Direct peptide-regulatable interactions between
MHC class I molecules and tapasin
Syed Monem Rizvi and Malini Raghavan†
Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, MI 48109-0620
Edited by Pamela J. Bjorkman, California Institute of Technology, Pasadena, CA, and approved October 10, 2006 (received for review June 21, 2006)
Tapasin (Tpn) has been implicated in multiple steps of the MHC
class I assembly pathway, but the mechanisms of function remain
incompletely understood. Using purified proteins, we could dem-
onstrate direct binding of Tpn to peptide-deficient forms of MHC
class I molecules at physiological temperatures. Tpn also bound to
M10.5, a pheromone receptor-associated MHC molecule that has
an open and empty groove and that shares significant sequence
identity with class I sequences. Two types of MHC class I–Tpn
complexes were detectable in vitro depending on the input pro-
teins; those depleted in ?2m, and those containing ?2m. Both were
competent for subsequent assembly with peptides, but the latter
complexes assembled more rapidly. Thus, the assembly rate of
Tpn-associated class I was determined by the conditions under
class I inhibited Tpn–class I-binding interactions, and peptide-
depletion enhanced binding. In combination with ?2m, certain
peptides induced efficient dissociation of preformed Tpn–class I
complexes. Together, these studies demonstrate direct Tpn–MHC
class I interactions and preferential binding of empty MHC class I
by Tpn, and that the Tpn–class I interaction is regulated by both
?2m and peptide. In cells, Tpn is likely to be a direct mediator of
peptide-regulated binding and release of MHC class I from the TAP
antigen presentation ? HLA ? TAP transporter
(?2m)] within the endoplasmic reticulum (ER) of cells and are
then transported to the cell surface, where they are available for
immune surveillance by cytotoxic T lymphocytes (1). The trans-
porter associated with antigen processing (TAP) is a peptide
transporter that translocates cytosolic peptides into the ER
lumen, for assembly with MHC class I HC and ?2m. Other ER
resident proteins that assist in MHC class I (class I) assembly
include the chaperones calnexin and calreticulin, the thiol-
disulfide isomerase ERp57, and the MHC-encoded transmem-
brane protein tapasin (Tpn). Individually or in combination,
these components ensure quality control of class I-peptide
For many human and mouse class I allotypes, Tpn increases
cell surface class I expression. How Tpn mediates this increase
has been a matter of considerable debate, and many functions
have been proposed for Tpn. Tpn stabilizes TAP, and increases
steady-state levels of TAP, thereby allowing more peptides to be
translocated into the ER (2, 3). Tpn also allows a physical link
between class I and the TAP (4), and by doing so, may increase
I binding. Tpn may also be important for preventing ER exit of
peptide-deficient class I (5–7), and may thereby indirectly pro-
mote the accumulation of more stable class I at the cell surface.
Soluble Tpn (sTpn) that does not stably bind TAP, enhance TAP
expression levels, or mediate the TAP–class I interaction, is able
that at least some of Tpn’s functions are independent of its
effects upon TAP. There are also indications from cell-based
experiments, that Tpn edits/optimizes the class I peptide reper-
eptide products of foreign and self-proteins bind to MHC
class I heavy chains (HC) and light chains [?2-microglubulin
toire (8), or directly facilitates peptide loading of class I mole-
The absence of a direct binding assay between Tpn and class
I has hampered a better understanding of the mechanisms of
Tpn’s function. Although cell-based experiments have demon-
strated class I-Tpn binding in the absence of other components
of the class I pathway (5, 12), there are no reports thus far of
direct Tpn–class I-binding interactions outside the context of a
cell. Here, we reconstituted the Tpn–class I interaction by using
purified proteins and investigated the effects of Tpn upon the
class I–peptide interaction, and the effects of peptide upon the
class I–Tpn interaction.
Tpn Can Bind to Peptide-Deficient Class I Molecules at Physiological
from CHO cells (Fig. 6, which is published as supporting
information on the PNAS web site), and soluble peptide-
deficient class I heterodimers were purified from insect cells (13,
14). Fluorescent peptides and native-PAGE-based assembly
assays (13) were used to ask whether sTpn binding influenced
peptide loading of soluble peptide-deficient HLA-A2 (A2) or
HLA-B*3501 (B35). Under the conditions that were examined,
no significant differences in peptide binding were observed in
the presence or absence of sTpn (Fig. 7 A–D, which is published
as supporting information on the PNAS web site).
Absence of a significant effect of sTpn on peptide loading
could arise because of absent or inefficient sTpn–class I-binding
interactions under these in vitro conditions, or because of
enhanced kinetics of class I peptide loading compared with sTpn
binding. Therefore, it was important to investigate binding
interactions between sTpn and A2. Equimolar amounts of A2
heterodimers and sTpn monomers were incubated for 2 h at 4°C
or 37°C. Samples were then immunoprecipitated with the anti-
FLAG antibody, proteins separated by SDS/PAGE, and visual-
ized by silver-staining to observe sTpn and any A2 that bound to
(IPs) after protein incubations at 37°C (Fig. 1A, lane 6), but not
4°C (Fig. 1A, lane 3).
To examine the specificity of the A2–sTpn interaction, we
related to class I. HFE (the protein mutated in hereditary
hemochromatosis) HC folds into a structure similar to class I
HC, and associates with ?2m, but does not bind peptides, and has
a closed peptide-binding groove (15). By IP assays similar to
Author contributions: S.M.R. and M.R. designed research; S.M.R. performed research;
S.M.R. and M.R. analyzed data; and S.M.R. and M.R. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS direct submission.
Abbreviations: HC, heavy chain; ER, endoplasmic reticulum; TAP, transporter associated
with antigen processing; ?2m, ?2-microglubulin; class I, MHC class I; Tpn, tapasin; sTpn,
soluble Tpn; A2, HLA-A2; B35, HLA-B*3501; IP, immunoprecipitation; M10, M10.5.
†To whom correspondence should be addressed at: Department of Microbiology and
Arbor, MI 48109-0620. E-mail: email@example.com.
© 2006 by The National Academy of Sciences of the USA
November 28, 2006 ?
vol. 103 ?
those described for A2, sTpn did not interact with HFE (Fig. 1B,
lane 6). M10.5 (M10), is a pheromone receptor-associated
protein with a class I-like fold. M10 binds ?2m, but the coun-
terpart of its peptide-binding groove is open and unoccupied
(16). Somewhat surprisingly, like A2, M10 bound sTpn at 37°C
(Fig. 1C Left, lane 2), but not at lower temperatures (Fig. 1C
Right, lane 7). Insect cells have previously been used to assess
TAP/Tpn/class I complex formation and to reconstitute class I
peptide loading (5, 17). Additionally, in insect cells, Tpn is the
obligate mediator of the TAP–class I interaction (12). To verify
that sTpn–A2 and sTpn–M10 interactions observed in vitro were
not nonspecific binding interactions induced by protein incuba-
tions at 37°C, we assessed A2 and M10 binding to Tpn in insect
cells. After metabolic labeling of baculovirus-infected cells,
Tpn/A2 and Tpn/M10 complexes could both be visualized by IP
with the anti-His antibody (both A2 and M10 are His tagged)
(Fig. 1D, lanes 5 and 4, respectively). Together, these observa-
tions indicated that Tpn–A2 and Tpn–M10 interactions were not
restricted to the in vitro incubation condition at 37°C, but were
also observable when the proteins were coexpressed in insect
The observation of M10-Tpn binding suggested preferential
Tpn recognition of open and empty class I molecules. This
500 ?M A2-specific peptide LLDVPTAAV (18) at 47°C for 2 h,
to obtain a peptide-loaded version. Unbound peptide was re-
moved from A2 by passage through a Biospin-30 column. Equal
amounts of untreated or peptide-loaded A2 (pA2) were com-
pared for tapasin binding in IP assays. In sTpn/pA2 incubations,
the signal intensity in the region corresponding to A2 HC was
significantly reduced compared with sTpn/untreated A2 incu-
bations (Fig. 1E, lane 6 compared with lane 4, and quantifica-
tions, Right). We also investigated Tpn binding to B35, a class I
allotype that has been described to be Tpn-dependent for its cell
surface expression (19). Soluble B35 was loaded with a specific
high affinity peptide LPSSADVEF (20) at 47°C for 2 h. Equal
amounts of untreated or peptide-loaded B35 heterodimers
(pB35) were incubated with sTpn at 37°C for 2 h, and IP assays
undertaken. Peptide-deficient B35 bound to sTpn, but peptide-
loaded B35 did not (Fig. 1F, lane 3 compared with lane 5).
Together, these analyses indicated that Tpn preferentially rec-
ognized peptide-deficient class I molecules, as well as the empty
and open class I like protein, M10.
When Added in Excess, ?2m Can Be Visualized as a Component of
Tpn–Class I Complexes. After IP of sTpn-A2 mixtures with the
anti-FLAG antibody, it was difficult to detect ?2m by direct
staining of gels (Fig. 2A Upper, lane 2). However, by immuno-
blotting with ?2m-specific antibodies, a faint signal was observed
for ?2m (Fig. 2A Lower, lane 2). We compared ?2m levels in the
sTpn–A2 complexes against different known amounts of puri-
fied heterodimeric A2. Relative to the ?2m/HC ratios in the
heterodimeric A2 samples (expected to be 1:1); a significantly
substoichiometric ?2m/HC ratio was observed in sTpn-
associated A2 HC (Fig. 2A Lower, ?2m blot). To examine
whether the presence of excess ?2m during sTpn–class I complex
formation could enhance the amount of sTpn-A2-associated
complexes are detectable by coimmunoprecipitation analyses. SDS/PAGE and silver staining analyses of direct protein loads of 0.2–0.5 ?g A2 (A, lane 1; B, lane
2), sTpn (B, lane 1, 0.5 ?g), M10 (C, lanes 1 and 5, 0.5 ?g) or HFE (B, lane 3, 1 ?g) and anti-FLAG IP of indicated proteins or buffer that had been incubated at
4°C (A, lanes 2–4), 30°C (C, lanes 6 and 7) or 37°C (A, lanes 5–8; B lanes 4–9; and C, lanes 2–4). (D) A2/Tpn and M10/Tpn complexes are detectable in insect cells.
SDS/PAGE phosphorimaging analyses of indicated samples. Insect cells cultured at 26°C were infected with baculoviruses encoding full length Tpn and indicated
proteins, metabolically labeled, lysed, proteins immunoprecipitated with Tpn-specific antibody (PaSta-1, lane 1) or anti-His to detect His tagged proteins (M10
or A2) (lanes 2–5). (E and F) Peptide loading of class I inhibits sTpn binding. (Left) Direct protein loads of A2 (E, lane 1, 0.25 ?g), LLDVPTAAV-loaded A2 (E, lane
2, 0.25 ?g), or B35 (F, lane 1, 0.5 ?g) and anti-FLAG IP (E, lanes 3–8; F, lanes 2–7) of indicated proteins. pA2 indicates LLDVPTAAV-loaded A2, and pB35 indicates
LPSSADVEF-loaded B35. (Right) Bar graph shows class I/sTpn intensity ratio comparisons for the indicated lanes. Binding of sTpn to peptide-deficient class I was
set at 100% to compare relative signals. Data are representative of numerous (A), two (B, C Right, E, and F) and four (C Left and D) independent analyses.
sTpn–A2 complexes were formed by incubating proteins (5 ?M each) at 37°C
either in the absence (A) or presence (B) of ?2m (50 ?M) and immunoprecipi-
tated with anti-FLAG. (C) sTpn (5 ?M) and ?2m (50 ?M), or ?2m alone were
incubated for 2 h at 37°C, then immunoprecipitated with anti-FLAG. (A–C
Upper) Silver-stained SDS/PAGE gels showing anti-FLAG IP of indicated pro-
teins (A, lane 2; B, lanes 1 and 2; C lanes 1 and 2) or buffer (A, lane 1; C, lane
3) and direct protein loads of indicated concentration of A2 heterodimers (A
and B, lanes 3–8). (A–C Lower) Anti-?2m blot of the above gels. Data are
representative of two independent analyses.
Tpn can form complexes with ?2m at physiological temperature.
Rizvi and RaghavanPNAS ?
November 28, 2006 ?
vol. 103 ?
no. 48 ?
?2m, we incubated A2 and sTpn in the presence of a ten-fold
excess of purified ?2m at 37°C for 2 h, followed by IP with the
anti-FLAG antibody. Indeed, when excess ?2m was present
during complex formation, the amount of sTpn-associated ?2m
was enhanced and equal or higher ?2m/HC ratios were observed
than in the heterodimeric A2 samples (Fig. 2B). This increase in
of sTpn-associated HC.
Direct incubation of sTpn with ?2m alone at 37°C for 2 h (Fig.
2C Upper and Lower, lanes 1) followed by anti-FLAG IP revealed
a signal for ?2m that was slightly above the nonspecific control
(Fig. 2C, lane 1 compared with lane 2; lane 2 was an IP of ?2m
alone with anti-FLAG), suggesting the possibility of a weak
?2m–sTpn interaction. Together, these observations indicate
that the primary site of Tpn-class I contact resides in the HC, but
that ?2m can be detected in complex with Tpn when it is present
in stoichiometric excess.
Tpn-Associated Class I Is Subsequently Assembly Competent. A2–
sTpn complexes were preformed by incubating both proteins (5
?M each) in the presence or absence of excess ?2m (50 ?M) at
37°C for 2 h (in 50 mM Tris/150 mM NaCl, pH 7.5). The
complexes were incubated with anti-FLAG beads overnight at
4°C. Proteins were eluted by using 100 ?M FLAG peptide, and
the eluates were found to contain both Tpn and A2 (Fig. 3A,
lanes 1 and 2).
The amounts of HC in the two anti-FLAG eluates were
estimated by titration against known amounts of A2 het-
erodimers (Fig. 3A Top, lanes 3–6). sTpn-A2 eluates in which
complexes were formed in the absence of excess ?2m contained
?80 ng A2 (Fig. 3A Top, compare lane 1 with lane 5 and 6),
which was reduced to ?40 ng when complexes were assembled
in the presence of excess ?2m (Fig. 3A Top, compare lane 2 with
lane 5 and 6). To assay for relative assembly competence of the
eluted A2, proteins recovered from the anti-FLAG column or
the different amounts of purified A2 heterodimers were incu-
bated with excess ?2m and fluorescently tagged LLDCFPTAAV
at 37°C for indicated time points (Fig. 3A Middle). Peptide
binding was quantified by native-PAGE and fluorimaging anal-
yses (13) (Fig. 3A Bottom). For eluates of the A2–sTpn com-
plexes formed in the presence of excess ?2m, the peptide-binding
signals were only slightly lower than the corresponding signals
obtained with the 40 ng A2 standard (Fig. 3A Middle, compare
lanes 2 and 5). We concluded that majority of this eluate was
assembly competent, with slightly reduced binding kinetics than
that of free class I. For eluates of the A2–sTpn complexes that
were formed in the absence of excess ?2m (complexes that were
depleted in ?2m), the peptide-binding signals obtained were
significantly lower than the corresponding signals obtained with
the 80 ng heterodimeric A2 standard (Fig. 3A Middle, compare
lanes 1 and 6). Thus, ?2m binding to HC may be rate limiting for
assembly of peptide-class I in these ?2m-depleted sTpn–A2
complexes. ?2m is a critical regulator of class I-peptide assembly
rates. Peptide loading of class I heterodimers is enhanced by the
presence of excess ?2m even in the absence of Tpn (Fig. 8, which
is published as supporting information on the PNAS web site).
A parallel set of results were obtained with B35. Under
conditions where sTpn–B35 complexes were formed in the
presence of excess ?2m, the extent of B35-peptide assembly
observed was similar to that observed with free B35 (Fig. 3B,
compare lane 2 with lane 3 and 4). Under conditions where
sTpn–B35 complexes were formed in the absence of excess ?2m,
B35-peptide assembly observed was significantly inhibited rela-
tive to that observed with free heterodimeric B35 (Fig. 3B,
compare lanes 1 and 5).
Tpn Binds Preferentially to Empty Class I Molecules. In the studies
described so far, sTpn–class I complexes were observable at
37°C, but not at lower temperatures (Figs. 1A and 4 A and C).
It was possible that the A2 and B35 purified from insect cells
were not completely empty, and that the 37°C incubations
promoted dissociation of peptides endogenous to insect cells. To
ask whether interactions of sTpn with completely ‘‘empty’’ class
I could occur at lower temperatures, A2 and B35 (100–300 ?g)
were dialyzed against 6 M guanidine hydrochloride (GnHCL) by
using Centricon (10-kDa membranes) to remove any associated
peptide, and proteins refolded by gel filtration chromatography
(Superose 6 column) in the presence of 100–300 ?g of ?2m.
Fractions corresponding to heterodimers were collected and
used directly in peptide-binding assays at room temperature to
compare their assembly competence in the presence or absence
of sTpn. Small enhancements, if any, were observed for peptide
binding to empty A2 and B35 in the presence of sTpn compared
information on the PNAS web site).
petent. (A and B Top) SDS/PAGE and silver-staining analyses of sTpn-A2 (A) or
sTpn-B35 (B) complexes eluted from an anti-FLAG beads. sTpn–class I com-
plexes formed in the absence or presence of excess ?2m are shown in lanes 1
and 2, respectively. Direct protein loads of indicated amounts of class I
standards are shown in lanes 3–6. (A and B Middle) Samples corresponding to
proteins shown in Top were incubated with 1 ?M LLDCFPTAAV (for A2, A) or
LPSCFADVEF (for B35, B) and 1 ?M ?2m at 37°C for 1, 2, or 4 h. Class I–peptide
complexes were separated from free peptide by native-PAGE, and protein–
peptide complexes were visualized by fluorimaging analyses. (A and B Bot-
tom) Peptide-binding signals from Middle were quantified by ImageQuant
and are represented as bar graphs. Data are representative of three indepen-
Class I isolated in complex with Tpn is subsequently assembly com-
SDS/PAGE showing direct protein loads of A2 (A, lane 1, 0.3 ?g), B35 (C, lane
2, 0.2 ?g), or sTpn (A, lane 2, 0.05 ?g; B, lane 1, 0.2 ?g; C, lane 1, 0.1 ?g) or
anti-FLAG IP (A, lanes 3 and 4; B, lanes 2 and 3; C, lanes 3–6) of indicated
proteins (3 ?M each) after incubations at 30°C for 6 h. Empty class I molecules
are represented as eA2 and eB35, and untreated class I molecules are repre-
sented as A2 and B35. Data are representative of two (A and B) or three (C)
Tpn preferentially binds empty class I molecules. (A–C) Silver-stained
www.pnas.org?cgi?doi?10.1073?pnas.0605131103Rizvi and Raghavan
Empty class I molecules were concentrated in the presence of
excess ?2m (300 ?g) and analyzed for binding to sTpn at lower
temperature (Fig. 4). Empty A2 and B35 but not the precursor
untreated proteins were observed to form complexes with sTpn
at 30°C (Fig. 4B, lane 3, and Fig. 4C, lane 6). The ability of
denatured/refolded class I (but not the precursor class I) to bind
binds to a conformation found in empty class I molecules. Empty
A2 and B35 that were complexed to Tpn at 30°C were purified
by using anti-FLAG beads, and proteins eluted with the FLAG
peptide as described in Fig. 3. Eluates containing A2 and B35
were both found to be assembly competent (Fig. 10, which is
published as supporting information on the PNAS web site).
Regulation of the Tpn–Class I Interaction by Peptide and ?2m. In the
binding analyses of Figs. 3 and 10, peptide-loaded class I
molecules migrated at the same position on a native gel regard-
less of whether the protein used in the assembly assays was free
class I or Tpn-class I eluates from the anti-FLAG column. These
results suggested that sTpn had dissociated from the peptide-
loaded class I either as a result of peptide loading, or as a result
of protein dilutions after protein elutions from anti-FLAG
beads. To directly investigate the effect of peptide on disassem-
bly of sTpn-class I, sTpn and A2 were incubated for 2 h in the
absence of excess ?2m after which, the mixture was divided into
three parts. One part was stored on ice and the remaining two
parts were each incubated with buffer or excess ?2m and A2
specific peptides (LLDVPTAAV) at 37°C for an additional 2 h
and IP analyses were undertaken (Fig. 5A). Compared with the
additional incubation with buffer (Fig. 5A, lane 5), and com-
pared with samples that were incubated for just 2 h (Fig. 5A, lane
4), additional incubation with peptide and ?2m resulted in a
reduction in the amount of coimmunoprecipitating A2 (Fig. 5A,
lanes 6). The FLPSDDFPSV peptide that bound to A2 with
2.5-fold higher affinity than LLDVPTAAV (Fig. 11A, which is
published as supporting information on the PNAS web site) also
induced dissociation of A2 from Tpn to similar levels as LLD-
VPTAAV (Fig. 11B). However, parallel analyses revealed more
efficient dissociation of Tpn-B35 induced by the high affinity
peptide LPSSADVEF compared with the lower affinity YPL-
HEQHGM (Fig. 5 B and C, compare lanes 6). LPSSADVEF was
estimated to bind B35 with ?6-fold higher affinity than YPL-
HEQHGM (Fig. 11C).
To further investigate and compare conditions for dissociation
of class I–Tpn, complexes were isolated by using anti-FLAG
beads, and then subject to different elution conditions. The first
set of elutions with the FLAG peptide verified that class I
binding to the anti-FLAG beads was indeed dependent on the
presence of tapasin (Fig. 5 D and E). In the next set of elutions
(Fig. 5F), anti-FLAG beads containing sTpn–class I complexes
formed in the presence of ?2m were incubated at room temper-
ature for 4 h with 0.1 ml buffer containing specific peptides
alone, ?2m and specific peptides, ?2m and nonspecific peptides,
or buffer alone, as indicated. Supernatants were collected and
analyzed for the presence of class I by immunoblotting analyses
with HC-specific antibodies. The analyses showed that specific
peptides alone could elute B35 to some extent, and low levels of
nonspecific peptides eluted more A2 and B35 compared with the
plus specific peptide combination was most efficient at elution of
between elutions with ?2m/nonspecific peptide and ?2m/specific
peptide were more pronounced for A2 than for B35, and
indicated more efficient elution of B35 with ?2m alone.
Anti-FLAG beads containing sTpn–class I complexes formed
in the absence of excess ?2m were incubated at room tempera-
ture for 4 h with 0.1 ml ?2m and specific peptides, ?2m and
nonspecific peptides, ?2m alone, or buffer alone, and the eluate
analyzed for class I by immunoblotting (Fig. 5G). Although some
elution was observed with ?2m alone or ?2m in combination with
nonspecific peptides (Fig. 5G, lanes 1–2 and 5–6), the
?2m?specific peptide combinations were more effective at
dissociating class I from tapasin (Fig. 5G, lanes 3 and 4).
If Tpn associates preferentially with the peptide-free form of
2) or anti-FLAG IP (lanes 3–7). (A and B) Indicated class I and sTpn (5 ?M each)
or class I alone were incubated for 2 h at 37°C (lanes 3 and 4, respectively),
and 500 ?M LLDVPTAAV (LLD) or LPSSADVEF (LPS) (lanes 6). (C) B35 and sTpn
were incubated for 2 h at 37°C (lane 5), followed by additional 2 h incubation
Samples were then processed for IP as described in Experimental Procedures.
(A–C Right) Bar graphs show class I/sTpn intensity ratio comparisons for the
indicated lanes, with the highest ratio in each gel set at 100%. Data in Right
panels are averaged over two independent analyses. (D and E) Class I binding
presence or absence of sTpn (5 ?M) and ?2m (50 ?M) as indicated at 37°C for
4 h. Proteins isolated with anti-FLAG beads were eluted with 100 ?M FLAG
peptide and analyzed by immunoblotting with anti-His (D; A2) or HC10 (E;
B35). (F and G) Class I–sTpn complexes were formed as described in D and E in
the presence (F) or absence (G) of excess ?2m, isolated with anti-FLAG beads,
and class I was eluted with combinations of specific peptide (S), nonspecific
These data are representative of three and four independent analyses, re-
spectively, for F and G.
Dissociation of sTpn–A2 and sTpn–B35 complexes by peptides and
Rizvi and Raghavan PNAS ?
November 28, 2006 ?
vol. 103 ?
no. 48 ?
class I, the expectation is that different peptides would inhibit
Tpn–class I complex formation to an extent that is determined
by the intrinsic affinity of the particular peptide–class I inter-
action. Indeed, this was the observed result. When present
during sTpn–class I complex formation, LLDVPTAAV was a
better inhibitor of the sTpn–class I interaction than the lower
as supporting information on the PNAS web site; see Fig. 7 for
comparisons of binding of the two peptides). Likewise for B35
peptides, the higher affinity LPSCFADVEF peptide was a better
inhibitor of the sTpn–class I interaction than the lower affinity
YPLHEQHGM peptide (Fig. 12 B and C).
Our data suggest two types of Tpn–class I complexes: those
containing predominantly HC and those containing both HC
and ?2m. ?2m seems to destabilize Tpn–HC interactions, as
indicated by the observations of inhibited HC-Tpn binding in the
presence of excess ?2m (Fig. 3), as well as ?2m-induced elution
of class I HC from Tpn (Fig. 5 F and G). Excess ?2m may simply
reduce the exposure of Tpn-binding residues by stabilization of
heterodimeric HC–?2m interactions (in the same manner that
peptides do), rendering HC–Tpn interaction weaker, but also
more dynamic and assembly competent (compared with the
HC–Tpn interaction formed in the absence of excess ?2m; Fig.
3). The observations that B35 was more easily dissociated by
?2m/peptide combinations and ?2m alone compared with A2
(Fig. 5) is reminiscent of previous findings that HLA-B*3501/
TAP interactions were not detectable in cell lysates under
conditions where A2/TAP interactions were readily detectable
(21). The stabilities of peptide-deficient forms of class I allotypes
may be variable, which in turn could be reflected in the extent
of steady-state Tpn/TAP binding. Additionally, for some class I
molecules such as A2, assembly of HC/?2m/peptide trimers may
be a cooperative event, whereas the heterodimeric intermediates
may be more stable for other class I allotypes.
That ?2m destabilizes HC–Tpn interactions may seem sur-
prising in light of reports that Tpn–HC interactions were signif-
icantly reduced in ?2m-deficient cells compared with ?2m-
sufficient cells (22). However, in cells, the absence of ?2m
markedly decreases levels of HLA class I HC, making it difficult
to directly quantify relative propensities of free HC and ?2m-
associated HC for Tpn binding (as was the case in insect cells; C.
Perria and M.R., unpublished observations). Alternatively/
additionally, in cells, in the absence of ?2m, free HC may be
sequestered from Tpn binding by other chaperones such as
Cell surface expression of many class I allotypes is markedly
increased by the presence of Tpn, and Tpn increases MHC class I
stability (11). How might the data described here explain these
assembly-promoting effects of Tpn? Our studies indicate prefer-
ential recognition of empty class I by Tpn. Furthermore, sampling
of the peptide environment by the MHC class I seems to be
permissive under conditions where Tpn binding was observed (Fig.
5), either because MHC class I is in dynamic equilibrium between
Tpn-free and Tpn-associated conformations, or because a Tpn-
loading. Higher affinity peptides were better inducers of Tpn-class
I dissociation (Fig. 5 B compared with C) and stronger inhibitors of
the Tpn class I binding (Fig. 12). It seems likely based on these data
that the presence of Tpn could allow for a more stringent class
I-peptide affinity checkpoint, consistent with models of Tpn-
mediated editing/optimization (8). Only peptides that are able to
with class I on the surface of Tpn-sufficient cells. Zarling et al. (11)
have suggested a role for Tpn as a peptide loading facilitator rather
than editor. This type of activity might have been reflected by an
increase in peptide loading by class I in Tpn-associated complexes,
but our studies revealed very small effects if any (Fig. 10). Addi-
tional conditions may need to be explored, varying protein con-
centrations and including Tpn-associated factors. Compared with
the in vitro experiments shown here, within the ER, assembly and
degradation of peptide-deficient class I are likely to be strongly
recruits ERp57 into the class I assembly complex (23), and it is well
known that ERp57 is found in physical association with the lectin
chaperones calreticulin/calnexin. Thus, the presence of Tpn may
serve to efficiently recruit a chaperone network around the assem-
interactions within the assembly complex are all cooperative to
some extent (reviewed in ref. 1). Assembly within this protective
chaperone network environment may be more highly favored than
outside this environment, where proteases and ER degradation
pathways could compete more effectively with assembly of class I
I, Tpn could also actively enhance dissociation of low affinity
peptides and promote peptide exchange, as previously observed
for HLA-DM in the MHC class II antigen presentation pathway
(reviewed in ref. 24). A detailed analysis of this possibility will
be important by using real time measurements of class I-peptide
dissociation and exchange in the presence and absence of Tpn.
Although our data indicate preferential recognition of empty
class I by Tpn (Fig. 4), Tpn may not be essential for ER retention
MHC class I molecules seem to be retained efficiently in the ER
even in the absence of Tpn (25). However, Tpn seems to be quite
important for ER retention of murine MHC class I (5–7). In the
absence of Tpn, increased ER escape and cell surface expression
of empty and suboptimally loaded class I could also, at least in
part explain the reduction in MHC class I surface expression in
some Tpn-deficient cells.
The interaction of Tpn with M10 was initially surprising in
light of the report that TAP is not expressed in cells that express
M10 (26), but subsequent comparison of the sequences of M10
and class I molecules revealed 40%, 40%, and 74% sequence
identity in the ?1, ?2, and ?3 domains, respectively. The extent
of sequence identity between HFE and class I was considerably
less significant (31%, 25%, and 41% sequence identity in the ?1,
?2, and ?3 domains, respectively). Thus, whereas Tpn may not
be a bona fide assembly factor for M10 in cells, the significant
sequence identity between class I and M10 HC, taken together
with its ‘‘open and empty groove’’ structure may promote
Tpn-M10 cross-reactivity in vitro (Fig. 1C), or under conditions
of coexpression (Fig. 1D). Residues that have been implicated in
29) in the ?2 domain of human class I. These residues are
conserved between A2 and M10, but not between HFE and A2,
supporting the possibility that this region forms a contact site for
Tpn binding. Also conserved in M10 are D227 and E229 in the
?3 domain, additional residues implicated in Tpn binding by
human MHC class I (27). It will be important to investigate
whether mutations within both the ?2 and ?3 domains disrupt
or destabilize Tpn-MHC class I binding interactions by the assay
A significant finding of these studies is that conditions that
promoted heterodimer assembly induced dissociation of MHC
class I-Tpn. Other investigators have reported difficulties in
demonstrating peptide-induced dissociation of class I from the
loading complex (30). Our data indicate that both ?2m (Figs. 3
and 5) and the type of peptide used (Fig. 5) are likely to be
important determinants of the extent of dissociation of class I
from a Tpn-associated complex. Furthermore, as illustrated in
Fig. 3, the specific conditions under which peptide loading
www.pnas.org?cgi?doi?10.1073?pnas.0605131103 Rizvi and Raghavan
complexes were isolated could determine the assembly kinetics
of Tpn-associated MHC class I molecules.
In summary, our studies demonstrate that Tpn binds to
peptide-deficient class I, that physiological temperatures pro-
mote binding, and that conditions favorable for class I assembly
dissociate Tpn–class I complexes. In cells, Tpn interaction with
network comprising Tpn, ERp57, and the lectin chaperones,
which together, may create an environment that favors peptide
loading and assembly over degradation.
Proteins and Peptides. Soluble A2/?2m, B35/?2m, and full-length
Tpn expression in insect cells has been previously described (13,
14, 31). Expression and purification of soluble tapasin and ?2m
and detection of class I/Tpn complexes in insect cells are
described in Supporting Materials and Methods, which is pub-
lished as supporting information on the PNAS web site. Fluo-
rescently labeled versions of class I-binding peptides were ob-
tained by cysteine substitutions at positions 4 or 5 of the parent
sequences, and labeling with iodoacetamidofluorescein, as pre-
viously described (13, 14). Purified human HFE (15), mouse
M10.5 (16), and a baculovirus encoding mouse M10.5/human
?2m (16) were obtained from the laboratory of P. J. Bjorkman
(California Institute of Technology, Pasadena, CA).
Detection of Tpn–Class I Complexes Using Purified Proteins. Soluble
class I, HFE, or M10 (2.5–5 ?M each) was incubated with sTpn
(2.5–5 ?M) in 50 mM Tris, 150 mM NaCl, pH 7.5, at indicated
temperatures for indicated time. After incubations, the samples
were centrifuged, diluted in the same buffer containing 1% Triton-
immunoprecipitation) overnight at 4°C. The supernatants were
further centrifuged and incubated for 2–4 h at 4°C with protein G
beads. The beads were washed three times with buffer containing
0.25% gelatin (10 mM Tris/10 mM phosphate buffer/130 mM
NaCl/1% Triton X-100, pH 7.5) and once with buffer without
gelatin. The beads were boiled in SDS/PAGE buffer, samples were
resolved by SDS/PAGE, and immunoprecipitated proteins were
visualized by silver staining. For ?2m detection, the anti-FLAG
immunoprecipitated samples or direct proteins loads were trans-
ferred to PVDF membrane, immunoblotted with anti-?2m antisera
(Roche, Nutley, NJ), HRP-conjugated secondary antibody, and
developed by ECL plus kit (Amersham Biosciences, Piscataway,
Isolation of Tpn–Class I Complexes with Anti-FLAG Beads. sTpn–class
I complexes were formed by incubation of proteins (5 ?M) in the
presence or absence of 50 ?M ?2m in a total volume of 40 ?l at
37°C for 2–4 h. Proteins were then diluted to 1 ml buffer (50 mM
Tris/150 mM NaCl/1% Triton X-100), incubated overnight at
4°C with 100-?l FLAG-beads, loaded onto an empty column,
and washed with 5–15 ml buffer to remove free class I. For some
analyses, beads were eluted with FLAG peptide (100 ?M in 100
?l). For other analyses, beads were then incubated with 100 ?l
of buffer (50 mM Tris/150 mM NaCl) containing 50 ?M ?2m, 50
?M ?2m plus 500 ?M specific or nonspecific peptides, or buffer
alone at room temperature for 4 h. Specific peptide used for A2
was FLPSDDFPSV and for B35 was LPSSADVEF and nonspe-
cific peptide for both was RRYQKSTEL. After incubations,
supernatants were collected and analyzed by SDS/PAGE and
immunoblotting analyses with anti-His (for A2) or HC10 (for
We thank Chris Perria for conducting the experiment shown in Fig. 1D;
Dr. Peter Cresswell (Yale University School of Medicine, New Haven,
of Technology, Pasadena, CA) for M10 and HFE constructs; the
University of Michigan Biomedical Research Core Facilities for DNA
sequencing and peptide syntheses and purification; the Hybridoma Core
for ascites production; and the Rheumatic Diseases Core Center and the
Michigan Diabetes Research and Training Centers for financial support.
This work was supported by National Institutes of Health Grant AI-
44115 and a Cancer Research Institute Investigator Award (to M.R.).
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Rizvi and Raghavan PNAS ?
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no. 48 ?