Structural and functional characterization of HIV-1 cell fusion inhibitor T20 : AIDS

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Structural and functional characterization of HIV-1 cell fusion inhibitor T20

Zhang, Xiujuana,b,*; Ding, Xiaohuia,*; Zhu, Yuanmeia; Chong, Huihuia; Cui, Shenga; He, Jinshengb; Wang, Xinquanc; He, Yuxiana,b

Author Information
doi: 10.1097/QAD.0000000000001979

Abstract

Introduction

In the early 1990s, several synthetic peptides derived from the N-terminal and C-terminal heptad repeat regions (NHR and CHR) of HIV-1 transmembrane glycoprotein gp41 were serendipitously discovered with potent antiviral activity [1–3], which triggered considerable efforts to explore the mechanisms of virus-mediated membrane fusion and to develop antiviral drugs that block viral entry process. As the second milestone, the core structure of gp41 was determined with the NHR and CHR peptides, which demonstrated a stable 6-helix bundle (6-HB) formed by three central NHR helices and three CHR helices packed antiparallelly [4–6]. Thus, it is believed that sequential binding of the surface subunit gp120 to cell receptor CD4+ and a chemokine receptor (CCR5 or CXCR4) induces a cascade of conformational changes in viral envelope (Env) complex, resulting in the N-terminal fusion peptide of gp41 being released and inserted into the cell membrane; subsequently, three CHR fold antiparallelly onto the trimeric coiled coil of the NHR to adopt a 6-HB structure, which pulls two membranes in close apposition required for fusion reaction. Peptides derived from the NHR or CHR can bind to the prehairpin intermediate of gp41 to block 6-HB formation, thereby inhibiting viral entry in a dominant negative manner.

The third milestone is that the peptide drug T20 (enfuvirtide) was approved in 2003 for clinical use as the first member of a new class of anti-HIV drugs: HIV entry inhibitors [7,8]. T20 is effective as a salvage therapy for HIV/AIDS patients who have failed to respond to antiretroviral therapeutics that include reverse transcriptase inhibitors (RTIs) and protease inhibitors, but the emergence of drug resistance has significantly limited its application [9–12]. In the last decade, there has been tremendous works paid to develop new fusion inhibitor peptides against HIV-1 and many other enveloped viruses; but unfortunately, T20 remains the only membrane fusion inhibitor available for treatment of viral infection. Actually, we are still confused by the mechanism of action of T20, as exemplified by its target sites being suggested on the NHR helices, the CHR helices, the fusion peptide, and the transmembrane domain (TMD) of gp41 [1,2,13,14] and the coreceptor binding site of gp120 [14–16]. Furthermore, a crystal structure specific for T20 alone or in complex with a target mimic sequence has never been determined. Therefore, we have dedicated our efforts to characterize the structural properties of T20 with aims to understand its mode of action and to develop novel HIV-1 fusion inhibitor peptides with improved pharmaceutical profiles.

Materials and methods

Peptide synthesis

NHR-derived (N39 and its mutants, N39ΔFPPR, N36) and CHR-derived (T20 and its mutants, T20ΔTRM, C34) peptides were synthesized on rink amide 4-methylbenzhydrylamine (MBHA) resin by using a standard solid-phase 9-flurorenylmethoxycarbonyl (Fmoc) method as described previously [17]. All the peptides were purified by reverse-phase high-performance liquid chromatography (HPLC) to more than 95% homogeneity and characterized by mass spectrometry. Concentrations of the peptides were measured by ultraviolet (UV) absorbance and a theoretically calculated molar extinction coefficient based on the tryptophan and tyrosine residues.

Assembly and crystallization of the T20/N39 complex

The 6-HBs were assembled by dissolving equal amounts (1 : 1 molecular ratio) of the peptides (T20 and N39) in denaturing buffer (100 mmol/l NaH2PO4; 10 mmol/l Tris–HCl, pH 8.0; and 8 mol/l urea). To refold the peptides, the mixture was dialyzed against buffer containing 50 mmol/l Tris–HCl (pH 7.5) and 100 mmol/l NaCl at 4 °C overnight. The dialyzed sample was concentrated by centrifugation and then subjected to the size-exclusion chromatography (Superdex 75 10/300 GL, GE Healthcare China, Beijing, China). Elutions corresponding to the molecular weight of a 6-HB were collected and concentrated prior to the crystallization trials. The T20/N39 complex was crystallized by mixing equal volumes (0.2 μl) of purified sample (∼10 mg/ml) and the reservoir solution in a sitting drop vapor diffusion system at 18 °C. The cryocooling for the crystals was achieved by soaking the crystal for 5 s in the reservoir solution containing 30% (v/v) glycerol, followed by flash freezing to 100 K in liquid nitrogen. All data sets were collected on beamline BL17U at the Shanghai Synchrotron Research Facility (SSRF) and processed with XDS. All data collection and processing statistics were listed in Table S1, https://links.lww.com/QAD/B336.

Structural determination and refinement

The crystal structure of T20/N39 was solved by molecular replacement with the crystallographic software PHASER [18]. The searching model was the crystal structure of LP-40 (PDB ID: 5Y14). The iterative refinement with the program PHENIX [19] and model building with the program COOT [20] were performed to complete the structure refinement. Structure validation was performed with the program PROCHECK [21], and all structural figures were generated with PyMOL [22].

Circular dichroism spectroscopy

A CHR-derived peptide was incubated with an equal molar concentration of an NHR-derived peptide at 37 °C for 30 min in PBS (pH 7.2). Circular dichroism spectra were acquired on a Jasco spectropolarimeter (model J-815) using a 1-nm bandwidth with a 1-nm step resolution from 195 to 260 nm. The α-helical content was calculated from the circular dichroism signal by dividing the mean residue ellipticity (θ) at 222 nm by the value expected for 100% helix formation (−33 000 deg cm2/dmol). Thermal denaturation was performed by monitoring the ellipticity change at 222 nm from 20 to 98 °C at a rate of 1.2 °C/min as described previously [23].

Native PAGE

Native PAGE (N-PAGE) was performed to determine the interactions between the NHR-derived and CHR-derived peptides, as described previously [17,24]. Briefly, a CHR peptide was mixed with an NHR peptide at a final concentration of 40 μmol/l and incubated at 37 °C for 30 min. The mixture was added with Tris–glycine native sample buffer at a ratio of 1 : 1 and then loaded onto a 10 × 1.0-mm Tris–glycine gel (20%) at 25 μl/per well. Gel electrophoresis was carried out with 100 V constant voltage at 4 °C for 3 h. The gel was then stained with Coomassie blue and imaged with a Bio-Rad imaging system (Bio-Rad, Hercules, California, USA).

Isothermal titration calorimetry

To characterize the interactions between the NHR-derived and CHR-derived peptides, isothermal titration calorimetry (ITC) assay was performed using an ITC200 Microcalorimeter instrument (MicroCal, Northampton, Massachusetts, USA) as described previously [25]. In brief, an NHR-derived peptide (N36, N39 or N39ΔFPPR) was dissolved in ddH2O at 1 mmol/l and injected into a chamber containing a CHR-derived peptide (C34, T20 or T20ΔTRM) at 100 μmol/l. The time between injections was 240 s, and the stirring speed was 500 rpm. The experiments were performed at 25 °C. Data acquisition and analysis were performed using the MicroCal Origin software (version 7.0).

Results

General features of T20/N39-based six-helical bundle structure

To anatomize the mechanism of action of T20, we assembled and crystallized the complex of T20 and N39, an NHR-derived target mimic peptide. Two peptides were equally dissolved in denaturing buffer, and the mixture was dialyzed to allow refolding of the peptides. The T20/N39 complex was purified by size-exclusion chromatography and then crystallized in commercial kits. The crystal of the T20/N39 complex belonged to the space group of P1211, contained three pairs of T20/N39 peptides (a complete 6-HB) per asymmetric unit, and diffracted X-ray to a resolution limit of 2.3 Å (Table S1, https://links.lww.com/QAD/B336). We could build most of the residues of T20/N39 peptides in the electronic density map with final sequences were as follows: from Thr-25, Val-28 or Thr-27 to Leu-55 on the three chains of N39, respectively; and from Tyr-127 to Trp-155, Leu-158 or Trp-159 on the three chains of T20 inhibitors, respectively.

As anticipated, the complex of T20 and N39 formed a typical 6-HB structure similar to many other gp41 core structures (Fig. 2). Three N39 helices formed an interior, trimeric coiled coil with three conserved, hydrophobic grooves, and three T20 inhibitor helices packed into each of the grooves antiparallelly in a left-handed direction. Different from the N36/C34 structure that has been considered a core 6-HB of gp41, it is observed that both the N-terminal segment of N39 and the C-terminal segment of T20 are highly splayed, resulting in incompact structure that disfavors their interactions through classic coiled coils. As a consequence, we failed to build the entire residues of tryptophan-rich motif (TRM) at the C-terminal of T20 and fusion peptide proximal region (FPPR) at the N-terminal of N39 because of a low-electronic density map.

F1-1
Fig. 2:
Crystal structure of T20/N39-based six-helical bundle.(a) A ribbon model of T20/N39 structure (left). Three N39 peptides are colored in grey and three T20 inhibitors are colored in cyan. The TRM in T20 is colored purple and marked, and the FPPR in N39 is colored orange and marked. Residues involving in hydrogen bonds on the T20 helix are shown as stick models with labels (right). Hydrogen bonds are indicated in dashed lines. The interactions in the N-terminal (b) and the C-terminal (c) of T20 are presented in ribbon models. Residues related to hydrophobic interactions are shown as stick models and marked in green. An acetyl group is colored purple and marked. The hydrogen bonds between residues are indicated in black dashed lines. The TRM sequences are colored in purple and the FPPR sequences are colored in orange. FPPR, fusion peptide-proximal region; 6-HB, six-helical bundle; TRM, tryptophan-rich motif.

Identification of intrahelical and interhelical interactions critical for the binding of T20

We identified four intrahelical hydrogen bonds that greatly stabilized the α-helices of T20 (Fig. 2a). In the middle site of T20, the Nε2 atom of Gln-141 donates a hydrogen bond to the Nζ atom of Lys-144, and simultaneously, the Oε1 atom of Gln-141 accepts a hydrogen bond from the Oδ1 atom of Asn-145, and the Nε2 atom of Gln-142 donates a hydrogen bond to the Oε2 atom of Glu-146. In the C-terminal TRM of T20, the Nζ atom of long side chain of Lys-154 donates a hydrogen bond to the Oγ atom of Ser-157.

The structure has verified that both the N-terminal and C-terminal of T20 are critical for its binding stability. In the N-terminal as shown in Fig. 2b, the first residue Tyr-127 not only interacts with His-53 on N39 by a hydrogen bond but also contacts with Leu-54 by hydrophobic force; the fifth residue Ile-131 of T20 also targets Leu-54, which is a key residue to form the deep gp41 pocket. Interestingly, the carbonyl group of the introduced acetyl group accepts hydrogen bonds from the NH group of Thr-128, Ser-129 and Leu-130, respectively, which stabilize the N-terminal of T20 and its binding with the target site. In addition, there are abundant hydrophobic interactions between Leu-130 and Ala-50, Ile-131 and Ala-50, Leu-134 and Ala-47, Ile-135 and Leu-45, which strengthen the N-terminal binding of T20. Near the C-terminal of the inhibitor (Fig. 2c), Leu-149 has hydrophobic interaction with Leu-33 and Leu-34 simultaneously, and notably, Trp-155 and Ala-156 mediate hydrophobic contacts with Leu-26, which locate at the TRM of T20 and the FPPR of N39, respectively.

We also identified plenty of interhelical hydrogen bonds, which form complicated networks thus markedly stabilizing the binding of T20. As shown in Fig. 3a–b, the Oε2 atom of Glu-137 forms a hydrogen bond with the Oδ1 atom of Asn-43; the O atom of Ser-138 accepts a hydrogen bond from the Nε2 atom of Gln-40; the Nε2 atom of Gln-139 donates a hydrogen bond to the Oε1 atom of Glu-49. Interestingly, the Oε1 atom of Gln-141, the O atom of Gly-36, the Nδ2 atom of Asn-145 and the N atom of Ile-37 link together through hydrogen bonds, whereas the Nδ2 atom of Asn-145 simultaneously donates a hydrogen bond to the Nε2 atom of Gln-41; the Oε1 atom and Nε2 atom of Gln-142 form hydrogen bonds with the Nε2 atom of Gln-40, the N atom of Asn-42, and the O atom of Val-38, respectively; the Oε1 atom of Glu-148 form hydrogen bonds with the O atom of Gln-32 and the N atom of Leu-33, respectively. Near the C-terminal of the inhibitor, the O atom of Leu-152 accepts a hydrogen bond from the Nη2 atom of Arg-31, whereas the negatively charged carboxyl group of Asp-153 attracts the positively charged amidogen group of Arg-31 to form a salt bridge.

F2-1
Fig. 3:
Identification of interhelical interactions critical for the binding of T20.(a) Ribbon models of T20/N39-based six-helical bundle (6-HB) structure. The N39 trimer is colored in grey and T20 inhibitors are colored in cyan. The residues involving hydrogen bonds between T20 and N39 are shown as stick models with labels. Hydrogen bonds and salt brides are indicated in dashed lines. (b) Sequence illustration of T20 binding. A single T20 peptide interacting with two N39 helices is shown in a sequence map. The dashed black lines indicate the interhelical hydrogen bonds and the dashed red line indicates a salt bridge.

The tryptophan-rich motif and fusion peptide-proximal region motifs are essential for the formation of T20/N39-based six-helical bundle structure

As illustrated in a hairpin model (Fig. 1), the C-terminal TRM of T20 is positioned to match the fusion peptide-proximal region (FPPR) of gp41; however, the crystal structure of T20 bound to N39 could not finely identify the interactions between the TRM and FPPR motifs because of the low electronic density. Thus, we decided to apply multiple biophysical and functional approaches to clarify the functions of the TRM and FPPR motifs. To this end, two truncated peptides, N39ΔFPPR and T20ΔTRM, were synthesized by deleting the TRM sequence from T20 and the FPPR sequence from N39, respectively. First, we used circular dichroism spectroscopy to compare the helical interactions of the peptide pairs N36/C34, N39/T20, and N39ΔFPPR/T20ΔTRM (Fig. 4a–b). As expected, an equimolar mixture of N36 and C34, which represent the core sequences of gp41 6-HB, displayed a typical secondary structure with an α-helical content of 98% and a thermal unfolding transition (Tm) value of 64 °C. In sharp contrasts, the N39/T20 complex only showed an α-helical content of 50% and a Tm value of 42 °C, and deletion of the TRM and FPPR motifs fully abolished the interactions between N39 and T20.

F3-1
Fig. 1:
Schematic illustration of HIV-1 gp41 and its peptide derivatives.(a) The functional domains of gp41. The gp41 numbering of HIV-1HXB2 is used. (b) A hairpin model illustrating the interactions between the NHR and CHR of gp41 and their peptide derivatives. The dashed lines between the NHR and CHR indicate the interaction between the residues located at the ‘e,’ ‘g’ and ‘a,’ ‘d’ positions in the NHR and CHR sequences, respectively. The FPPR and TRM sequence are respectively marked in orange and purple; the pocket-forming sequence and pocket-binding domain are respectively marked in blue and red. CHR, C-terminal heptad repeat; CT, cytoplasmic tail; FP, fusion peptide; NHR, N-terminal heptad repeat; TM, transmembrane domain; TRM, tryptophan-rich motif.
F4-1
Fig. 4:
Biophysical interactions of N-terminal heptad repeat-derived and C-terminal heptad repeat-derived peptides.(a) The α-helicity and thermostability of T20 and control peptides complexed with the NHR-derived counterpart peptides were determined by circular dichroism (CD) spectroscopy. The final concentration of each peptide was 10 μmol/l. (b) Visualization of the interactions between the NHR-derived and CHR-derived peptides by native PAGE. Each of the peptides was used at a final concentration of 40 μmol/l. The positively charged NHR-derived peptides N36, N39, and N39ΔFPPR migrated up and off the gel thus, no bands appeared, whereas the CHR-derived peptides C34, T20, and T20ΔTRM gave specific bands because of net negative charges. The N36/C34 and N39/T20 complexes displayed specific bands corresponding to the six-helical bundle (6-HBs), whereas N39ΔFPPR and T20ΔTRM failed to form a complex in the gel. The experiments were repeated at least two times, and representative data are shown. CHR, C-terminal heptad repeat; NHR, N-terminal heptad repeat.

We also used a N-PAGE-based method to visualize the interactions between the peptide pairs. As shown in Fig. 4c, the positively charged peptides N36, N39, and N39ΔFPPR might migrate up and off the gel thus no bands appeared, whereas C34, T20, and T20ΔTRM gave specific bands because of their properties with net negative charges. Although the N36/C34 and N39/T20 complexes displayed the specific bands corresponding to the 6-HB conformation, N39ΔFPPR and T20ΔTRM failed to form a complex in the gel. Taken together, the data demonstrated that the TRM and FPPR motifs play essential roles in the formation of T20/N39-based 6-HB structure.

The interactions between tryptophan-rich motif and fusion peptide proximal region are critical for the binding and inhibitory activities of T20

As the circular dichroism spectroscopy determined the α-helical content and thermostability of the preformed peptide complexes rather than their instantaneous interactions, we further used ITC to compare the thermodynamic profiles of the molecular interactions between three peptide pairs, in which the released or absorbed heat during the interaction allowed an accurate measurement of the binding constant (K), reaction stoichiometry (N), enthalpy (ΔH) and entropy (ΔS). As shown in Fig. 5, both C34 and T20 interacted with their counterpart peptides (N36 and N39) in a typical enthalpy-driven reaction in which a large amount of heat was released. Very surprisingly, although the reaction of N36/C34 showed a K value of 4.6 × 105/mol/l, the reaction of N39/T20 exhibited a K value of 1.9 × 106/mol/l, which indicated a 4.1-fold increase for the interacting affinity of N39/T20 over that of N36/C34; however, N39ΔFPPR and T20ΔTRM exhibited a dramatically decreased interaction, resulting in the reaction parameters could not be precisely defined. Therefore, the measurement suggested that the TRM and FPPR motifs might mediate interactions with higher affinity over that by the pocket sites.

F5-1
Fig. 5:
Binding affinities of N-terminal heptad repeat-derived and C-terminal heptad repeat-derived peptides.Thermodynamic profiles of the molecular interactions between N36 and C34 (a), N39 and T20 (b), and N39ΔFPPR and T20ΔTRM (c) were determined by ITC technology. The titration traces are shown at the top, and the binding affinities are shown at the bottom. The experiments were repeated two times, and representative data are shown. CHR, C-terminal heptad repeat; ITC, isothermal titration calorimetry; NHR, N-terminal heptad repeat.

To identify the residues critical for the TRM–FPPR interactions, we synthesized a large panel of N39 and T20 mutant peptides, which carry a single mutation in the FPPR or TRM (Fig. S1, https://links.lww.com/QAD/B336). The circular dichroism spectroscopy was used to examine the effects of the mutations on the α-helicity and thermostability of the N39/T20 complex. As shown in Table S2, https://links.lww.com/QAD/B336, the A22G and T27A mutations in the FPPR abolished the interaction between N39 and T20, whereas the L26A mutation in the FPPR and the A156G, W159A, and W161A mutations in the TRM could significantly reduce the thermostability of the complexes. In contrasts, the FPPR's T25A and Q29A mutations resulted in increased α-helical contents and Tm values, suggesting that they might enhance the binding stability of T20. Furthermore, we determined the effects of the mutations on the antiviral activity of T20. As shown in Table S3, https://links.lww.com/QAD/B336, T20W155A showed significantly decreased activities in inhibiting HIV-1NL-4-3 Env-mediated cell fusion and viral entry, whereas T20W159A, T20W161A, and T20F162A showed significantly decreased inhibitory activities on both HIV-1NL-4-3 and HIV-1SF162, suggesting the importance of the mutated amino acids for T20. It is conceivable that combinations of the two or more mutations would lead to more dramatic changes in the binding and inhibition of T20.

Structural insights into the mechanism of T20-induced resistance

Previous studies identified a panel of T20-induced resistance mutations on the inhibitor-binding site of gp41 NHR, including G36D, G36V, I37T, V38A, V38E, Q40H, N43K, N43D, and L45M. Herein, we attempted to elucidate the molecular mechanism of T20 resistance based on the crystal structure of T20/N39 complex. As shown in Fig. S2a, https://links.lww.com/QAD/B336, Gly-36 is located at the interface between N39 and T20 helices, and it mediates huge contacts with Gln-141, Lys-144, and Asn-145 on T20. Substitutions of Gly-36 by a negatively charged residue (Asp-36) or a hydrophobic residue (Val-36) might introduce ‘large amino acid-mediated steric obstruction’ or ‘basic amino acid-mediated electrostatic attraction’ as described by Eggink et al.[26]. As shown in Fig. S2b, https://links.lww.com/QAD/B336, three Ile-37 residues are located at the center of three-fold symmetry axis of the trimeric N39 coiled coil, where the electron densities are well defined and the side chains of Ile-37 approach each other closely by hydrophobic force forming ‘a hydrophobic core’ in the center of 6-HB. Substitution of Ile-37 by a polar residue threonine can break the hydrophobic core thereby destabilizing the structure of trimeric N39 helices and afterwards decreasing the binding efficacy of T20. Three Val-38 side chains point to the hydrophobic region composed of Leu-149, Glu-146, and Asn-145 on T20. Substitution of Val-38 by a less hydrophobic residue alanine or a charged acidic residue glutamic acid apparently reduces the hydrophobic contacts or introduces ‘acidic amino acid-mediated electrostatic repulsion.’

As shown in Fig. S2c, https://links.lww.com/QAD/B336, Gln-40 and Asn-43 are deeply buried in the 6-HB, where the electron densities involve a group of highly conserved residues forming a hydrogen bond network that mediates the extensive interactions between N39 and T20 helices. Specifically, the Nδ2 atom of Asn-43 and Nε2 atom of Gln-141 on T20 helices coordinate a water molecule stabilizing the interaction between T20 and N39 helices. The Nε2 atom of Gln-40 donates a hydrogen bond to the O atom of Ser-138 and Oε1 atom of Gln-142, respectively. The Oε1 atom of Gln-142 also forms a hydrogen bond with a water molecule enhancing the flexibility of the hydrogen bond network. Moreover, the Oε1 atom of Gln-40 also accepts a hydrogen bond from the Nδ2 atom of Asn-43 further fortifies the stability of the 6-HB structure. Therefore, as a member of the remarkable glutamine-rich polar layer, Gln-40 plays a very important role for the stability of 6-HB structure. Substitution of Gln-40 by histidine can increase steric obstruction and destroy the nearby hydrogen bond network of the layer, thus resulting in loosening of N39 helices itself and diminishing the binding of NHR and CHR. Similarly, the side chain of Asn-43 also contributes to keep the stability of hydrogen bond network. Substitution of Asn-43 by lysine would generate a steric obstruction in the core of 6-HB leading to the disruption of the hydrogen bond network. Alternatively, substitution of Asn-43 by a negatively charged residue (Asp-43) not only interferes with the hydrogen bond network but also increases the electrostatic repulsion with the negatively charged residue Glu-137, thus leading to the instability of 6-HB. As shown in Fig. S2d, https://links.lww.com/QAD/B336, Leu-45 is a member of another nonpolar layer in the 6-HB structure of T20/N39, and its side chain faces the hydrophobic region composed of Ser-138 and Gln-139 on T20. Substitution of Leu-45 by the less hydrophobic residue methionine would not only increase steric obstruction but also reduces the hydrophobic interactions between T20 and N39 helices.

Discussion

In this study, we dedicated our efforts to understand the structural basis of T20. The crystal structure of T20 complexed with the target mimic peptide N39 was determined, which revealed the intrahelical and interhelical interactions underlying the mechanism of action of T20. Specifically, the structure identified multiple intrahelical hydrogen bonds that greatly stabilized the α-helices of T20, and plenty of interhelical hydrogen bonds that form complicated networks thus markedly stabilizing the binding of T20. Both the N-terminal and C-terminal of T20 were verified to be critical for the binding stability of the inhibitor through extensive hydrophobic interactions and hydrogen bonds. By applying circular dichroism spectroscopy, native PAGE and ITC-based biophysical approaches as well as mutational analysis, we demonstrated that the interactions between the TRM motif of T20 and the FPPR motif of N39 play essential roles for the binding affinity of the inhibitor and for the formation of 6-HB structure between T20 and N39. On the basis of structural information of T20/N39, we also analyzed the mechanism of T20-induced resistance.

Discovery of T20 did trigger tremendous efforts to explore the mechanism of viral membrane fusion and to develop antiviral therapeutics. In the early stage, T20 was focused in two diverged directions: the development as a new class of antiviral drug and the research towards understanding its mechanism of action. Although it was successfully licensed a decade ago, the inhibitory model of T20 remains elusive [27]. First, it is widely thought that T20 targets the NHR helices to prevent the formation of 6-HB structure, similar to other CHR-derived peptides [1,28]. Second, T20 may inhibit the self-interaction of the N-terminus of gp41while it is extended in the early fusion steps and prevent the interaction of the membrane proximal external region (MPER) with the N-terminus of gp41 in the late fusion steps [2,29,30]. Third, it was proposed that T20 may have two binding sites with different binding affinities; the lower affinity site is the NHR, but the higher affinity site is still unknown [13,31,32]. Although T20 may bind to a parallel endogenous region to prevent aggregation of several Env trimers needed to enlarge the fusion pore [13], it may also target the TMD of gp41 as its anti-HIV activity could be blocked by TMD-derived peptides [14]. More impressively, several studies suggested that T20 may also target the sites on gp120 [33–36]. As T20 could block the interaction of gp120-CD4+ complexes with the CXCR4 co-receptor and peptides derived from the co-receptor binding site of gp120 blocked the inhibitory activity of T20 competitively, thus the co-receptor binding site on gp120 has been considered its molecular target [14–16]. Lastly, a body of evidence shows that T20 can bind to the target cell membrane through its C-terminal TRM, which critically determines its inhibitory activity [37–40]. Therefore, these data highlight that T20 may act with a multifaceted mechanism that needs to be further characterized. More impressively, a crystal structure of T20 in the absence or presence of a target surrogate had never been determined, even if it was discovered over 20 years. Indeed, we experienced huge difficulties to assembly the T20/N39 complex and to grow the crystals, possibly because of their unstable features in the denatured buffer and/or crystallization conditions. Finally, we were somewhat lucky to obtain the only T20/N39 crystal applicable for the determination of structure. For the first time, we described the intrahelical and interhelical interactions underlying the mechanism of action of T20 and its resistance mutations.

Differing from a number of C34-based fusion inhibitor peptides such as T2635, SC34EK, and sifuvirtide [41–43], T20 has no pocket-binding domain at its N-terminus but it is characterized by a C-terminal 8-amino acid TRM sequence [14,39,40,44]. Currently, it is considered that the TRM serves as a ‘lipid-binding domain,’ which can anchor the inhibitor to the target cell membrane. In agreement with this conclusion, the substitution of the TRM by fatty acids could largely recover or improve the antiviral activity of T20 [39,45]. However, it is unclear whether the TRM of T20 interacts with the FPPR in the upstream of gp41 NHR thus determining the antiviral activity of T20. Although the earlier gp41 structures were mainly determined by using N36/C34 peptides that contained the pocket-forming site and pocket-binding domain but missed the FPPR and TRM motifs, a new crystal structure based on a recombinant gp41 protein that included both fusion peptide and membrane proximal external regions was described [46]. The structure revealed that the TRM and FPPR motifs lacked regular coiled coil interactions because of their splayed helices; instead, hydrophobic contacts, such as Ala-22/Trp-159 (Ala-533/TRp-670), Met-24/Asn-160 (Met-535/Asn-671), Thr-25/Leu-26/Trp-155 (Thr-536/Leu-537/Trp-666), and one hydrogen bond between the carbonyl of Ala-22 and NE1 of Trp-159 could stabilize the TRM–FPPR interactions. In this study, we could not finely define how the TRM of T20 inhibitor interacted with the FPPR of N39 because of the low-density electronic map that might be caused by their flexibilities, in addition to the structure verified the hydrophobic interactions between Leu-26/Trp-155/Ala-156. Regardless, we were surprised by the data from additional biophysical approaches (circular dichroism spectroscopy, N-PAGE, ITC), which demonstrated the essential roles of the TRM–FPPR interactions in the formation of T20/N39-based 6-HB structure. As compared to the interactions in the deep pocket sites, the interactions between the TRM and FPPR motifs might contribute to the binding of the inhibitors more efficiently. Therefore, we conclude that the TRM of T20 critically determines the inhibitory activity by interacting with the FPPR of gp41 rather than serving only as a membrane anchor. Definitely, more work should be pursued to answer whether and how the TRM of T20 is involved in these two aspects. A comprehensive analysis by integrating the structural and functional information from many other related characterizations will be also appreciated [47–49].

Acknowledgements

We thank the scientists at the SSRF BL17U beamline for assistance in diffraction data collection. This work was supported by grants from the Natural Science Foundation of China (81630061, 81473255) and the CAMS Innovation Fund for Medical Sciences (2017-I2M-1-014).

Author contributions: X.Z., X.D., Y.Z., and H.C. performed the experiments. X.Z., S.C., J.H., and X.W. analyzed the data. Y.H. conceived and designed the study and drafted the manuscript. All authors read and approved the final manuscript.

Conflicts of interest

There are no conflicts of interest.

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* Xiujuan Zhang and Xiaohui Ding contributed equally to this work.

Keywords:

crystal structure; fusion inhibitor; HIV-1; six-helical bundle; T20

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