
is essential for a functional signal and that it is not a continuous
signal but a ``signal patch'' that depends upon the secondary
structure of the protein.
Met-Asp-Asp-Gln-Arg
-Asp-Leu-Ile-Ser-Asn
-Asn-Glu-Gln-Leu-Pro
-Met-Leu-Gly-Arg-Arg
-Pro-Gly-Ala-Pro-Glu
-Ser-Lys
Scheme I. Amino acid sequence of the Ii-(1-27) peptide corresponding to the cytosolic tail of human invariant chain. The di-leucine-like signals are in italics.
Fig. 1reports the summary of NOE information
for Ii-(1-27) in water at 283 K and pH 7.4. Together with the
strong CH
-NH
connectivities, a
number of NOEs were observed between the NH resonances of sequential
amino acids in the regions before and after Pro
, the
strongest being observed in the Asp
-Asn
region and between Met
and Leu
.
Proximity of NHs of adjacent residues requires a kink in the backbone,
a conformation associated with a
turn or with an
helix(42) . Accordingly, the NOEs between pairs of NHs in the
region Arg
-Glu
suggest that consecutive
turns are present in this part of the peptide and that
Met
-Leu
is part of a turn. An ensemble of
consecutive turns resembles a helix-like conformation, since distance
constraints in tight turns are similar to those in helical
segments(29) . This is confirmed by
CH
-NH
connectivities
(Arg
-Leu
, Asp
-Ile
,
Leu
-Ser
, Ile
-Asn
, and
Ser
-Asn
), and by
CH
-NH
cross-peaks
(Gln
-Arg
, Leu
-Ile
,
Ser
-Asn
, and
Glu
-Gln
). However, the contemporary presence
of
CH
-NH
and
NH
-NH
NOEs (Fig. 1)
indicates that the local helix-like structure in the region at the
amino-terminal side of Pro
dynamically transforms into
extended conformations.
Figure 1:
Diagrammatic
representation of sequential and secondary structural interresidues
NOEs observed for Ii-(1-27) in water at 283 K and pH 7.4. Atoms
involved in the NOEs are listed in the text. Strong, medium, and weak
NOEs are indicated by thick, medium, and thin
lines, respectively. Empty bars refer to effects to the
protons of Pro residues.
The absence of a stable helical structure is
confirmed by the lack of characteristic NOEs and by the J
coupling constants values. We
did not observe loop to loop interproton NOEs (e.g.
of
residue i to the NHs of residues i+3 and i+4), although several residues show the NH to NH
interaction. On the other hand, only the amides of residues Leu
and Ser
have coupling constants (5.9 Hz) similar to
that expected for a stable helical structure(43) , while the
J
coupling constants for the
remaining residues in that region all are > 7 Hz. These results
(stretch of sequential and short range NOEs, the absence of
helix-defining NOEs within a conformation ensemble giving rise to
largely averaged coupling constants) argue for the presence of
``nascent helix'' structure(44) , including the
Leu
-Ile
sorting signal of invariant chain. The
term nascent helix refers to an ensemble of interconverting extended
chain and turn-like structures existing over a peptide sequence at the
earliest stages of helix initiation.
From Pro onward,
the peptide also assumes turn-like conformations up to
Ala
, as suggested by
NH
-NH
,
CH
-NH
and
CH
-NH
NOE connectivities (Fig. 1). In particular, the presence of strong NOEs between the
amide protons of Met
and Leu
and between
CH of Pro
and NH of Leu
suggest that the
second signal Met
-Leu
is part of a turn.
A
nascent helix can be stabilized by small amounts of organic
co-solvent(44) . Upon addition of methanol at a concentration
of 20% (v/v) to the Ii-(1-27) aqueous solution, all resonances
needed to be reassigned because one-to-one comparison between the two
solvents was not possible. Complete sequential assignment was made as
described above. Stabilization of the helical structure was confirmed
by the sequential (CH
-NH
and NH
-NH
) and
medium-range (
CH
-NH
, n
2, and
CH
-
CH
)
NOEs(45) , slowly exchanging amide protons(29) , and
J
coupling constants (43) . Fig. 2summarizes the observed NOEs, the relative
exchange rates of amide protons and the apparent
J
coupling constants for
Ii-(1-27) at 283 K and pH 7.4 in the presence of 20% methanol.
The fact that in the region Gln
-Leu
the
NH
-NH
NOEs are intense, while
the
CH
-NH
NOEs are much
weaker, implies a generally helical structure(42) . The
observation of several unambiguous
CH
-NH
,
CH
-
CH
and a single
CH
-NH
cross-peaks (Fig. 2) supports the presence of a helix.
Figure 2:
J
coupling constants, NOEs, and hydrogen-deuterium exchange rate
observed for Ii-(1-27) in 20% methanol, 80% water, 283 K and pH
7.4. Coupling constants are labeled as follows:
, parent
J
< 5.0 Hz;
, measured
J
> 7.0 Hz;
, 6.0 Hz
J
7.0 Hz. NOE intensities
are indicated by the height of the bars, and empty bars refer to effects to the
protons of Pro residues. Filled
circles also label residues for which slow exchange of amide
protons with deuterons was observed.
Further
corroborative data come from slowly exchanging amides; except for
Gln, all the amide protons in the
Gln
-Leu
region are in slow exchange. The
slow exchange most likely indicate hydrogen bonding, since it is
unlikely that a slowly exchanging proton is buried in the interior of a
biomolecule as small as the cytosolic tail of Ii.
J
< 6 Hz in the Gln
to Leu
region also supports the presence of a
helix(43) . Furthermore,
CH
-NH
cross-peaks,
suggestive of a 3
helix(45) , were not detected in
the middle region of the peptide and we conclude that the
Gln
-Leu
region of Ii-(1-27) forms
an
helix.
From Pro onward, we observed strong
CH
-NH
NOE
connectivities, meaning that the conformation is essentially extended.
However, the presence of
NH
-NH
,
CH
-NH
, two
CH
-NH
(Pro
-Leu
and
Met
-Gly
) and a single small
CH
-NH
(Leu
-Leu
) NOE connectivities (Fig. 2) is indicative of local structures and short range
order.
CH
-NH
connectivities are commonly observed in type I or type III
turns(42) , but while type III turns are very similar to type
I, the presence of a Pro in the trans isomer is compatible with both
type I and type II classes of turns(46) . In principle, they
can be distinguished on the basis of
J
coupling constants at positions 2 and 3 of the turns, and NOE
connections(42, 45) . Except for Met
and
Lys
, for which we measured a
J
> 7 Hz, and Gly
and Ser
(both with a
J
< 6 Hz), all the residues
of the carboxyl-terminal region showed coupling constants of
approximately 6.5 Hz. Furthermore, the amides of Leu
,
Ala
, and Ser
are slowly exchanging, thus
suggesting the formation of hydrogen bond (45) and the possible
formation of consecutive turns in the regions Leu
to
Leu
, Arg
to Ala
, and Ala
to Ser
. The NMR data slightly favor a type I and two
type II turns, respectively, for the above segments. In fact, a
CH-NH NOE connectivity between Pro
and Met
suggested that the region Leu
to Leu
forms a type I
turn, since the
CH-NH distance is
between 0.19 and 0.35 nm (47) . The presence of an
CH
-NH
NOE between
Leu
and Leu
suggests that the type I turn can
actually prolong the helix up to Leu
. For Arg
to Ala
and Ala
to Ser
, the
observation of
CH-
CH NOE connectivities between Arg
and Pro
, and Ala
and Pro
for the trans isomer of both regions, suggests the presence of
two type II
turns.
Methanol is known to induce structure in peptides (see, for example, (48) ). However, at the used ratio of 80% water/20% methanol (v/v), the mixture has a helix-promoting ability slightly higher than that of pure water(48) , indicating that the structure is only stabilized by the presence of methanol. The possibility that the secondary structure arises through aggregation was ruled out by investigating a 10-fold diluted sample of Ii-(1-27). No differences in chemical shift and line width of the NH resonances were observed in one-dimensional spectra, and NOESY experiments confirmed all the connectivities, and thus the structure, described above.
From NMR data in water/methanol, 100 randomly
selected starting conformations were generated by means of
distance-geometry calculations. The best nine structures, in terms of
smallest target function values, were subjected to restrained energy
minimization. Before minimization, they fulfilled quite well the whole
set of NMR restraints with no violations of the upper bounds of the
distance restraints greater than 0.05 nm, and of dihedral angle
restraints greater than 5°. The energy of the refined structures
were all in the narrow range from -1877 to -2080
kJ
mol
; the maximal distance constraint
violation was 0.050 ± 0.001 nm, and the average sum of distance
constraint violations was 0.293 ± 0.001 nm. None of the selected
structures presented additional short interproton distances not
experimentally observed. Fig. 3A shows a superposition of
all nine structures for the region covering residues
Met
-Pro
, all compatible with NOEs
data, since calculations of structures from NOEs is a means to assess
possible and favored conformational states and not single structures.
The convergence achieved over the well defined
helix from
Gln
-Leu
was good, with a root mean
square deviation for its backbone atoms of 0.090 nm. The region
Pro
-Glu
(Fig. 3B) did
not converge to a consensus structure, reflecting the absence of
structurally significant NOEs for this region of the polypeptide and
the variability in its relative position with respect to the helix. The
average root mean square deviation for backbone atoms of residues
16-25 is 0.293 nm. No unique conformation could be determined for
the Met
-Asp
-Asp
and the
Ser
-Lys
segments.
Figure 3:
View of the nine calculated NMR structures
of Ii-(1-27). The structures are superimposed for minimum root
mean square deviation of backbone atoms (N, C, C`, and O) for the
region Met
-Pro
(overlaid over residues
4-14) (A) and region from Pro
to Lys
(overlaid over residues 16-25) (B). For clarity
only side chains of the Pro residues are
shown.
Figure 4: Cellular distribution and internalization rates of INA and its constructs. Mutations of amino acids into alanine are indicated by A, while dashes refer to conserved residues. Empty space indicates deletion within each construct. The peptide studied by NMR and corresponding to Ii-(1-27) is indicated by a continuous line under the sequence numbers. Localization in vesicular staining or plasma membrane is indicated by V and PM, respectively. For the internalization time, NI means not internalized, indicating that less than 15% of construct molecules internalized after 10-min chase at 310 K. Each value of internalization rate is the mean of at least three independent experiments.
Regarding the Leu-Ile
signal, point mutation
of Arg
and Asp
to Ala (construct 6, Fig. 4), no change of the internalization rate was detected,
whereas both Gln
(construct 5) and Asp
(construct 4) prevented internalization. Alteration of amino acids at
the carboxyl terminus of the signal can be changed to alanine without
affecting the internalization signal; mutation of the potentially
phosphorylatable Ser
(construct 9), and Asn
and Asn
(construct 10), did not change the rate of
internalization in line with earlier studies(15) .
For the
second signal, Met-Leu
, mutation of
Pro
to Ala (construct 21) abolished internalization,
whereas neither mutation of Gln
nor Leu
(constructs 19 and 20, respectively) altered the internalization
efficiency. The negative residue Glu
(construct 15),
however, reduced internalization to background level. For all the INA
mutations, immunofluorescence studies showed a strong plasma membrane
staining for the constructs that were not internalized and vesicular
staining (V, localization column in Fig. 4)
corresponding to endosomes for the constructs that were actively
internalized. When native Ii harboring the identical cytosolic tail
mutations as INA was expressed in COS cells, the corresponding Ii
construct accumulated on the plasma membrane or in endosomes like the
INA molecule (data not shown), verifying that the tetrameric NA is a
reporter molecule that reflects the endosomal sorting properties of the
trimeric native Ii molecule(15) .
Deletion of the first 11 residues (construct 18) reached 25% internalization in 4.0 min, whereas elimination of the first signal by point mutations (construct 14) reached 25% internalization in 2.0 min. This may indicate that additional amino-terminal residues than those provided in construct 17 modulate the efficiency of the Met-Leu signal. Our biological data thus confirm that the cytosolic tail of Ii comprises two autonomous endosomal sorting signals that function in internalization (15, 16, 39) and in addition point out that a functional Leu-based sorting signal requires specific geometrically neighboring residues.
We have applied NMR spectroscopy to study the solution structure of a synthetic peptide corresponding to the cytosolic region of the protein Ii and containing the two internalization signals.
In
aqueous solution at pH 7.4 we detected NOEs characteristic of a nascent
helix involving the membrane distal motif
Leu-Ile
, while the membrane proximal motif
Met
-Leu
is part of a turn. In the presence of
20% methanol, the nascent helix was stabilized. We observed a regular
helix in the region Gln
-Leu
and a
segment of consecutive
turns between Leu
and
Ser
, namely a type I
(Leu
-Leu
) and two type II
(Arg
-Ala
and
Ala
-Ser
)
turns. The presence of a
small NOE between the
CH of Leu
and the NH of
Leu
suggests that the turn is actually part of the helix.
Inspection of the calculated backbone
and
dihedral angles
of the segment
Leu
-Pro
-Met
-Leu
shows that except for minor variation in Leu
, all residues
retain values appropriate for an
helix. The calculations thus
indicate that the type I turn can be accommodated in the helix with
minor conformational changes on Leu
without the rest of
the helical residues being perturbed. The resulting structure is an
helix from Gln
to Leu
with a bend at
Pro
. Previous calculations on model helical polypeptides
containing proline also confirm that it is sufficient to introduce a
conformational change of only one residue in order to accommodate
proline in a distorted helix(49, 50) . Kinked proline
helices with minor conformational changes and minimal disruption
of the helix hydrogen bonding have also been observed in crystal
structures of proteins(51) .
Comparison of the cytosolic
tails of Ii from human, mouse, and rat shows that Pro is
conserved(12) , while in the chicken (
)a Pro is
present at site 17. This suggests that the proline conserved in that
area might have a definite structural/functional role. It is noteworthy
that mutation of Pro
to Ala (construct 21, Fig. 4)
abolishes internalization. Considering that Ala is the most helix
favoring of the 20 commonly occurring amino acids (52, 53) such a substitution is expected to preserve
the helix while avoiding the bend. In fact, energy minimization
calculations (not shown) on Ala
Ii-(1-27) peptide
have found a regular helix from Gln
to Leu
.
Accordingly, it is tempting to speculate that a kinked helix is
required for internalization of Ii.
Our biological data show that
leucine motifs are influenced by residues located at their
amino-terminal side as Ser, Asn
, and
Asn
can be changed to alanine without affecting the
internalization signal. This is in line with other studies showing that
the signals work independently from the carboxyl-terminal residues (8, 9, 10) . In addition, amino acids
spatially close to the signals are fundamental for their correct
functioning and this can be rationalized by referring to the
three-dimensional structure of Ii-(1-27). The presence of Ala
instead of Gln
hampers efficient internalization (Fig. 4). The helical wheel diagram (Fig. 5) indicates
that Gln
is positioned on the same side of the helix as
Leu
and Ile
, so that its mutation alters the
surroundings of the signal. Accordingly, if residues pointing away from
the signal are changed, the mutation is expected to be irrelevant. In
fact, mutation of Arg
and Asp
, which are found
on the opposite side of the helix (Fig. 5), does not alter the
sorting capabilities of the molecule. Mutation of the negatively
charged Asp
into Ala also abolishes internalization. This
may be related either to the need of negatively charged residue to be
located at the amino-terminal side of the signal (see below) and/or to
the specific ability of Asp to stabilize
helical structures when
flanking the amino terminus(54, 55) . The side chain
of Ala at site 3 cannot form a hydrogen bond with the free NH groups at
the amino terminus of the helix as does Asp residue, resulting in a
strong decrease of helicity, which may prevent internalization. An
indirect confirmation of the relevance of the second hypothesis would
be the finding that Asp
becomes part of the helix when the
concentration of Ii-(1-27) is increased. (
)Altogether
our results suggest that for a correct functioning, the first signal
requires the specific structural context generated by the helix and the
amino-terminal residues with a specific spatial arrangement around
Leu
-Ile
.
Figure 5:
Helical wheel representation of the
Met-Leu
region of the Ii-(1-27)
peptide. Leu
and Ile
are boxed.
The segment
Leu-Pro
-Met
-Leu
takes up a turn and forms the kinked part of the helix. Point mutations
reveal that except for Leu
(construct 20 in Fig. 4), it is essential to have all the other residues
unaltered for a functional second signal. By mutating Pro
,
Met
, and Leu
(constructs 21, 22, and 23,
respectively), no internalization was observed. It must be noted that
substitution of residues within the helix with Ala does not modify the
helix, rather it alters the chemical nature of the environment of the
signal. In fact, only the substitution Ala for Pro has a structural
explanation, since it destabilizes the turn (56) and/or removes
the kink from the helix (see above), while both Ala and the native
residues Met and Leu at sites 16 and 17 have low preferences for
turns(56) . As a partial confirmation to the relevance of the
chemical properties of the side chains, we observed reduced
internalization to background level through the second signal for the
substitution Glu
to Ala. This finding points out the
necessity of a negatively charged residue on the amino-terminal side of
the second signal, suggesting as a putative consensus signal for Ii two
hydrophobic residues and the negatively charged amino-terminal residue:
Asp
(Glu
)-Xaa-Xaa-Xaa-Leu
(Met
)-Ile
(Leu
).
The two signals might work differently at various stages of the
intracellular pathway, requiring specific structural contributions. For
Ii, and the fusion protein INA, it is not clear whether it is sorted to
the endosomal pathway solely via the plasma membrane and/or directly to
endosomes from TGN. The most likely interpretation of the accumulated
data is that both pathways are functional(2) . Since endosomal
localization is not detected when active internalization is destroyed,
this would lead to the conclusion that both pathways are affected by
the same point mutations. The sorting machinery at TGN and plasma
membrane might thus see elements of the same sorting signal or require
common structural elements.
The Ii protein assembles as a
trimer(57) , and the luminal domain encoded by exon 6 is
essential for this process both in vitro(58) and in vivo. ()Recently, Arneson and Miller (59) suggested that a single Ii tail is needed for
internalization from the plasma membrane, whereas at least two intact
tails in the complex are needed for efficient TGN to endosomal
targeting. The NMR structure reported above is based on the Ii
monomeric form and appears to be biologically relevant although sorting
at some stages of the intracellular pathway may require more than one
tail. Preliminary NMR results (
)on more concentrated samples
of Ii-(1-27) indicate that the peptide starts to self-associate
at a concentration 30-fold higher than that used in the present study
and that the overall structure of the monomeric Ii-(1-27)
described above is preserved. Further studies are thus required to
elucidate whether the tail forms multimers and whether this influences
the structure of the single molecule; however, the study presented here
is certainly of interest for future modeling of the oligomer.
Similar to our results, NMR data on a 22-residue peptide corresponding to the amino-terminal cytosolic tail of the native low density lipoprotein receptor(4) , containing a Tyr in the internalization signal, indicate a nascent helix upstream of a turn containing a Pro at position 2. Comparing our results with those of Bansal and Gierasch (4) and Wilde et al.(7) on Tyr-containing signals, we suggest that Tyr and Leu signals might share a common structural model. Although the nature of the specific molecules that are able to recognize leucine signal is not known, the structural similarity of the helix suggests that the site of interaction may be similar for the two signals. The sorting signals can be found in both type I and type II membrane proteins and can satisfactorily be transplanted from one molecule to another (for reviews, see Refs. 1, 6, and 8), suggesting that they are possibly recognized by cytosolic proteins that are not membrane bound or otherwise have a distinct direction with regard to the plasma membrane.