Structure and Dynamics of Membrane-associated
ICP47, a Viral Inhibitor of the MHC I
Antigen-processing Machinery

Christopher Aisenbrey, Christina Sizun, Joachim Koch, Meike Herget, Rupert Abele, Burkhard Bechinger, Robert Tampé

► To cite this version:

Christopher Aisenbrey, Christina Sizun, Joachim Koch, Meike Herget, Rupert Abele, et al.. Structure and Dynamics of Membrane-associated ICP47, a Viral Inhibitor of the MHC I Antigen-processing Machinery. Journal of Biological Chemistry, 2006, 281 (41), pp.30365-30372. 10.1074/jbc.M603000200 .
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# Structure and Dynamics of Membrane-associated ICP47, a Viral Inhibitor of the MHC I Antigen-processing Machinery*

Received for publication, March 29, 2006, and in revised form, June 26, 2006 Published, JBC Papers in Press, July 11, 2006, DOI 10.1074/jbc.M603000200

Christopher Aisenbrey<sup>±51</sup>, Christina Sizun<sup>§</sup>, Joachim Koch<sup>¶</sup>, Meike Herget<sup>¶</sup>, Rupert Abele<sup>¶</sup>, Burkhard Bechinger<sup>±52</sup>, and Robert Tampé<sup>¶3</sup>

From the <sup>‡</sup>Institut/Faculté de Chimie, Université Louis Pasteur/CNRS LC3-Unité Mixte de Recherche 7177, 4 Rue Blaise Pascal, Strasbourg 67070, France, <sup>§</sup>Max-Planck-Institute of Biochemistry, Am Klopferspitz 18A, 82152 Martinsried, Germany, and <sup>¶</sup>Institute of Biochemistry, Biocenter, Johann Wolfgang Goethe-University, Max-von-Laue-Strasse 9, 60438 Frankfurt am Main, Germany

To evade the host's immune response, herpes simplex virus employs the immediate early gene product ICP47 (IE12) to suppress antigen presentation to cytotoxic T-lymphocytes by inhibition of the ATP-binding cassette transporter associated with antigen processing (TAP). ICP47 is a membrane-associated protein adopting an α-helical conformation. Its active domain was mapped to residues 3–34 and shown to encode all functional properties of the full-length protein. The active domain of ICP47 was reconstituted into oriented phospholipid bilayers and studied by proton-decoupled <sup>15</sup>N and <sup>2</sup>H solid-state NMR spectroscopy. In phospholipid bilayers, the protein adopts a helix-loop-helix structure, where the average tilt angle of the helices relative to the membrane surface is ~15° (±7°). The alignment of both structured domains exhibits a mosaic spread of ~10°. A flexible dynamic loop encompassing residues 17 and 18 separates the two helices. Refinement of the experimental data indicates that helix 1 inserts more deeply into the membrane. These novel insights into the structure of ICP47 represent an important step toward a molecular understanding of the immune evasion mechanism of herpes simplex virus and are instrumental for the design of new therapeutics.

Survival of vertebrates is strongly dependent on the adaptive immune system, which confers protection against pathogens or cancer. On the cellular level, the major histocompatibility complex (MHC)<fnref n="4" /> class I-dependent pathway of antigen processing allows for presentation of antigenic peptides at the cell surface and can trigger the elimination of virus-infected or malignantly transformed cells by cytotoxic T lymphocytes (1–3). The efficiency of antigen presentation depends on the transporter associated with antigen processing (TAP), a member of the ATP-

binding cassette protein family that translocates peptides generated by proteasomal protein degradation into the endoplasmic reticulum for loading onto MHC class I molecules (4). This process requires a macromolecular peptide-loading complex of ~1 MDa comprising the transporter subunits TAP1 and TAP2, the MHC class I heavy chain, the non-covalently associated β₂-microglobulin, the adaptor protein tapasin, and several auxiliary factors (5–9).

Herpes simplex virus type 1 is a highly abundant human pathogen that achieves lifelong persistence in the ganglia of the nervous system. Upon exogenous stimuli, it can be repeatedly reactivated and infect related mucosal tissues leading to clinical symptoms (10). To escape immune surveillance, herpes simplex virus compromises the host's cytotoxic T lymphocyte response via ICP47, an 88-amino-acid immediate early gene product (IE12) that blocks TAP function (11–15). In the absence of a functional TAP transporter (within 3 h of infection with herpes simplex virus), peptide loading onto MHC class I molecules is inhibited, and as a consequence, empty MHC I molecules are retained in the endoplasmic reticulum and ultimately directed to proteasomal degradation. By binding with nanomolar affinity to the heterodimeric TAP complex, ICP47 blocks peptide binding but not ATP binding to the ATP-binding cassette transporter (12, 15). Functional studies with N- and C-terminally truncated variants of ICP47 demonstrate that the N-terminal domain of ICP47 is sufficient for TAP inhibition (residues 1–53 (16), 2–35 (17), and 3–34 (18)). Moreover, by alanine scanning mutagenesis, three regions (residues 8–12, 17–24, and 28–31) were identified within the active domain of ICP47, which are critical for TAP inhibition (17). The active domain of ICP47 (residues 3–34) displays an identical ability to inhibit TAP function when compared with the full-length protein, illustrating preservation of the functional properties (18).

The secondary structure and membrane association of ICP47 have been analyzed by circular dichroism spectroscopy and tryptophan fluorescence quenching (16). Both, full-length ICP47 as well as the active domain are mainly unstructured in aqueous solution independent of the protein concentration or pH value (16), which is in accordance with the observation that purification in organic solvent or heating to 100 °C has no effect on its ability to inhibit TAP (15, 18). However, after binding to phospholipid membranes, a transition to helical conformations is induced in the active domain as well as in the full-length protein, confirming a tight membrane interaction (16). Multidimensional solution NMR spectroscopy indicates that the

<footnote>* This work was supported by the Agence Nationale pour la Recherche contre le Syndrome d'Immunodéficience Acquise and the Deutsche Forschungs-gemeinschaft (SFB 628). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.</footnote>

<footnote n="1">¹ Present address: Dept. of Biophysical Chemistry, Umeå University, 90187 Umeå, Sweden.</footnote>

<footnote n="2">² To whom correspondence may be addressed. Fax: 33-390-24-51-51; E-mail: bechinger@chimie.u-strasbg.fr.</footnote>

<footnote n="3">³ To whom correspondence may be addressed. Fax: 49-69-798-29495; E-mail: tampe@em.uni-frankfurt.de.</footnote>

<footnote n="4">⁴ The abbreviations used are: MHC, major histocompatibility complex; ICP, infected cell protein; TAP, transporter associated with antigen processing; ppm, parts/million.</footnote>
Structure and Dynamics of Membrane-bound ICP47

active domain of ICP47 adopts a helix-loop-helix conformation
in the presence of detergent micelles, where helix 1 comprises
residues 4–15 and helix 2 residues 22–32. However, it should
be noted that considerable differences have been observed in
the past when the structural details of polypeptides obtained in
the presence of detergents or when associated with bilayers are
compared with each other. For example, the possibility exists
that the helix-turn-helix structure observed in micelles is an arti-
fact of the geometry and the high curvature of such environments.
Furthermore, it would be of interest to know how the helical
domains align relative to the bilayer surface. The answer to such
questions can only be obtained by investigating the structure of
ICP47 when associated with planar phospholipid bilayers.

Solid-state NMR spectroscopy is an emerging technique in
the study of the structure and dynamics of membrane-associ-
ated proteins and peptides (19–22). Although a priori NMR
interactions strongly depend on the alignment of the molecules
relative to the magnetic field direction, in isotropic solutions,
fast rotational diffusion results in averaging, and therefore only
the isotropic chemical shift values as well as direct spin-spin
couplings are observed. In contrast, in static solid samples or in
samples where averaging is anisotropic, the most pronounced
features of the spectra can be attributed to this orientational
dependence of NMR interactions. When uniaxially oriented
samples are investigated by static solid-state NMR spectros-
copy, advantage is taken of this orientation dependence of
NMR interactions, which allows extraction of angular con-
straints for structural analysis. Proton-decoupled ¹⁵N solid-
state NMR spectroscopy has been shown to be particularly use-
ful in providing detailed information on the helical tilt angles
and therefore the topology of membrane-associated polypep-
tides in a direct manner (23). In the case of polypeptides that
have been isotopically labeled with ¹⁵N at one of their amide
bonds and reconstituted into bilayers oriented with their nor-
mal parallel to the magnetic field direction, ¹⁵N chemical shifts
in the 200-ppm range are indicative of transmembrane helix
alignments, whereas in-plane-oriented helices resonate at
<100 ppm (24). This is illustrated in the top panel of Fig. 2.

Deuterium solid-state NMR spectroscopy provides comple-
mentary angular restraints, which in combination with the ¹⁵N
chemical shift allow for the accurate determination of the tilt
and azimuthal angles of membrane-associated helices (24). The
deuterium quadrupole splitting of alanines carrying three deu-
terons at the methyl group has been shown to be a very sensitive
indicator of helix alignments, which permits the tracking of
even small topological changes (24). Furthermore, the ²H NMR
technique has recently been developed to monitor the mosaic
spread of helix alignments and the rotational diffusion of mem-
brane polypeptides (24, 25).

Here ²H and proton-decoupled ¹⁵N solid-state NMR spec-
troscopy have been combined to investigate in considerable
detail the alignment, limits, and mosaic spread of the secondary
structural elements of reconstituted ICP47 in oriented phos-
pholipid bilayers. By positioning at strategic places eight differ-
ent isotopic labels within the ICP47 sequence, it was not only
possible to test for the existence of a helix-loop-helix confor-
mation also in phospholipid bilayers, but even more important,
the alignment of the helical domains relative to the membrane

surface could be determined. Furthermore, a novel Edmundson
helical wheel-based “energy minimization” strategy was devel-
oped for this study to select, among a number of topological
states, the one that agrees with the oriented solid-state NMR
data. The structural and topological information derived from
these measurements provide detailed insights into the inhibi-
tion mechanism of ICP47.

EXPERIMENTAL PROCEDURES

*Peptide Transport Assays*—Assays were performed with microsomes isolated from Raji cells (human Burkitt lymphoma). Microsomes (50 µg of total protein) were pre-incubated with 10 µM ICP47-(2–34) for 20 min on ice. After the addition of MgATP (3 mM) or apyrase (1 unit) and radiolabeled peptide (460 nM, RRYQNSTEL; N-core glycosylation-targeting site underlined), transport was performed for 2 min at 32 °C. The reaction was stopped by the addition of 1 ml of cold phosphate-buffered saline buffer supplemented with 10 mM EDTA. After centrifugation, microsomes were solubilized in 1 ml of lysis buffer (50 mM Tris/HCl, 150 mM NaCl, 5 mM KCl, 1 mM CaCl₂, 1 mM MnCl₂, 1% Nonidet P-40, pH 7.5) for 20 min on ice. Insoluble debris was removed by centrifugation, and glycosylated peptides in the supernatant were bound to concanavalin A-Sepharose beads for 16 h at 4 °C. After washing three times with lysis buffer, radioactivity associated with the beads was quantified by gamma counting (26). Peptides were radiolabeled with Na¹²⁵I as reported previously (27).

ICP47 Binding Assays—TAP-containing membranes or purified TAP were incubated with increasing concentrations of ICP47-(2–34) for 60 min at 4 °C in 50 µl of binding buffer A (20 mM Hepes, 140 mM NaCl, 15% glycerol, pH 7.4) or binding buffer B (20 mM Hepes, 140 mM NaCl, 15% glycerol, 0.1% digitonin, pH 7.4), respectively. Radiolabeled peptide RR(¹²⁵I)YQKSTEL was added to a final concentration of 200 nM and incubated for 15 min at 4 °C. Unbound peptides were removed by washing the membranes or purified protein with 200 µl of ice-cold binding buffers using a vacuum manifold with 96-well filter plates (MultiScreen, 1-µm glass fiber Type B filter, Millipore). The amount of peptide bound to the filters was quantified by gamma counting. Background binding was determined in the presence of a 200-fold excess of unlabeled peptide RRYQKSTEL. The binding constant of ICP47-(2–34) was derived from the inhibition data by using heterologous competition with ligand depletion as the fitting procedure with the program Prism® (GraphPad Software, Inc.). The affinity of RRYQKSTEL was set to 30 nM as determined by equilibrium binding; the specific activity of the peptide was 40 counts/min/fmol, and the 200 nM radiolabeled peptide in 50 µl of reaction volume had an activity of 400,000 counts/min.

*Peptide Synthesis and Isotope Labeling*—ICP47-(2–34), Ac-SWALEMADTFLDNMRVGPRTYADVRDEINKRGR-NH₂, was synthesized by solid-phase synthesis using Fmoc (N-(9-fluorenyl)methoxycarbonyl) chemistry. At the underlined positions, isotope-labeled amino acids were incorporated in the following manner. In a first set, polypeptides labeled at a single site with ¹⁵N at either position Ala⁸ or Ala²³ were prepared. To test in more detail the alignment of the second helix encompassing residues 22–31 (28), the functionally active mutant D27A (18)
was labeled with [¹⁵N]alanine at the modified position. In a second set, ICP47 analogues that are simultaneously enriched at the backbone with ¹⁵N and with a [²H₃]alanyl residue were prepared. The double labeling patterns were [¹⁵N]Gly¹⁸/ [²H₃]Ala²⁷ (D27A) and [¹⁵N]Val¹⁷/[²H₃]Ala⁸. The synthetic products were purified by a C₁₈ reversed-phase high pressure liquid chromatography column (Vydac). The identity of the products was confirmed by analytical high pressure liquid chromatography and matrix-assisted laser desorption ionization mass spectrometry.

*NMR Sample Preparation*—Phospholipids were purchased from Avanti Polar Lipids (Birmingham, AL). 10 mg of peptide and 200 mg of 1-palmitoyl-2-oleoyl-*sn*-glycero-3-phosphatidyl choline (or 1-palmitoyl-2-oleoyl-*sn*-glycero-3-phosphatidylcholine/1-palmitoyl-2-oleoyl-*sn*-glycero-3-phosphatidylserine 3:1 mol/mol; spectra not shown) were co-dissolved in 2.5 ml of trifluoroethanol and 0.2 ml of water. The mixtures were applied onto 30 ultrathin cover glasses (9 × 22 mm; Paul Marienfeld GmbH & Co. KG, Lauda-Königshofen, Germany) first dried in air and thereafter in high vacuum overnight. After equilibration at 93% relative humidity, the glass plates were stacked on top of each other. The stacks were stabilized and sealed with Teflon tape and plastic wrappings. The sample alignment was routinely controlled using proton-decoupled ³¹P solid-state NMR spectra as described previously (24, 29).

*NMR Measurements*—Solid-state NMR spectra were recorded on a Bruker Avance wide bore NMR spectrometer operating at 9.4 teslas. A commercial double-resonance solid-state NMR probe modified with flattened coils of dimensions 15 × 4 × 9 mm was used (30). The spectra of samples rotated by 90° relative to the magnetic field directions were acquired using a second coil of similar geometry. Proton-decoupled ¹⁵N solid-state NMR spectra were acquired using cross-polarization (31, 32). Typical acquisition parameters were the following: spin lock time 1.6 ms, recycle delay 3 s, ¹H B₁ field 31 kHz, 256 data points, 20,000–40,000 acquisitions, and spectral width 40 kHz. Before Fourier transformation, an exponential apodization function corresponding to a line broadening of 300 Hz was applied. NH₄Cl (41.5 ppm) was used as a reference corresponding to ~0 ppm for liquid NH₃.

Deuterium solid-state NMR spectra were recorded using a quadrupolar echo pulse sequence (33) with the following parameters: ²H B₁ field 50 kHz, inter-pulse delay 50 µs, spectral width 100 kHz, 4096 data points, 40,000 scans, and a repetition time of 1.5 s. The spectra were referenced relative to ²H₂O (0 ppm). An exponential apodization function corresponding to a line broadening of 300 Hz was applied before Fourier transformation.

*Calculation of Orientational Restraints*—To evaluate the peptide orientations, a Cartesian coordinate system was defined. The long axis of the α-helix defines the z-axis, and the y-axis resides within the plane dividing hydrophobic and hydrophilic sections of amphipathic helices, such as those found in the ICP47 structure in micellar environments (28). The computer program Insight II (BIOSYM, Molecular Simulations, San Diego, CA) was used to generate Protein Data Bank (PDB) data sets of the oriented helices. The PDB structural data file was used to extract the coordinates of the labeled ¹⁵N atoms and its neighbors (NH and C atom of the previous amino acid).

<figure><img src="image_2.png" /></figure>

<figure><img src="image_3.png" /></figure>

**FIGURE 1.** Viral inhibition of TAP-dependent peptide binding and transport. A, microsomes isolated from Raji cells were used in transport assays with radiolabeled peptide (RRYQNSTEL) in the presence of ATP, apyrase, or ATP and a 20-fold molar excess of non-labeled and [¹⁵N]Val¹⁷/[²H₃]Ala⁸-labeled ICP47-(2-34), respectively. Peptide translocation was performed for 2 min at 32 °C. Transported peptides were quantified by gamma counting in triplicate experiments. B, the binding affinity of the active domain of ICP47 (ICP47-(2-34)) to TAP within the membrane (open circles) or purified, digitonin-solubilized TAP (closed circles) derived from insect cells was determined in competition experiments with a radiolabeled peptide epitope. TAP was pre-incubated with increasing concentrations of ICP47-(2-34) for 60 min at 4 °C. Subsequently, binding was assayed by adding 200 nM radiolabeled peptide (RRYQKSTEL) for 15 min at 4 °C. The amount of peptide bound to the TAP complex was quantified by gamma counting in triplicate measurements. cpm, counts/min.

The coordinates of the labeled peptide bonds were needed to calculate the ¹⁵N chemical shift tensor in this reference frame using information on ¹⁵N chemical shift tensors reported in the literature (34), where the main tensor elements exhibit values of 223, 75 and 61 ppm. By successively rotating the peptide molecule around the z- (pitch angle) and the y-axis (tilt angle) the three-dimensional orientational space was systematically screened in 50 × 50 steps using a program written in MATH-EMATICA (24). Contour plots mark the angular pairs that agree with the experimental results. The simulation of the ²H solid-state NMR spectra was performed on the same principles and has been described previously (24).

<figure><img src="image_4.png" /></figure>
<figure><img src="image_5.png" /></figure>

<figure><img src="image_6.png" /><figcaption>FIGURE 3. ²H solid-state NMR spectra of ICP47-(2–34) labeled with ²H₃ at position Ala⁸ (A) or Ala²⁷ (B). The samples are aligned with the membrane normal parallel to the magnetic field direction. Underneath the spectra are simulations of the deuterium line shapes resulting from alignments of the C₂–C₃ bonds relative to the magnetic field direction of 45 or 55° for Ala⁸ and 62° for Ala²⁷, respectively. The mosaic spread is ±10° for both sites.</figcaption></figure>

Similarly, the coordinates of the Cα atoms were extracted from oriented PDB files. From these coordinates, the center of the mass of the Cα atoms for each of the respective helices 5–16 and 22–31 was calculated to define the center of a new coordinate system. The coordinates thus established were also used for an analysis of the positioning of amino acid residues relative to the hydrophobic-hydrophilic interface. During these calculations, the insertion depth dins was simulated by a corresponding shift of the z coordinates. The z coordinates were then scaled with the following factor.

$$C_z = 0.5 - \frac{1}{1 + 10^{2-15.75}} \quad (\text{Eq. 1})$$

In this manner, it was possible to model a membrane of 27 Å hydrophobic thickness and an interface extending over 4–5 Å (35). The values thus obtained are multiplied with the hydrophobic index of the individual amino acid side chains (36) and summed up to provide a total index for the hydrophobic effect Ihydro that is associated with the peptide alignment and insertion depth. The graphs (see Fig. 4, A and B, and Fig. 5) represent the configurations of penetration depth, tilt, and pitch angle where Ihydro is minimal.

<figure><img src="image_7.png" /></figure>
<figure><img src="image_8.png" /></figure>

FIGURE 4. Shown are the contour plots of the tilt and rotation pitch angles of helices 1 (A) and 2 (B). The contours are calculated from the orientation-dependent $^{15}$N chemical shift and $^{2}$H quadrupolar splitting data of Ala$^{8}$ (A) as well as the Ala$^{23}$ and Ala$^{27}$ positions (B). The lines in dark blue represent angular restraints obtained from the $^{15}$N chemical shift measurements (±5 ppm) of position 8 and 27, and the green lines represent that of position 23. Angular restraints obtained from $^{2}$H NMR measurements are shown in light blue (Ala$^{8}$, 55 ± 10°; and Ala$^{27}$, 62 ± 10°) (Fig. 3). The red circles are indicative of the tilt and rotational pitch angles that result from a three-dimensional hydrophobic index analysis (see "Experimental Procedures" for details). For a bilayer of 31.5 Å hydrophobic thickness and an interface of 4.5 Å, the positioning is optimized when the distance from the membrane interior to the center of the mass of helix 1 is 15.5 Å and that of helix 2 is 17.5 Å. C, model illustrating the tilt angle $\delta$ and the rotational pitch angle $\gamma$.

## RESULTS AND DISCUSSION

Solution NMR investigations have shown that the active domain of ICP47 adopts α-helical secondary structures encompassing residues 4–15 and 22–32 in the presence of SDS micelles (28). To investigate for the first time the structure of this potent immune evasin in the bilayer-associated state, ICP47-(2–34) was synthesized by solid-phase chemistry, and $^{15}$N and $^{2}$H isotopic labels were incorporated at single sites.

The isotope-labeled active domain of ICP47-(2–34) ($[^{5}N]Val^{17}/[^{2}H_3]Ala^{8}$) was first examined for its ability to inhibit TAP-dependent peptide translocation in microsomes isolated from human Burkitt lymphoma (Raji) cells. As expected, the isotope-labeled active domain of ICP47 displayed the same ability to inhibit TAP when compared with its non-labeled counterpart, demonstrating that the labeling had no adverse effects on the properties of the domain (Fig. 1A).

<figure><img src="image_9.png" /></figure>

FIGURE 5. Edmundson helical wheel diagrams of ICP47 helices. Residues 5–16 (helix 1) and 22–31 (helix 2) are shown in their classical representation. The side view projections indicate the distribution of hydrophobic and hydrophilic residues when the helices are inclined relative to the membrane surface (dotted lines). The coordinate system used during analysis of solid-state NMR spectra is indicated *next to* helix 1.

We next examined whether the active domain of ICP47 requires a lipid environment to interact efficiently with the TAP complex. We thus performed competition binding experiments with purified, digitonin-solubilized TAP or membrane-bound TAP employing the active domain of ICP47 and the radiolabeled peptide RR($^{125}$I)YQKSTEL. Solubilized TAP, as well as TAP within the membrane, displays the same affinity ($K_D$ = 30 nM).<fnref n="5" /> The TAP samples were pre-incubated with graded amounts of ICP47-(2–34) and subsequently probed with radiolabeled peptide. Peptides bound to TAP were quantified by $\gamma$-counting. Strikingly, the inhibition constant ($K_i$) of ICP47 for the solubilized TAP complex ($K_i$ = 5093 nM) is 65 fold higher than that for TAP within the membrane ($K_i$ = 79 nM) (Fig. 1B). These results demonstrate that ICP47 requires a lipid environment to bind with high affinity to the TAP heterodimer. Notably, the amount of functional heterodimeric TAP complexes was identical in both sets of experiments as indicated by the same peptide binding value in the absence of ICP47 (Fig. 1B, see converging intersection).

To determine the structure of the active domain of ICP47 in the membrane-bound state, isotopically labeled ICP47-(2–34) was reconstituted into oriented phospholipid bilayers and investigated by $^{2}$H- and proton-decoupled $^{15}$N solid-state NMR spectroscopy. The macroscopic phase properties and the membrane alignment were routinely controlled by proton-decoupled $^{31}$P solid-state NMR spectroscopy on the same samples (data not shown). Fig. 2 shows the proton-decoupled $^{15}$N solid-state NMR spectra of ICP47-(2–34) in oriented phospholipid bilayers. The Ala$^{8}$, Ala$^{23}$, and Ala$^{27}$ positions all show chemical shift values between 85 and 89 ppm (Fig. 2; A, D, and E) indicative of helix alignments approximately parallel to the mem-

<figure><img src="image_10.png" /></figure>

<footnote n="5">⁵ M. Herget, R. Abele, and R. Tampé, unpublished data.</footnote>
Structure and Dynamics of Membrane-bound ICP47

<figure><img src="image_11.png" /><figcaption>FIGURE 6. Model of the active domain of ICP47 in phospholipid bilayers. A, the bilayer normal (arrow) and the tilt angle δ for helix 2 are indicated. MOLMOL software was used to prepare this figure (38). B, after binding to the cytosolic face of the endoplasmic reticular (ER) membrane, ICP47 adopts a helix-loop-helix conformation. Subsequent association with the peptide-loading complex at the lipid-TAP interface blocks the peptide supply to MHC class I molecules. The peptide-loading complex consists of the ATP-binding cassette half-transporter subunits TAP1 and TAP2, the adaptor protein tapasin, the MHC class I heavy chain (hc), the non-covalently associated β₂-microglobulin (β₂m), and several auxiliary factors (e.g. calnexin and the thiol-oxidoreductase ERp57) (8).</figcaption></figure>

brane surface (23). In contrast, the [15N]Val¹⁷ position exhibits a well defined ¹⁵N chemical shift of 108 ppm, a value close to the isotropic chemical shift position (i.e. indicative of a lack of orientation; Fig. 2B).

To investigate the orientation and dynamics of this helix in further detail, the same sample was rotated by 90° (25). In this arrangement, the long axis of an in-plane-oriented α-helix can adopt all angles between 0 and 360° relative to the magnetic field direction. *A priori*, the ¹⁵N chemical shift is therefore expected to vary over a wide range within the limits of 60 and 230 ppm. However, in the case of fast rotational diffusion around the membrane normal, the observed ¹⁵N chemical shift exhibits a mean value corresponding to σ⊥ of the averaged chemical shift tensor (23, 25). The value observed for the Val¹⁷ position was 138 ppm (Fig. 2F) and different from the ¹⁵N chemical shift observed at alignments of the sample normal

parallel to the magnetic field direction (Fig. 2B). Therefore, the ensemble of ¹⁵N chemical shift data indicates a significant amount of non-isotropic averaging at this site. Relatively weak signal intensities were obtained for the Gly¹⁸ position at ~117 ± 5 ppm (Fig. 2C). In the presence of fast motions, the concomitant reduction of the active ¹H-¹⁵N dipolar interactions reduces the efficiency of the cross-polarization technique, which allows for the transfer of magnetization from the abundant ¹H to the less sensitive ¹⁵N nuclei. As a result, weak ¹⁵N signal intensities close to the isotropic values were observed for these mobile sites. The solid-state NMR data thereby reflect well the structure observed in micellar environments, where Ala⁸ is part of the first helix followed by a flexible loop region encompassing Val¹⁷ and Gly¹⁸ and a second helix including Ala²³ and Glu/Ala²⁷. However, when the chemical shift positions are analyzed in more detail, it becomes obvious that, in lipid bilayers, the Val¹⁷ site is less flexible when compared with Gly¹⁸. Collectively, the solid-state NMR data confirm a helix-loop-helix conformation, where the helices are aligned approximately parallel to the membrane surface.

Additional deuterium NMR spectra were obtained from the ²H₃-labeled alanines at positions 8 or 27 (Fig. 3). It has been shown previously that the deuterium NMR quadrupole splitting is very sensitive to the alignment

of the Cα–Cβ vector relative to the magnetic field direction (24). The data therefore provide additional orientational restraints (Fig. 4). Furthermore, the deuterium NMR line shape is sensitive to the orientational mosaic spread of helix domains. By comparison to theoretical line shapes, a mosaic spread of the deuterium-labeled alanine Cα–Cβ²H₃ bond of 10° is estimated, indicating that the peptide helices exhibit a considerable degree of motion that results in a broadened distribution of tilt and rotational pitch angles. A quantitative evaluation of the ¹⁵N chemical shift data indicates a tilt angle in the range between 64 and 88° for helix 1 encompassing Ala⁸ (Fig. 4A). The ²H data provide valuable information not only about the helix mosaic spread (see above), but the resulting line shape also limits the range of possible helix alignments relative to the membrane normal (Fig. 3A). The combination of both data sets therefore confines the helix alignment to six regions on the tilt/pitch angle topological map shown in Fig. 4A.
When the data of helix 2 are analyzed in a quantitative manner, the orientational restraints of positions 23 and 27 overlap, thereby defining four regions where the orientational pairs (tilt/ pitch angle) agree with both ¹⁵N chemical shift measurements. These are located around 63/166, 106/118, 74/295, and 117/ 348° (Fig. 4B). Of those pairs, the first and last as well as the second and third are symmetry-related, corresponding to heli- ces associated with either the top or the bottom monolayer. Of those results, only the second and third angular pair also fit the ²H solid-state NMR data.

In a next step, we refined these results by evaluating the ener-
gies associated with the insertion of the helices in the hydro-
phobic-hydrophilic environment of the membrane interface
using an extension of the Edmundson helical wheel analysis.
The helical wheel diagram allows one to view the amphipathic
distribution in the projection of the helix long axis and thus to
evaluate the rotational pitch angle and the penetration depth of
a given helix. In this work, a third dimension representing the
peptide tilt angle was introduced for the analysis. Such an
energy minimization protocol indicates a tilt angle of 84° and a
rotational pitch angle of 106° for helix 1 (Fig. 4A) in proximity of
the region around 75/72°, which was identified by our experi-
mental data. The small deviation is probably a result of the
calculations not taking into account atomic models of phos-
pholipids or peptide conformational flexibility. When the same
analysis is performed with the structural coordinates of helix 2,
which have previously been obtained from the NMR structural
analysis of ICP47 in detergent micelles and deposited in the
PDB data base (28), the most favorable alignment is 95/102°
(Fig. 4B). This topological coordinate corresponds closely to the
second angular pair identified experimentally (106°/118°). The
data therefore indicate that the average tilt angle of the helices
deviates from perfect in-plane alignments by ~15° (±7°). These
findings are represented by helix projection diagrams in Fig. 5.
Notably, the calculations also suggest that the penetration
depth of the two helices, when considering their centers of
mass, is different by 2 Å.

In agreement with these solid-state NMR data, several
lines of biochemical evidence demonstrate that this α-helical
conformation of ICP47 in the membrane-bound state is a
prerequisite for TAP inhibition. First, truncation of the helix
1 leads to a complete inactivation of the inhibitor and failure
to induce an α-helical conformation (16). These data show
that key sequence elements in the active domain are essential
for both membrane association and TAP inhibition. Second,
insertion of a proline at position 11 breaking helix 1 causes a
drastic loss of activity, illustrating the functional impact of
the formation of an α-helical conformation (18). Third, flu-
orescence quenching experiments suggest that the single
tryptophan located at position 3 is in contact with the lipid
environment, thus demonstrating that the active domain of
ICP47 interacts with the membrane (16). Most importantly,
within the current study, we have demonstrated, based on
competition binding experiments, that a lipid environment
is a prerequisite for high affinity binding of ICP47 to the
heterodimeric TAP complex (Fig. 1B). Without a membrane,
the binding affinity of ICP47 was reduced by almost two
orders of magnitude.

A structural model summarizing the data discussed above is shown in Fig. 6. The solid-state NMR data reveal a flexible loop region separating two helical domains, which exhibit small deviations from perfect in-plane alignments and considerable mosaic spread. Theoretical considerations suggest that helix 1 inserts more deeply into the hydrophobic interior of the membrane (Fig. 6A). This first structural model of ICP47 in its bilayer-associated state provides important insights into the structure and topology of membrane-bound ICP47, which are instructive in understanding how and where ICP47 binds to the heterodimeric TAP complex. Based on the data presented, the active domain interacts with TAP at the subunit-membrane interface within the lipid head group region and blocks peptide translocation into the endoplasmic reticular lumen (Fig. 6B). Previous studies have demonstrated that both TAP subunits are required for ICP47 binding (12, 15, 26). In combination with similar observations for the partitioning at various peptide hormone receptors (37), the N-terminal region, with particular emphasis on helix 1 of ICP47, might serve as a membrane anchor to locally concentrate the inhibitor at the TAP complex, thus increasing the efficiency of the inhibition process.

In the membrane-bound state, ICP47 escapes proteasomal
degradation, which otherwise occurs rapidly in the membrane-
free state.<fnref n="6" /> The presented structural model of ICP47 corre-
sponds to a conformation, which is adopted just before or con-
currently with the functional association with the TAP
heterodimer, circumventing a disfavored soluble intermediate.
This represents an elegant and so far unforeseen strategy of
peptidic effectors and unstructured regions in avoiding degra-
dation by the proteasome.

Acknowledgments—We thank Dr. Karl-Heinz Wiesmüller (EMC microcollections, Tübingen, Germany) for support in peptide synthesis and Dr. Clemens Glaubitz for helpful comments on the manuscript.

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