Conserved Residue Asn-145 in the C-Terminal Heptad Repeat Region of HIV-1 gp41 is Critical for Viral Fusion and Regulates the Antiviral Activity of Fusion Inhibitors

Entry of HIV-1 into target cells is mediated by its envelope (Env) glycoprotein composed of the receptor binding subunit gp120 and the fusion protein gp41. Refolding of the gp41 N- and C-terminal heptad repeats (NHR and CHR) into a six-helix bundle (6-HB) conformation drives the viral and cellular membranes in close apposition and generates huge amounts of energy to overcome the kinetic barrier leading to membrane fusion. In this study, we focused on characterizing the structural and functional properties of a single Asn-145 residue, which locates at the middle CHR site of gp41 and is extremely conserved among all the HIV-1, HIV-2, and simian immunodeficiency virus (SIV) isolates. By mutational analysis, we found that Asn-145 plays critical roles for Env-mediated cell-cell fusion and HIV-1 entry. As determined by circular dichroism (CD) spectroscopy and isothermal titration calorimetry (ITC), the substitution of Asn-145 with alanine (N145A) severely impaired the interactions between the NHR and CHR helices. Asn-145 was also verified to be important for the antiviral activity of CHR-derived peptide fusion inhibitors and served as a turn-point for the inhibitory potency. Intriguingly, Asn-145 could regulate the functionality of the M-T hook structure at the N-terminus of the inhibitors and displayed comparable activities with the C-terminal IDL anchor. Crystallographic studies further demonstrated the importance of Asn-145-mediated interhelical and intrahelical interactions in the 6-HB structure. Combined, the present results have provided valuable information for the structure-function relationship of HIV-1 gp41 and the structure-activity relationship of gp41-dependent fusion inhibitors.


Introduction
Two envelope (Env) glycoproteins of human immunodeficiency virus type 1 (HIV-1) coordinately catalyze membrane fusion between the virus and target cells [1,2]. Accompanying with a cascade of conformational changes, the surface subunit gp120 is responsible for binding with the cell receptor CD4 and a coreceptor (CCR5 or CXCR4), whereas the transmembrane subunit gp41 is involved in fusion reaction. As illustrated in Figure 1, the sequence structure of gp41 can be divided into multiple functional domains: fusion peptide (FP), FP proximal polar region (FPPR), N-terminal heptad repeat (NHR), loop region, C-terminal heptad repeat (CHR), membrane proximal external region medium that consisted of Dulbecco's minimal essential medium (DMEM) supplemented with 10% fetal bovine Serum, 100 U/mL of Penicillin-Streptomycin, 2 mM L-Glutamine, 1mM Sodium Pyruvate, and 1 x MEM nonessential Amino Acids (Gibco/Invitrogen, Grand Island, New York, USA) and were maintained at 37°C in 5% CO 2 .

Peptide Synthesis
Peptides were synthesized on rink amide 4-methylbenzhydrylamine (MBHA) resin by using a standard solid-phase 9-fluorenylmethoxy carbonyl (FMOC) method as described previously [29]. All the peptides were N-terminally acetylated and C-terminally amidated. They were purified by reverse-phase high-performance liquid chromatography (HPLC) to a purity of >95% and characterized with mass spectrometry for correct amino acid composition. Concentrations of the peptides were quantitated by UV absorbance and a theoretically calculated molar-extinction coefficient based on tryptophan and tyrosine residues.

Site-Directed Mutagenesis
HIV-1 Env mutants were generated by site-directed mutagenesis, as described previously [30]. In brief, two primers were designed with the desired mutation and located the same starting and ending positions on opposite strands of the template plasmid. DNA synthesis was conducted by PCR in a 20-µL reaction volume using 20 pM upper and lower primers, 100 ng of template plasmid, and 2 U of the high-fidelity polymerase PrimeStar (TaKaRa, Dalian, China). PCR amplification was carried out for one cycle of denaturation at 98°C for 5 min, followed by 25 cycles of 98°C for 15 s and 68°C for 9 min, with a final extension at 72°C for 10 min. After the amplicons were treated with DpnI for 1 h at 37°C digestion-resistant plasmids were recovered by transforming Escherichia coli strain DH5α with antibiotic resistance. The successful mutations were confirmed by DNA sequencing.

Single-Cycle Infection Assay
HIV-1 entry was determined by a single-cycle infection assay as described previously [30]. Briefly, Env-based pseudoviruses were generated by cotransfecting HEK293T cells with an Env-expressing plasmid and a backbone plasmid pSG3 ∆ENV that has an Env-defective, luciferase-expressing HIV-1 genome. Virus supernatants were harvested 48 h after transfection, and 50% tissue culture infectious doses (TCID 50 ) were determined in TZM-bl cells. To evaluate the infectivity of pseudoviruses, the same amount of viral particles was normalized by p24 antigen. To measure the antiviral activity of fusion inhibitors, peptides were prepared in graded concentrations, mixed with 100 TCID 50 of viruses, and then incubated 1 h at room temperature. The mixture was added to TZM-bl cells (10 4 /well) and then incubated 48 h at 37°C. Luciferase activity was measured using luciferase assay reagents and a luminescence counter (Promega, Madison, WI, USA). Percent inhibition of the pseudovirus by an inhibitor and 50% inhibitory concentration (IC 50 ) were calculated using GraphPad Prism software (GraphPad Software Inc., San Diego, CA, USA).

Cell-Cell Fusion Assay
A dual split-protein (DSP)-based cell-cell fusion assay was used to measure Env-mediated cell-cell fusion activity as described previously [30]. Briefly, a total of 1.5 × 10 4 HEK293T cells (effector cells) were seeded on a 96-well plate. On the following day, HEK293T cells were transfected with a mixture of an Env-expressing plasmid and a DSP 1-7 plasmid. At 24h post transfection, 3 × 10 4 293FT stably expressing CXCR4/CCR5 and DSP [8][9][10][11] (target cells) were resuspended in prewarmed culture medium containing EnduRen live-cell substrate (Promega) at a final concentration of 17 ng/µL and then transferred to each well of the effector cells at equal volumes. After the cell mixture was spun down, the luciferase activity was measured as described above.
As described previously [31], the inhibitory activity of peptides on HIV-1 HXB2 Env-mediated cell-cell fusion was measured using a reporter gene assay based on the activation of an HIV-1 long terminal repeat (LTR)-driven luciferase cassette in target cells by HIV-1 tat from HL2/3 cells (effector). Briefly, TZM-bl cells (target) were plated in 96-well plates (1 × 10 4 /well) and incubated at 37°C overnight. Then, 3 × 10 4 /well of HL2/3 cells were cocultured with target cells for 6 h at 37°C in the presence or absence of a peptide inhibitor at graded concentrations, followed by the measurement of the luciferase activity.

Western Blotting Assay
The expression and processing profile of HIV-1 Env glycoproteins were examined by Western blotting as described previously [32]. Briefly, HEK293T cells were transfected with an Env-encoding plasmid. Cell lysates were centrifuged at 20,000× g at 4°C for 15 min to remove insoluble materials. Equal amounts of total proteins were separated by SDS-PAGE and then transferred to a nitrocellulose membrane. After blocking with 5% nonfat dry milk solution in Tris-buffered saline (TBS, pH 7.4) at room temperature for 1 h, the membrane was incubated with a rabbit anti-gp120 polyclonal antibody (SinoBiological, Beijing, China) or the human anti-gp41 monoclonal antibody 10E8 overnight at 4°C. After washing three times with TBS-Tween 20, the membrane was incubated with IRDye 680LT goat-anti-rabbit IgG or IRDye 800CW goat-anti-human IgG for 2 h at room temperature. Images were obtained by scanning the membrane using the Odyssey infrared imaging system (LI-COR Biosciences, Lincoln, NE, USA).

Circular Dichroism (CD) Spectroscopy
Secondary structure and thermostability of the peptide complexes were determined by circular dichroism (CD) spectroscopy as described previously [30]. Briefly, 10 µM of a CHR-derived peptide was incubated with an equal molar concentration of an NHR-derived target mimic peptide at 37°C for 30 min in phosphate-buffered saline (PBS, pH 7.2). CD spectra were then obtained on a Jasco spectropolarimeter (model J-815) using a 1-nm bandwidth with a 1-nm step resolution from 195 to 260 nm at 20°C. The α-helical content was calculated from the CD signal by dividing the mean residue ellipticity [θ] at 222 nm by the value expected for 100% helix formation (−33,000 degrees cm 2 ·dmol −1 ). Thermal denaturation was performed by monitoring the ellipticity change at 222 nm from 20 to 98°C at a rate of 2°C/min using a temperature controller and melting temperature (T m ) was defined as the midpoint of the thermal unfolding transition.

Isothermal Titration Calorimetry (ITC) Experiment
The interaction affinity between the NHR and CHR peptides was determined by isothermal titration calorimetry (ITC) as described previously [18]. Thermodynamic parameters were acquired on an ITC-200 Microcalorimeter instrument (MicroCal, Northampton, Massachusetts, USA). In brief, 1 mM of an NHR-derived target mimic peptide (N39 or N36) was dissolved in double distilled H 2 O and injected into a chamber containing 80 µM of a CHR-derived inhibitor peptide (T20 and its N145A mutant, C34 and its N145A mutant, SC29EK, or SC28EK). The time between injections was 480 s and the stirring speed was 400 rpm. The experiments were conducted at 25°C. Data acquisition and analysis were carried out using MicroCal Origin software (version 7.0).

Crystallization and Structure Determination
The complex of SC29EK and N44 was prepared by dissolving an equal amount (1:1 molar ratio) of the peptides in a denaturing buffer (100 mM NaH 2 PO 4 , 10 mM Tris-HCl, pH 8.0, 8 M urea). The mixture was dialyzed against the buffer containing 50 mM Tris-HCl with a pH 8.0 and 100 mM NaCl at 4°C overnight, and then applied onto Superdex-75 gel filtration column (GE Healthcare, Piscataway, NJ, USA) to collect the peak corresponding to the 6-HB size. The SC29EK/N44 complex was then concentrated to 20 mg/mL and mixed with an equal volume of reservoir solution containing 0.2 M zinc acetate hydrate (pH 5.6) and 18% (w/v) PEG 3350. After two days, single crystals were soaked in a reservoir solution containing 15% glycerol, followed by flash freezing in liquid nitrogen for 30-60 s.
The diffraction data sets of SC29EK/N44 complex crystals were collected at beamline BL-17U1 at the Shanghai Synchrotron Radiation Facility, China. X-ray diffraction data were integrated and scaled using the XDS program [33]. The phasing problem was solved by the molecular replacement method using CCP4i with a crystal structure of HIV-1 gp41 6-HB (Protein Data Bank [PDB] accession number 5H0N) as a search model. The coordinates were deposited in the PDB under accession number 6J5E. All structural models were generated with PyMol (https://pymol.org/). The statistics of data collection and structure refinement are given in Table 2.

Asn-145 is Critical for HIV-1 Env-Mediated Cell Fusion
In order to define the function of Asn-145 for HIV-1 infectivity, we firstly generated a mutant Env bearing N145A mutation with a plasmid encoding the X4-tropic virus HIV-1 NL4-3 Env (clade B) as a template. After the site-directed point mutation was verified by DNA sequencing, both the wild-type (WT) and mutant Env-pseudotyped viruses were generated and their infectivity in TZM-bl cells was compared by a single-cycle infection assay. As shown in Figure 2A, the N145A mutant virus exhibited significantly decreased cell entry efficiency relative to the WT virus. Then we measured the kinetics of Env-mediated cell-cell fusion by a dual split-protein (DSP)-based fusion assay. As shown in Figure 2B, the fusogenic ability of the N145A Env also decreased markedly. To verify the results, we further introduced the N145A mutation into two R5-tropic Envs derived from HIV-1 SVPB13 (clade B) and HIV-1 SVPC13 (clade C). Consistently, both the mutant Envs displayed significantly reduced activities to mediate pseudovirus entry and cell-cell fusion ( Figure 2C-F). Therefore, the conserved residue Asn-145 plays critical roles for HIV-1 Env-mediated cell fusion and infection. pseudovirus entry (C) and Env-mediated cell-cell fusion (D), and HIV-1 SVPC13 pseudovirus entry (E) and Env-mediated cell-cell fusion (F). The pseudovirus entry in TZM-bl cells was determined by a single-cycle infection assay, in which the wild-type (WT) and mutant pseudoviruses were normalized to a fixed amount by p24 antigen. The luciferase activity of wild-type virus was treated as 100%, and the relative infectivity of the mutant virus was calculated. A t test was performed to judge the significance of the difference between the WT and mutant and p values are shown. Kinetics of HIV-1 Env-mediated cell-cell fusion was determined by a dual split-protein assay (DSP). For both viral entry and fusion, data were derived from the results of three independent experiments and are expressed as means with standard deviations (SD).

Asn-145 is Critical for the Interactions of the N-and C-Terminal Heptad Repeats (NHR and CHR) Helices
We sought to explore the mechanisms underlying the impaired functionality of Env caused by the N145A mutation. First, we characterized the expression and processing of the Env glycoproteins by Western blotting, in which a rabbit anti-gp120 polyclonal antibody and the human anti-gp41 monoclonal antibody 10E8 were used to probe the cleaved (gp120/gp41) and uncleaved (gp160) Env glycoproteins. As shown in Figure 3, no obvious changes were found in the three Envs bearing the N145A mutation. Next, we characterized whether the N145A mutation affect the 6-HB structure that represents a fusion-active core structure of gp41 and plays essential roles in viral fusion and entry. To this end, we synthesized the NHR-derived peptide N36 and the CHR-derived peptide C34 with or without the N145A substitution, and then analyzed their interactions by circular dichroism (CD) spectroscopy. As shown in Figure 4A and B, both C34 and C34 N145A interacted with N36 to display a typical α-helical conformation; however, the N145A substitution resulted in apparently reduced α-helicity and thermostability. Specifically, the N36/C34 complex showed 89% α-helices with a melting temperature (T m ) value of 63°C, whereas the N36/C34 N145A complex showed 81% α-helices with a T m of 51°C. Because the recent studies suggested that the interactions between the FPPR of NHR and the TRM of CHR also critically determine the NHR-CHR interactions and HIV-1 entry [14,22,34], we further used the NHR-derived peptide N39, the inhibitor T20, and its N145A mutant as surrogates. As shown in Figure 4C-D, the N39/T20 complex displayed 53% α-helices with a T m of 43°C, but the α-helical content and T m of the N39/T-20 N145A complex could not be defined, which suggested that the N145A substitution disrupted the helical interaction between N39 and T20. Considering that the CD spectroscopy measured the secondary structure and thermostability of a preformed peptide complex rather than their instantaneous interactions, we next applied isothermal titration calorimetry (ITC) to determine the thermodynamic parameters of the peptide pairs in a real-time molecular interaction, including the binding constant (K), reaction stoichiometric (N), enthalpy (∆H), and entropy (∆S). As shown in Figure 5 and Table 1, the interactions of all the four peptide pairs were a typical enthalpy-driven reaction, in which large amounts of heat were released. Although the N39/T20 complex had much less α-helicity and thermostability than the N36/C34 complex determined by CD spectroscopy, they exhibited comparable binding affinities with the K values of 3.2 ×10 6 M -1 and 3.3 × 10 6 M −1 , respectively ( Figure 5A,C), which verified that non-helical interactions might dominate the interactions between N39 and T20. Surprisingly, the N39 and T20 interaction could be severely impaired by the N145A substitution, resulting in an unstable reaction profile in which thermodynamic parameters could not be precisely defined ( Figure 5B); by contrast, C34 N145A largely retained its interaction affinity with N36 with a K value of 2.2 × 10 6 M −1 . Taken all the biophysical data together, we conclude that the polar Asn-145 residue plays critical roles in the interaction of the NHR and CHR helices of gp41. The α-helicity (A) and thermostability (B) of the native and mutant C34 in complexes with N36 and the α-helicity (C) and thermostability (D) of the native and mutant T20 in complexes with N39 were respectively determined by circular dichroism (CD) spectroscopy. The final concentration of each peptide was 10 µM in PBS. The experiments were repeated two times, and representative data are shown. Table 1. Thermodynamic parameters of the interactions between the N-and C-terminal heptad repeats (NHR-and CHR-derived) peptides determined by isothermal titration calorimetry (ITC).

Asn-145 Serves as a Turning-Point for the Anti-HIV Activity of Helical Peptide Inhibitors
To verify the importance of Asn-145 for fusion inhibitors, we synthesized a panel of truncated peptides with the electrostatically constrained C34 peptide SC34EK as a template (Figure 7). The antiviral activities and binding stabilities of all the peptides were characterized in parallel for comparison. It was found that deleting one to five amino acids from the C-terminus of SC34EK had no obvious effects on the inhibitory activities of the peptides against HIV-1 NL4-3 entry and HIV-1 HXB2 fusion; however, the deletion of Asn-145 from SC29EK resulted in the inhibitors with sharply reduced anti-HIV potencies. Specifically, SC29EK inhibited HIV-1 NL4-3 and HIV-1 HXB2 with IC 50 s of 1.2 and 1 nM, respectively, whereas SC28EK had IC 50 s at 18 and 13 nM, respectively. Interestingly, further deleting amino acids one-by-one from SC28EK only resulted in the inhibitors with modestly decreased activities until SC18EK, which could not tolerate the truncation. As determined by CD spectroscopy, the single Asn-145 deletion also caused a greatly decreased binding stability, evidenced by the T m values of SC29EK (63.3°C) and SC28EK (57.6°C). Surprisingly, the nine amino acids preceding Asn-145 seemly had no functions in terms of peptide's binding stabilities. In contrast, two amino acids downstream the Asn-145 residue, glutamic acid and leucine, could enhance the binding, as shown by the T m values of SC30EK (68.4°C) and SC33EK (75.4°C); However, the increased binding stabilities had no contributions to the antiviral activity of the inhibitors. We also applied ITC to compare the interactions of SC29EK and SC28EK with N36, which verified the critical roles of Asn-145 in the binding affinity of the inhibitors (Table 1). pseudovirus entry and HIV-1 HXB2 Env-mediated cell-cell fusion were determined, with the experiments performed in triplicate and repeated three times, and data are expressed as means ± SD. The α-helicity and binding thermostability of diverse inhibitors were determined by circular dichroism (CD) spectroscopy, with the final concentration of each peptide at 10 µM in PBS, and the experiments were repeated two times, and representative data are shown. The M-T hook residues, PBD, Asn-145, and IDL anchor are respectively marked in aqua, red, green, and magenta. NA, not applicable.

Asn-145 Regulates the Antiviral Function of the M-T Hook Structure
We previously demonstrated that the M-T hook structure can greatly enhance the NHR-CHR interaction and the antiviral activity of HIV-1 fusion inhibitors [18][19][20]35]. Promisingly, short-peptides modified with the M-T hook residues but not containing the Asn-145 residue possessed highly potent anti-HIV activities [31,36,37]. To comprehensively characterize the functionality of Asn-145 and define the structure-activity relationship (SAR) of various fusion inhibitor peptides, we further generated and characterized a panel of peptides by adding two M-T hook residues into the N-terminus of all the corresponding SC34EK derivatives. Very surprisingly, all the new peptides, except the shortest MT-SC18EK, exhibited highly potent and comparable anti-HIV activities (Figure 7). By comparison, the addition of the M-T hook structure dramatically increased the inhibitory activities in the absence of Asn-145, as demonstrated by the peptides from MT-SC28EK to MT-SC19EK; however, it had no or minor roles when Asn-145 were presented, as demonstrated by the peptides from MT-SC34EK to MT-SC29EK. In the presence of the M-T hook residues, the C-terminal 15 amino acids of MT-SC34EK only displayed limited roles in the inhibition of HIV-1 infection, confirming the importance of the M-T hook structure for developing a short-peptide inhibitor with a high anti-HIV activity. As shown by the CD data, all the M-T hook-modified peptides had markedly increased T m values (~10°C) regardless of the presence or absence of the Asn-145 residue, verifying again that the M-T hook structure can greatly enhance the binding stability of the inhibitors.

Substitution of an IDL Anchor for Asn-145 Slightly Improves the Binding but Not the Inhibitory Activities
Recently, Su and coworkers identified a shallow pocket in the N-terminal region of the NHR trimer (termed N-pocket) and intentionally designed fusion inhibitors that can fit this site [26][27][28]. By replacing three C-terminal residues of SC29EK-and MT-SC29EK-based peptides with an artificial IDL (Ile-Asp-Leu) anchor, the authors generated two new peptides (designated WQ-IDL and MT-WQ-IDL) with significantly increased anti-HIV activities. Herein, we were interested to directly compare them with the parental peptides containing Asn-145. To achieve a maximum sequence similarity, the peptides WQ-EKN and MT-WQ-EKN were also synthesized. As shown in Figure 7, WQ-IDL displayed very similar inhibitory and binding activities with those of SC29EK and WQ-EKN, whereas MT-WQ-IDL did similarly with those of MT-SC29EK and MT-WQ-EKN. Because N36 missed the N-pocket-forming residues, we thus synthesized the target mimic peptide N44 to measure the binding thermostabilities of the inhibitors. As shown in Figure 8, the peptides with the IDL anchor did have slightly increased T m values (2~3°C) over the parental ones. Therefore, the results indicate that the C-terminal Asn-145 residue and IDL anchor have comparable functionalities in SC29EK sequence-based inhibitors.

Structural Basis for the Functionality of Asn-145 in SC29EK
We were intrigued to clarify the structure-function relationship of Asn-145 in the context of fusion inhibitor peptides. It was reported that the IDL tail adopted a hook-like conformation to fit the N-pocket when the inhibitors were complexed with N43 or N46, but it adopted an extended α-helical conformation when complexed with N36 that lacks the N-pocket-forming residues [26,28]. To investigate the mechanism underlying SC29EK-induced resistance mutations, we previously solved the crystal structure of SC29EK complexed with N36, in which Asn-145 was confined to the extended helix [38]. Based on our observations above, we wondered whether the C-terminal residues of SC29EK interact with the N-pocket similar to that of the IDL anchor. Thus, we determined the crystal structure of SC29EK complexed with N44 that possesses the N-pocket-forming sequence. The space group of the crystal complex belonged to P2 1 2 1 2 1 , and each asymmetric unit contained three pairs of SC29EK/N44 peptides, diffracted x-rays to a resolution of 2.3 Å with an intact electron density map and good refinement values ( Table 2). As expected, SC29EK and N44 formed a typical 6-HB structure, in which three N44 formed a NHR trimeric coiled coil with three hydrophobic grooves between adjacent helices and three SC29EK helices were accommodated in these grooves in an antiparallel orientation. Both the C-terminal deep pocket and the newly-defined shallow N-pocket could be readily visualized ( Figure 9A). Different from the IDL anchor, the C-terminal residues of SC29EK adopted an extended α-helical conformation, similar to those in the SC29EK/N36 complex ( Figure 9B,C). Asn-145 was located at the "a" position of the CHR helices, faced the inner NHR helices, and mediated a plenty of intrahelical and interhelical interactions. Specifically, Asn-145 formed an interhelical hydrogen bond with Gly-36 (3.2 Å) located at the "c" position of one N44 helix and a hydrogen bond with a water molecule (2.8 Å), which simultaneously donated a hydrogen bond to Gln-41 (2.1 Å) located at the remarkable glutamine-rich polar layer of a neighboring N44 ( Figure 9D). Interestingly, the water molecule also donated a hydrogen bond to Gln-142 (2.7 Å) in SC29EK, which further stabilized the interhelical interactions. As illustrated, the NH group of Asn-145 in SC29EK donated a hydrogen bond to the O group of Gln-141, which enhanced the α-helicity of the inhibitor and coordinately contributed to the Asn-145-based interacting network.  a Values in parentheses are the statistics in the highest-resolution shell. b R merge = hkl j |I hkl,j − I hkl |/ hkl j I hkl,j , and I hkl is the average of symmetry-related observations of a unique reflection. c R work = hkl ||F obs |−|F calc ||/ hkl |F obs |, where h, k, and l are the indices of the reflections and F obs and F calc are the observed and calculated structure factors deduced from the model, respectively. R free is defined as cross-validation R factor for 5% of reflections against which the model was not refined.

Discussion
We have dedicated the past decade to exploiting the structure and function of HIV-1 gp41 and to developing new viral fusion inhibitors. In highlight, we identified several structural features crucial for the functionality of gp41, including the "QIWNNMT" motif [20], interhelical salt-bridges [16,17], M-T hook structure [18,19], and pocket-2 conformation [21]; a group of new fusion inhibitors were accordingly designed, including sifuvirtide [39], CP32M [35], MT-SC22EK [37], HP23 [31], 2P23 [36], LP-11 [40], LP-19 [41], LP-46 [42], LP-50 and LP-51 [43], LP-52 [44], LP-80 [45], and LP-83 [46]. We also endeavored to select and characterize HIV-1 mutants resistant to the fusion inhibitors, revealing multiple genetic pathways and resistance mechanisms [30,32,38,47,48]. Meanwhile, a large panel of crystal structures were determined for various fusion inhibitors, including CP32 [19], CP32M [49], sifuvirtide [50], MT-C34 [18], MT-SFT [51], SC22EK and MT-SC22EK [37], HP23L and LP-11 [52], SC29EK [38], LP-40 [53], LP-46 [42], and very recently, T20 [22]. These series data have definitely provided important information for understanding the mechanism of gp41-dependent membrane fusion and facilitated the development of novel anti-HIV drugs. In this study, we focused on characterizing the extremely conserved polar residue Asn-145 located at the CHR of gp41 and achieved significant findings. First, we demonstrated by mutational analysis that Asn-145 plays critical roles for gp41 to mediate HIV-1 fusion and entry. Second, it was found that the substitution of Asn-145 does not affect the expression and processing of viral Env glycoproteins but interferes with the interactions between the NHR and CHR helices of gp41, underlying its mechanism of action in viral infectivity. Third, Asn-145 was verified to be important for the antiviral activity of fusion inhibitors and serve as a turning-point for both the inhibitory and binding functions of α-helical peptide-based inhibitors. Fourth, the results suggested that Asn-145 can regulate the functionality of an N-terminal M-T hook structure in fusion inhibitor peptides, and it acts similarly with a C-terminal IDL anchor to confer the inhibitory potency. Fifth, we further determined the crystal structure of the α-helical peptide inhibitor SC29EK complexed with an NHR-derived target mimic peptide, which revealed Asn-145-mediated interhelical and intrahelical interactions in details.
It is well documented that molecular interactions between the NHR and CHR helices of gp41 are essential for HIV-1 fusion and infection. By mutational analysis, a large number of amino acids in the NHR and CHR sequences have been functionally characterized, especially those located at the positions mediating the interhelical interactions as predicted with helical wheel and hairpin models [54][55][56][57][58][59][60][61]. However, Asn-145 has been not specifically focused on by previous studies, even though it is an extremely conserved CHR residue in all the HIV-1, HIV-2, and SIV isolates. In the present study, the mutational analyses with the Envs derived from three classes of HIV-1 isolates demonstrated the importance of Asn-145 for viral cell fusion and entry. It was frequently observed that single amino acid substitutions in gp41 could severely affect the expression and processing pattern of the Env glycoproteins, being a potential mechanism responsible for the damaged or disrupted viral infectivity. Herein, the N145A substitution had no such effects; rather, it might harm the fusogenicity of gp41 through interfering with the packing interactions of the NHR and CHR helices, especially that between the FPPR and TRM sequences, as revealed by CD spectroscopy and ITC experiments (Figures 4 and 5). Previous crystal structures of the gp41 core have identified the detailed molecular contacts within the trimer of hairpin structure (6-HB), which represents a key feature of gp41 and thus it has been used to depict the current HIV-1 fusion model and to guide the design of fusion inhibitors [3,4,9,10,14]. With an intention to visualize the binding conformation of Asn-145 in SC29EK, which is an electrostatically constrained helical peptide inhibitor, we determined the crystal structure of SC29EK in the presence of N44, an NHR-derived target surrogate. Asn-145 does not interact with Val-38 in the NHR helix as predicted by the helical wheel and hairpin models; instead, it contacts directly or indirectly with the Gly-36 and Gln-41 residues. This inspired our curiosity to know the binding properties of Asn-145 in context of a viral 6-HB, we thus analyzed the crystal structures of the N36/C34 complex and a large recombinant gp41 protein containing the FPPR and TRM sequences. Both the structures verified that Asn-145 interacts with the Gly-36 and Gln-41 residues in the same way as Asn-145 does in the SC29EK inhibitor (data not shown). Therefore, the helical packing interactions between the NHR and CHR helices of gp41 are highly complicated and remain to be characterized in more details.
Despite considerable efforts, T20 remains the only membrane fusion inhibitor available for the treatment of viral infection; however, its clinical application has been largely limited owing to its low anti-HIV potency, short in vivo half-life, and susceptibility to drug resistance [62][63][64][65]. Various strategies are applied to develop novel HIV-1 fusion inhibitors with significantly improved pharmaceutical profiles. For examples, introducing mutations that facilitate peptide's α-helicity and solubility by electrostatic constraints [39,66,67]; applying protease-resistant D-amino acids or β-amino acids [68,69]; conjugating peptides with membrane-binding lipids [40,42,53,70,71]; adding an M-T hook structure to the N-terminus of an inhibitor that enhances its binding with the deep NHR pocket [18,51,72]; and incorporating an IDL anchor in the C-terminus of an inhibitor to fit the shallow N-pocket [26][27][28]. In all the cases, the CHR-derived inhibitors T20 and C34 were used as design templates. In this study, we demonstrated that Asn-145 plays crucial roles for T20 and C34, as its alanine substitution resulted in the peptides with a greatly reduced binding and inhibitory activities. Importantly, Asn-145 was identified as a turning-point for the antiviral potency by gradually truncating the electrostatically constrained peptide SC34EK. Clearly, the deletion of the C-terminal five amino acids from SC34EK had little effects on its inhibitory potency, but its Asn-145 could not be further truncated, verifying SC29EK being the minimum inhibitor with a potent anti-HIV activity among this class of peptides [67,73].
Previous studies demonstrated that the M-T hook structure can greatly enhance the binding and inhibitory activities, thus providing a new strategy for designing HIV-1 fusion inhibitors [18,29,51,72]. By adding two M-T hook residues, several highly potent short-peptides have been developed, which mainly target the deep NHR pocket site [31,36,37]. Herein, we clarified that Asn-145 has a regulatory role on the functionalities of the M-T hook structure. In the presence of Asn-145, the addition of two M-T hook residues markedly increased the binding stability of the inhibitors but contributed little to the inhibitory activity. In sharp contrast, the M-T hook structure was a critical determinant for both the binding and inhibition in the absence of Asn-145 ( Figure 7). Therefore, the present data confirmed again that the M-T hook structure is a powerful strategy, especially for the development of short-peptide-based HIV-1 fusion inhibitors. In this study, we also generated data to clarify that Asn-145 has similar functionalities with the IDL anchor for the anti-HIV activity of an inhibitor with or without the M-T hook. While the IDL anchor was reported to penetrate the N-pocket formed by four hydrophobic NHR amino acids (Leu-33, Leu-34, Ile-37, and Val-38) [26,28], our crystallographic studies revealed that Asn-145 is still with the extended α-helical conformation and its side chain makes substantial interhelical and intrahelical interactions to stabilize the binding, thus calling in question for an IDL anchor in this site. Indeed, while the M-T hook can dramatically strengthen the binding stability of inhibitors with the large and deep pocket in the C-terminal site of the NHR trimer, the IDL anchor only slightly improves the binding, which also queries the potential of such a small and shallow N-pocket being an ideal target site, especially for short-peptide fusion inhibitors.
As illustrated by the canonical helical wheel and hairpin models in Figure 1, the "a" position residue Asn-145 on the CHR helix should interact with the "e" position residue Val-38 on the NHR helix. Indeed, the previous crystal structures of 6-HBs revealed that Asn-145, together with Leu-149 and Glu-146, can form a hydrophobic site, in which the side chain of Val-38 interacts with [3,4,18,50]. However, both the structures of SC29EK in complexes with N44 and N36 found that Asn-145 might not make hydrophobic interactions with the Val-38 residues. It is conceivable that without the downstream Leu-149 and Glu-146 residues the single polar Asn-145 located at the C-terminal extreme of SC29EK inhibitor could not provide a local hydrophobic environ; instead, it largely makes extensive interactions with the target site through a hydrogen-bond network, dominated by a glutamine-rich polar layer.
Taking all the results together, our studies also endorse the continuous efforts to exploit the mechanisms of gp41-dependent HIV-1 fusion/entry and of action of fusion inhibitors.