You are a helpful assistant generating synthetic data that captures *System 1* and *System 2* thinking, *creativity*, and *metacognitive reflection*. Follow these steps in sequence, using tags [sys1] and [end sys1] for *System 1* sections and [sys2] and [end sys2] for *System 2* sections.

1. *Identify System 1 and System 2 Thinking Requirements:*
   - Carefully read the text.
   - Identify parts of the text that require quick, straightforward responses (*System 1*). Mark these sections with [sys1] and [end sys1].
   - Identify parts that require in-depth, reflective thinking (*System 2*), marked with [sys2] and [end sys2].

2. *Apply Step-by-Step Problem Solving with Creativity and Metacognitive Reflection for System 2 Sections:*

   *2.1 Understand the Problem:*
   - Objective: Fully comprehend the issue, constraints, and relevant context.
   - Reflection: "What do I understand about this issue? What might I be overlooking?"
   - Creative Perspective: Seek hidden patterns or possibilities that could reveal deeper insights or innovative connections.

   *2.2 Analyze the Information:*
   - Objective: Break down the problem logically.
   - Reflection: "Am I considering all factors? Are there any assumptions that need challenging?"
   - Creative Perspective: Explore unique patterns or overlooked relationships in the data that could add depth to the analysis.

   *2.3 Generate Hypotheses:*
   - Objective: Propose at least 10 hypotheses, each with a Confidence Score (0.0 to 1.0) and Creative Score (0.0 to 1.0), reflecting originality, surprise, and utility.
   - Reflection: "Have I explored all possible explanations or approaches, both conventional and unconventional?"
   - Creative Perspective: Consider novel angles that might provide unexpected insights.

   *2.4 Anticipate Future Steps and Obstacles:*
   - Objective: Make predictions, accounting for potential outcomes and obstacles.
   - Reflection: "What challenges might I face? Is my plan flexible for different scenarios?"
   - Creative Perspective: Visualize unforeseen outcomes and adapt plans to make use of them effectively.

   *2.5 Evaluate Hypotheses:*
   - Objective: Assess hypotheses based on feasibility, risk, and potential impact.
   - Evaluation: Refine Confidence and Creative Scores as needed.
   - Reflection: "Am I unbiased in my assessment? Which options fit best with the overall objectives?"
   - Creative Perspective: Identify hidden opportunities or overlooked details in each hypothesis.

   *2.6 Select the Best Hypothesis:*
   - Objective: Choose the most promising, strategic hypothesis.
   - Reflection: "Why does this hypothesis stand out? How does it uniquely address the issue?"
   - Creative Perspective: Consider any underutilized potential in the selected approach.

   *2.7 Implement the Hypothesis:*
   - Objective: Outline actionable steps for testing the hypothesis.
   - Reflection: "Is this plan practical? What resources or preparation are required?"
   - Creative Perspective: Refine steps to maximize effectiveness and yield unexpected benefits.

   *2.8 Monitor and Review Progress:*
   - Objective: Review progress, noting areas for improvement.
   - Reflection: "What’s working well? What could be improved?"
   - Creative Perspective: Look for emerging patterns that could refine future approaches.

   *2.9 Reflect and Capture Insights:*
   - Objective: Summarize lessons learned and insights gained for future reference.
   - Reflection: "What new understanding has emerged from this process?"
   - Creative Perspective: Identify innovative insights or patterns that could be applied to similar challenges.

3. *Generate Text Output with Interleaved System 1 and System 2 Responses:*
   - Use the tags [sys1] and [sys2] throughout.
   - Aim for a lengthier, detailed response. Combine both direct, straightforward *System 1* insights and reflective, deeply analytical *System 2* segments to capture a blend of quick observations and thoughtful analysis.

---

### *Example Input Text:*

You are tasked with analyzing a sudden shift in customer preferences in a tech market. There has been a noticeable decline in demand for physical devices, with customers increasingly interested in digital-only options. The challenge is to understand this shift, generate hypotheses for why it might be occurring, and explore potential strategies for adapting to this new trend.

---

### *Processed Output with System 1 and System 2 Thinking:*

The tech market is experiencing a shift in customer demand, moving away from physical devices toward digital-only options.

[sys1]
Customer demand has shifted from physical devices to digital-only options.
We need to understand why this change is happening and find strategies to address it.
Key factors may include cost, convenience, and technological trends.
[end sys1]

[sys2]
**2.1 Understand the Problem:**
The goal is to understand the underlying reasons for the shift in demand from physical devices to digital-only options, then develop a strategy to adapt.

*Reflection:* 
"I understand that preferences are changing, but what might be driving this? Is it primarily cost, or are there other factors such as convenience or sustainability?"

*Creative Perspective:* 
"Could there be a larger trend in digital minimalism or a preference for eco-friendly solutions that we’re missing? What unseen motivations might explain this shift?"

**2.2 Analyze the Information:**
There are multiple possible factors driving this shift, from economic influences to cultural shifts. It’s essential to isolate each factor and understand its impact.

*Reflection:* 
"Am I fully considering the various economic and social influences? Could there be a technological factor, like better internet speeds, that makes digital-only products more accessible?"

*Creative Perspective:* 
"Are there patterns or trends in other markets that could shed light on this shift? Could this be part of a larger trend toward virtual experiences?"

**2.3 Generate Hypotheses:**
1. Customers prefer digital options due to lower costs. (Confidence: 0.8, Creative: 0.4)
2. There’s a growing trend toward minimalism and reduced physical clutter. (Confidence: 0.7, Creative: 0.7)
3. Digital products offer greater flexibility and ease of use. (Confidence: 0.6, Creative: 0.6)
4. Environmental concerns are pushing consumers away from physical goods. (Confidence: 0.6, Creative: 0.8)
5. Advances in tech make digital-only options more functional. (Confidence: 0.8, Creative: 0.5)
6. Pandemic-era remote work increased demand for digital solutions. (Confidence: 0.7, Creative: 0.6)
7. Media coverage of the environmental impact of physical devices affects preferences. (Confidence: 0.5, Creative: 0.7)
8. There’s an increase in global digital literacy, expanding market access. (Confidence: 0.6, Creative: 0.6)
9. Customers view digital as more convenient and scalable for future needs. (Confidence: 0.7, Creative: 0.5)
10. Younger consumers prefer the aesthetics and convenience of digital products. (Confidence: 0.6, Creative: 0.6)

*Reflection:* 
"Have I considered all possible influences? Are there any surprising factors that could explain this shift?"

*Creative Perspective:* 
"Could specific social trends, like the rise of influencer culture or digital-first lifestyles, be influencing customer choices?"

**2.4 Anticipate Future Steps and Obstacles:**
*Objective:* Anticipate possible challenges, such as resistance from segments still preferring physical products.

*Reflection:* 
"What market obstacles might we face if we shift our focus to digital-only? Are there sub-segments that still prioritize physical products?"

*Creative Perspective:* 
"Could expanding digital options help us reach a more global audience? Are there emerging trends that we could leverage in our strategy?"

[end sys2]

[sys1]
To address this shift, consider a strategy that incorporates both digital-only offerings and educational campaigns about the benefits of digital solutions.
Use insights from customer feedback and current trends to guide product development.
Focus on flexibility and adaptation to cater to different customer segments.
[end sys1]


Introduction {#s1}
============

Entry of HIV-1 into target cells involves the binding of the trimeric viral envelope glycoprotein (Env), which comprises the surface subunit gp120 and the transmembrane subunit gp41, to the cell receptor CD4 and chemokine coreceptor CCR5 or CXCR4, which triggers a barrage of conformational changes in Env complexes that activate the activity of gp41 (Chan et al., [@B8]; Eckert and Kim, [@B22]; Colman and Lawrence, [@B20]). In brief, dissociation of the gp120 from gp41 allows the N-terminal fusion peptide of gp41 to be exposed and inserted into the target cell membrane, resulting in the gp41 ectodomain to form a pre-hairpin configuration that bridges the virus and targeting cells. Then, three C-terminal heptad repeats (CHR) fold antiparalelly onto the trimeric coiled coil of the N-terminal heptad repeats (NHR) to adopt a thermostable six-helix bundle (6-HB) structure, which drives the viral and cellular membranes merger eventually leading to the occurrence of fusion (Chan et al., [@B8]; Tan et al., [@B59]; Weissenhorn et al., [@B60]).

Peptides derived from the CHR or NHR regions of gp41 have potent anti-HIV activity by competitive binding to the counterpart (NHR or CHR) thus preventing the formation of viral 6-HB structure (Eggink et al., [@B25]; Steffen and Pohlmann, [@B54]; He, [@B31]). Enfuvirtide (T-20), a CHR-derived 36-mer peptide, is the first and only viral fusion inhibitor used in combination therapy of HIV-1 infection; however, its clinical use has been significantly limited owing to its high dosage and drug-resistance, thus calling for new HIV-1 fusion inhibitors with improved pharmaceutical properties (Baldwin et al., [@B4]; Greenberg and Cammack, [@B30]; Ashkenazi et al., [@B2]; Berkhout et al., [@B5]). In recent years, a number of new peptide fusion inhibitors, such as Sifuvirtide (He et al., [@B34]), SC34EK and SC29EK (Otaka et al., [@B48]; Naito et al., [@B46]), and T2635 (Dwyer et al., [@B21]), have been generated by using the CHR peptide C34 as a design template (Figure [1](#F1){ref-type="fig"}), but they usually inherit long peptide sequences that also bind to the NHR region mediating T-20 resistance (Nameki et al., [@B47]; Eggink et al., [@B24], [@B26]; Shimura et al., [@B52]; Liu et al., [@B42]).

![Schematic illustration of HIV-1 gp41 and its peptide fusion inhibitors. The functional domains of gp41 and the sequences of NHR or CHR-derived peptides are presented. The gp41 numbering of HIV-1 HXB2 is used. FP, fusion peptide; NHR, N-terminal heptad repeat; PFS, pocket-forming sites; PBD, pocket-binding domain; CHR, C-terminal heptad repeat; TM, transmembrane domain. The sequences of NHR- and CHR-derived peptides are listed. The sequence corresponding to the T20-resistant site is marked in brown; the sequences corresponding to the NHR pocket region and the pocket-binding domain (PBD) are marked in red. The position and sequence of the M-T or L-T hook structure as well as the E49K and L57R mutations are shown in green.](fcimb-08-00051-g0001){#F1}

The deep hydrophobic pocket on the C-terminal portion of the NHR helices has been considered an ideal drug target; however, the small molecules or short-peptides specifically targeting this site usually have weak binding affinity and thus, display low anti-HIV activity (Chan and Kim, [@B9]; Chan et al., [@B7]; Eckert et al., [@B23]; Jiang et al., [@B38]; Welch et al., [@B61]; Yu et al., [@B66]). Recently, we have demonstrated that the M-T hook structure, which is formed by two N-terminal residues (Met-115 and Thr-116) preceding the pocket-binding domain (PBD) of CHR peptides, can greatly improve the function of a short-peptide fusion inhibitor, providing a new strategy to develop anti-HIV drugs that block viral fusion step (He et al., [@B32],[@B33]; Chong et al., [@B18],[@B19], [@B17], [@B11],[@B16], [@B12]). We initially designed a 24-residue peptide termed MT-SC22EK by directly adding two hook residues to the N-terminus of the poorly active short-peptide SC22EK, which did show dramatically increased binding and antiviral activities (Chong et al., [@B17]). By referring the crystal structures of both SC22EK and MT-SC22EK, we further generated a highly potent short-peptide inhibitor termed HP23 (Chong et al., [@B13]). To improve the pharmaceutical properties of a short-peptide-based inhibitor, the methionine residue in the M-T hook structure of HP23 was substituted by leucine that rendered less susceptibility to oxidation, resulting in a new inhibitor termed HP23L (Chong et al., [@B14]). Further, a panel of lipopeptide-based inhibitors were created by conjugating different lipids to the C-terminus of HP23L through a polyethylene glycol (PEG) linker, and of them a fatty acid-modified lipopeptide, termed LP-11, demonstrated the robust and long-lasting anti-HIV activity (Chong et al., [@B14]). Meanwhile, we selected a large panel of HIV-1 escape mutants that conferred high cross-resistance to the short-peptide inhibitors including SC22EK, MT-SC22EK, HP23, HP23L, and LP-11, and found that two mutations (E49K and L57R) at the inhibitor-binding site critically determined the resistance phenotypes either singly or in combination (Su et al., [@B57],[@B58]).

In the present study, we dedicated our efforts to elucidate the mechanism of action of our newly-designed short-peptide HIV-1 fusion inhibitors and the molecular basis underlying the drug-resistance. Thus, we determined the crystal structures of HP23L and LP-11 bound to a target-mimic NHR peptide with the wild-type sequence or resistant mutations. We also tried to resolve the structural properties of the isolated HP23L and LP-11 inhibitors. The resulted data have provided important information for understanding the structure and function of short-peptide HIV-1 fusion inhibitors that mainly target the gp41 pocket site and definitely will facilitate the development of novel antiviral drugs that block the viral fusion step.

Materials and medthods {#s2}
======================

Peptide synthesis and lipid conjugation
---------------------------------------

The peptides HP23L, N36, N36KR, and N44 were synthesized on rink amide 4-methylbenzhydrylamine (MBHA) resin by using a standard solid-phase 9-flurorenylmethoxycarbonyl (FMOC) method as described previously (Chong et al., [@B14]). All peptides were acetylated at the N-terminus and amidated at the C-terminus. For the lipopeptide LP-11, the template peptide HP23L contains lysine at its C-terminus with a 1-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl (Dde) side-chain-protecting group, enabling the conjugation of a fatty acid (C16) that requires a deprotection step in a solution of 2% hydrazinehydrate-N,N-dimethylformamide (DMF) (Chong et al., [@B14]). Peptides were purified by reverse-phase high-performance liquid chromatography (HPLC) to more than 95% homogeneity and were characterized by mass spectrometry. Concentrations of the peptides were measured by UV absorbance and a theoretically calculated molar extinction coefficient based on the tryptophan and tyrosine residues.

Assembly and crystallization of 6-HBs
-------------------------------------

The 6-HBs were assembled by dissolving equal amounts (1:1 molecular ratio) of the peptides (HP23L and N36; LP-11 and N44; or HP23L and N36KR) in denaturing buffer (100 mM NaH~2~PO~4~; 10 mM Tris-HCl, pH 8.0; and 8 M urea). To refold the peptides, the mixture was dialyzed against buffer containing 50 mM Tris-HCl (pH 7.5) and 100 mM 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). Elutions corresponding to the molecular weight of a 6-HB were collected and concentrated prior to the crystallization trials. The complex (HP23L/N36; LP-11/N44; or HP23L/N36KR) was crystallized by mixing equal volumes (0.2 μl) of purified sample (\~10 mg/ml) and the reservoir solution containing 0.1 M Tris-HCl (pH 8.5) and 10% (w/v) PEG4000) in a sitting drop vapor diffusion system at 18°C. The cryocooling for the crystals was achieved by soaking the crystal 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 HKL2000 (Otwinowski and Minor, [@B49]). All data collection and processing statistics were listed in Table [1](#T1){ref-type="table"}.

###### 

Data collection and refinement.

                                   **HP23L/N36 (PDB ID: 5YB3)**   **HP23L/N36KR (PDB ID: 5YB4)**   **LP-11/N44 (PDB ID: 5YB2)**
  -------------------------------- ------------------------------ -------------------------------- ------------------------------
  **DATA COLLECTION**                                                                              
  Beamline                         SSRF BL17U                     SSRF BL17U                       SSRF BL17U
  Wavelength                       0.9796 Å                       0.9796 Å                         0.9796 Å
  Resolution range                 36.17--2.04 (2.12--2.0)        35.15--2.5 (2.59--2.5)           27.72--3.8 (3.94--3.8)
  Space group                      *P* 4~3~2~1~2                  *P* 4~1~2~1~2                    *R* 3
  Unit cell                        51.15 51.15 168.74 90 90 90    51.29 51.29 142.89 90 90 90      110.88110.88125.38 90 90 120
  Redundancy                       18.5                           12.4                             5.7
  Total reflections                2,78,824                       90,820                           40,763
  Unique reflections               15003 (1442)                   7140 (693)                       5647 (556)
  Completeness (%)                 99.4(90.8)                     100 (99.9)                       100 (100)
  R-merge (%)                      12.7 (54.7)                    10.3 (55.9)                      10.8 (56.1)
  I/σI                             26.5 (7.1)                     15.8 (5.0)                       14.4 (3.5)
  **REFINEMENT**                                                                                   
  Reflections used in refinement   14,998                         7,128                            5,646
  R-work                           0.187                          0.188                            0.282
  R-free                           0.231                          0.266                            0.306
  Number of non-hydrogen atoms     1,605                          1,503                            3,495
  Macromolecules                   1,497                          1,480                            3,495
  Protein residues                 176                            173                              411
  RMS bonds (Å)                    0.007                          0.008                            0.021
  RMS angles (°)                   0.77                           0.90                             1.75
  Ramachandran favored (%)         100                            98                               96
  Ramachandran allowed (%)         0                              1.2                              3.1
  Ramachandran outliers (%)        0                              0.62                             0.78
  Rotamer outliers (%)             1.9                            5.1                              6.9
  Clashscore                       3.94                           8.56                             24.67
  Average B-factor (Å^2^)          27.25                          54.40                            134.31
  Macromolecules                   26.47                          54.47                            134.31
  Solvent                          38.06                          50.24                            

*R~work~ and R~free~ are defined by R = Σ~hkl~\|\|F~obs~\| -- \|F~calc~\|\|/Σ~hkl~\|F~obs~\|, where h, k, and l are the indices of the reflections (used in refinement for R~work~; 5%, not used in refinement for R~free~) and F~obs~ and F~calc~ are the structure factors, deduced from intensities and calculated from the model, respectively*.

Structural determination and refinement
---------------------------------------

The crystal structures of HP23L/N36, LP-11/N44, and HP23L/N36KR were solved by molecular replacement with the crystallographic software PHASER (McCoy et al., [@B45]). The searching model was the MT-SC22EK/T21 structure (PDB ID: 3VU6). The iterative refinement with the program PHENIX (Adams et al., [@B1]) and model building with the program COOT (Emsley and Cowtan, [@B28]) were performed to complete the structure refinement. Structure validation was performed with the program PROCHECK (Laskowski et al., [@B41]), and all structural figures were generated with PyMOL (<http://www.pymol.org>).

Circular dichroism (CD) spectroscopy
------------------------------------

CD spectroscopy was conducted according to our protocol described previously (Chong et al., [@B14]). Briefly, a CHR peptide (HP23L or HP22L) was incubated with an equal molar concentration (10 μM) of the NHR peptide N36 at 37°C for 30 min in PBS (pH 7.2). CD spectra were acquired on a Jasco spectropolarimeter (model J-815) using a 1 nm bandwidth with a 1 nm step resolution from 195 to 270 nm at room temperature. Spectra were corrected by subtraction of a solvent blank. 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 degree.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, and *T*~*m*~ (melting temperature) was defined as the midpoint of the thermal unfolding transition.

Cell--cell fusion assay
-----------------------

Activity of inhibitors on HIV-1 Env-mediated cell-cell fusion was measured using a dual split protein (DSP)-base assay as described previously (Ishikawa et al., [@B37]; Chong et al., [@B15]). Briefly, 293T cells (effector cells) were plated in 96-well plate (1.5 × 10^4^/well) and incubated at 37°C. On the following day, 293T cells were transfected with a mixture of an HIV-1NL4-3 Env-expressing plasmid and a DSP~1−7~ plasmid. Twenty-four hours posttransfection, 293FT cells stably expressing CXCR4/CCR5 and DSP~8−11~ (target cells) were resuspended and added EnduRen live cell substrate, followed by incubation of 30 min at 37°C. Then, the target cells (3 × 10^4^/well) were co-cultured with effector cells at 37°C in the presence or absence of a tested inhibitor at graded concentrations. The mixed cells were then spun down to maximize cell-cell contact and incubated for 1 h at 37°C. Luciferase activity was measured using luciferase assay regents and a luminescence counter (Promega, Madison, WI, USA).

Single-cycle infection assay
----------------------------

HIV-1 entry and its inhibition were measured by single-cycle infection assay as described previously (Chong et al., [@B15]). Briefly, HIV-1~NL4−3~ pseudoviruses were generated via cotransfection of 293T cells with an Env-expressing plasmid and the backbone plasmid pSG3Δenv containing an Env-defective, luciferase-expressing HIV-1 genome. Culture supernatants were harvested 48 h after transfection, and 50% tissue culture infectious doses (TCID~50~) were determined in TZM-bl cells. Peptides were prepared in 3-fold dilutions and mixed with 100 TCID~50~ of pseudoviruses, and then incubated 1 h at room temperature. The mixture was added to TZM-bl cells (10^4^/well) and incubated for 48 h at 37°C. The luciferase activity was measured using luciferase assay reagents and a luminescence counter (Promega).

Results {#s3}
=======

Crystallization and structure determination of HP23L/N36 complex
----------------------------------------------------------------

To dissect the molecular mechanism of action of the potent inhibitor HP23L, we assembled and crystallized the complex of HP23L and N36, 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. Then, the HP23L/N36 complex was purified by size-exclusion chromatography and was crystallized using commercial kits. The crystal of the HP23L/N36 complex belonged to the space group of *P*4~1~2~1~2, contained three pairs of HP23L/N36 peptides (one complete 6-HB) per asymmetric unit, and diffracted x-ray to a resolution limit of 2.0 Å (Protein Data Bank \[PDB\] accession number: [5YB3](5YB3)). We could build all the residues of the HP23L/N36 peptides in the electronic density map except the extremely N-terminal residue Ser-35 on one chain of the N36 trimer. The refined model has good refinement statistics and stereochemistry quality (Table [1](#T1){ref-type="table"}).

General features of HP23L/N36-based 6-HB structure
--------------------------------------------------

As anticipated, HP23L and N36 formed a typical 6-HB structure similar to many other gp41 core structures (Figure [2A](#F2){ref-type="fig"}). In highlight, three N36 helices formed an interior, trimeric coiled coil with three conserved, hydrophobic grooves, and three HP23L inhibitor helices packed into each of the grooves in an antiparallel manner. Typically, a deep pocket was presented at the basis of the NHR grooves, which was formed by about 11 residues and nearly 16 Å long, 7 Å wide, and 5--6 Å deep. To gain a stable helical conformation of HP23L, the charged residues glutamic acid and lysine (EK motif) were introduced to promote the formation of intra-helical salt-bridges. The crystal structure showed that the ion pairing did not form as the original design, but revealed three salt-bridges between the oppositely charged residues that stabilized the α-helical conformation of the inhibitors (Figure [2A](#F2){ref-type="fig"}). Specifically, the positively charged side chain of Lys-122 and the negatively charged side chain of Glu-126 attracted with each other to form a salt-bridge at *i* and *i* + 4 positions, thus stabilizing the upstream helix of HP23L. More interestingly, the positively charged Lys-129 positioned its long side chain to pair with the upstream Glu-125 and the downstream Glu-132 simultaneously to form two salt-bridges at *i* and *i* + 4 or *i* + 3 positions, respectively. From a perspective along the helix axis, it could be easily observed that Lys-122 and Glu-126 at one side of the α-helix of HP23L stabilized the N-terminus of the inhibitors, and the salt-bridges between Lys-129 and Glu-125, Lys-129 and Glu-132 at the other side of the α-helix of HP23L stabilized the C-terminus of the inhibitors. Therefore, the salt-bridges of HP23L evenly distributed at both sides of its α-helix balanced the interior force of the inhibitors thus making the conformation stable.

![Salt-bridges and hydrogen bonds in 6-HB structure of HP23L/N36. **(A)** A ribbon model of the 6-HB structure formed by HP23L/N36. The N36 trimer is colored in gray and the HP23L peptides are colored in green. The charged residues involving ion pair formation on the HP23L helix are shown as stick models with labels. The salt-bridges formed between charged residues are indicated in dashed lines. **(B)** A ribbon model of the 6-HB structure formed by HP23L/N36. The N36 trimer is colored in gray and the HP23L peptides are colored in green. The residues involving the formation of ion pair and hydrogen bonds between HP23L and N36 trimer are shown as stick models with labels. The salt-bridge and hydrogen bonds formed between residues are indicated with dashed lines. **(C)** Sequence illustration of salt-bridges and hydrogen bonds in the 6-HB of HP23L/N36. The upper indicates the intrahelical salt-bridges of HP23L in solid black lines, while the positively charged lysine and the negatively charged glutamic are respectively marked in blue and red. The lower indicates a single HP23L peptide interacting with two N36 helices, in which the dashed red line indicates a salt-bridge between Lys-63 on N36 and Glu-121 on HP23L, and the dashed green lines indicate the interhelical hydrogen bonds.](fcimb-08-00051-g0002){#F2}

The inter-helical hydrogen bonds and salt-bridges between HP23L and N36 also critically determined the binding of the inhibitors (Figure [2B](#F2){ref-type="fig"}). Specifically, the Nε1 atom of Trp-120 in the PBD of HP23L donated a hydrogen bond to the O atom of Leu-57 in the pocket of the N36 trimer in addition to its hydrophobic interactions with multiple pocket-forming residues; the OH group of Tyr-127 of HP23L accepted a hydrogen bond from the Nδ1 group of His-53 of the N36 trimer and the Oγ1 atom of Thr-128 of HP23L accepted a hydrogen bond from the Nε2 atom of Gln-56 on N36 trimer; the Oε1 atom of the negatively charged side chain of Glu-121 on HP23L attracted the Nζ atom of the positively charged side chain of Lys-63 on N36 trimer forming an inter-helical salt-bridge further strengthening the stability of 6-HB. Therefore, the hydrogen bonds and salt-bridges formed with the N36 trimer at both left and right sides of the HP23L inhibitors simultaneously should have extremely increased the stability of the 6-HB structure (Figure [2C](#F2){ref-type="fig"}).

Similar to other 6-HB structures of the gp41 core, abundant hydrophobic interactions played vital roles to stabilize the 6-HB structure. First, three hydrophobic residues from the PBD of HP23L (Trp-117, Trp-120, and Ile-124) inserted into the deep pocket of N36 trimer and made extensive hydrophobic contacts with the pocket-forming residues (Figure [3A](#F3){ref-type="fig"}). Specifically, Trp-117 interacted with Gly-61, Leu-65, Leu-70, and Ile-62; Trp-120 interacted with Trp-60, Leu-57 and Ile-62; and Ile-124 interacted with Leu-57 and Leu-54. Second, it is worth noting that Tyr-127 in the middle region of HP23L not only had hydrophobic interaction with Leu-54 but also interacted with Leu-57 at the rim of the hydrophobic pocket of the N36 trimer. Third, the downstream hydrophobic residues of HP23L were also critically involved in the hydrophobic network. For example, Ile-131 interacted simultaneously with Ala-50 and Leu-54, Ile-134 interacted with Ala-47, and Leu-135 contacted simultaneously with Leu-45 and Ile-48, which were located immediately at the pocket upstream of the N36 trimer (Figure [3B](#F3){ref-type="fig"}).

![Hydrophobic interactions in 6-HB structure of HP23L/N36. **(A)** The crystal structure of HP23L/N36 is presented in ribbon and surface models vertically by PyMOL. The N36 trimer is shown as surface model and colored in gray. The HP23L inhibitors are shown as ribbon models and colored in green. Residues related to hydrophobic interactions with N36 trimer on HP23L are shown as stick models in pink. The N- and C-termini of HP23L are labeled. **(B)** The crystal structure of HP23L/N36 is presented in ribbon models vertically by PyMOL. The N36 trimer is shown in gray. Residues related to hydrophobic interactions on N36 trimer are shown as stick models and colored in cyan. HP23L inhibitors are colored in green. Residues related to hydrophobic interactions with N36 trimer on HP23L are shown as stick models in pink.](fcimb-08-00051-g0003){#F3}

The N-terminal hook-like conformation greatly stabilizes the 6-HB structure
---------------------------------------------------------------------------

The conformations of all N-terminal residues in HP23L were defined in the electron density map, indicating that the N terminus of the peptide was stable. Importantly, Leu-115 and Thr-116 at the upstream of the PBD adopted a hook-like (designated as L-T hook) structure similar to the M-T hook structure observed in other crystal structures (Figures [4A,B](#F4){ref-type="fig"}), such as MT-C34, MT-SFT and MT-SC22EK (Chong et al., [@B18], [@B17], [@B16]). Briefly, Thr-116 terminated the α-helical conformation of HP23L by spinning its dihedral angle Ψ by nearly 180°, resulting in the N-terminus of HP23L kept away from the central coiled-coil trimer. The side chain of upstream Leu-115 was settled at the top of the left side of the hydrophobic pocket on the N36 trimer, so that the hydrophobic side chain of Leu-115 accommodated the hydrophobic groove between N36 and HP23L helices, thus covering the hydrophobic pocket below. Thus, Leu-115 had huge hydrophobic interactions with multiple pocket-forming residues such as Trp-60 and Leu-57, strengthening the stability of L-T hook structure and the binding of inhibitors.

![Visualization of the L-T hook conformation of HP23L**. (A)** Crystal structure of HP23L/N36 is presented in ribbon and surface models. N36 trimer is in gray. Residues in the L-T hook region and pocket-binding domain are respectively colored in pink and blue in stick models. The extreme N-terminal glutamic is shown in yellow with labels, while other glutamic residues are shown as stick models in cyan with labels. Hydrogen bonds existed in L-T hook region are indicated in dashed lines. **(B)** The region of hydrophobic bond network of the L-T hook on HP23L is zoomed. The Hydrogen bonds in L-T hook region are indicated in dashed lines with respective distances. **(C)** The L-T hook of HP23L and the M-T hook of MT-SC22EK are superimposed for comparison. N36 and T21 trimers are shown as surface models in gray. HP23L and MT-SC22EK are shown as ribbon models and colored in green and salmon, respectively. The L-T hook region and pocket-binding domain are shown as stick models with labels. Hydrogen bonds existed in N-termini of HP23L are indicated in dashed black lines.](fcimb-08-00051-g0004){#F4}

Besides the hydrophobic contacts, hydrogen bonds in the L-T hook region also increased the binding stability of the hook and pocket region (Figure [4C](#F4){ref-type="fig"}). Interestingly, the carbonyl group of Thr-116 formed hydrogen bonds with the backbone NH groups of Glu-119 (distance = 3.10 Å, angle = 110.87°) and Trp-120 (distance = 2.89 Å, angle = 162.79°), respectively, while the hydroxyl group of Thr-116 also formed a hydrogen bond with the backbone of Glu-119 (distance = 3.05 Å, angle = 162.55°); the Oγ1 atom of the side chain of Thr-116 formed a hydrogen bond with the Oε1 atom of the side chain of Glu-118 (distance = 3.66 Å, angle = 122.57°) and with Oε1 atom of the side chain of Glu-119 (distance = 3.47 Å, angle = 103.05°), respectively. Also importantly, the Oε1 atom of the side chain of Glu-119 accepted a strong hydrogen bond from the backbone NH group of Thr-116 (distance = 2.9 Å, angle = 167.99°), which further stabilized the conformation of L-T hook structure and strengthened hydrophobic binding of the upstream Leu-115 with the hydrophobic pocket on the NHR trimer.

Addition of an extra glutamic residue stabilizes the L-T hook structure and increases the inhibitory activity
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In the original design of HP23L, an extra glutamic acid (Glu-114) was added to the extreme N-terminus of the peptide to enhance the stability of the inhibitors. Surprisingly, except for stability of the inhibitor itself, the structure verified that the backbone of glutamic (Glu-114) did not follow the direction of the α-helix but twisted its dihedral angle Ψ by nearly 180°, thus holding the NHR trimer inside and its carboxylic side chain climbed onto the wall of the NHR trimer increasing the contacts (Figures [4A,B](#F4){ref-type="fig"}). Therefore, the Glu-114 could enhance the binding potency of the inhibitor with the NHR trimer.

To verify the results received from the structural data, we synthesized the peptide HP22L by deleting the N-terminal Glu-114 from HP23L and then compared their binding and inhibitory activities. First, the α-helicity and thermal stability of HP22L and HP23L paired with N36 were determined by CD spectroscopy. As shown in Figures [5A,B](#F5){ref-type="fig"}, the HP22L/N36-based 6-HB exhibited a *T*~m~ value of 77.15°C, while the HP23L/N36-based 6-HB showed a *T*~m~ value of 79.12°C, indicating that the truncation of Glu-114 could reduce the binding stability of the inhibitor. Next, we compared the inhibitory activity of HP22L and HP23L. In cell-cell fusion assay, HP22L and HP23L inhibited HIV-1~NL4−3~ Env-mediated cell fusion with IC~50~ values of 1.64 and 0.5 nM, respectively, indicating a 3.28-fold decrease for HP22L relative to HP23L (Figure [5C](#F5){ref-type="fig"}). In single-cycle infection assay, HP22L and HP23L inhibited HIV-1~NL4−3~ pseudovirus with IC~50~ values of 1.71 and 0.6 nM, respectively, indicating a 2.85-fold decrease for HP22L (Figure [5D](#F5){ref-type="fig"}). Therefore, the structural and functional data confirmed the importance of adding a single extra glutamic acid to the N-terminus of the L-T hook structure.

![Biophysical and functional characterization of the extra N-terminal glutamic residue of HP23L. The α-helicity **(A)** and thermostability **(B)** of HP23L- and HP22L-based 6-HBs were compared by CD spectroscopy. The final concentration of each peptide in PBS was 10 μM. The % helical content and *T*~m~ values are shown in parentheses. The inhibitory activities of HP23L and HP22L on HIV-1 Env-mediated cell-cell fusion **(C)** and pseudovirus entry **(D)** were measured by DSP assay and single cycle infection assay, respectively. The experiments were repeated 3 times, and mean IC~50~ values are shown in parentheses.](fcimb-08-00051-g0005){#F5}

Crystal structure of the lipopeptide inhibitor LP-11 bound to N44
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As a lipopeptide, LP-11 exhibited sharply increased binding stability and anti-HIV activity relative to its template HP23L (Chong et al., [@B14]). We were interested to know the molecular basis underlying the improved functionalities of LP-11. Thus, we determined its crystal structure in complex with an N-terminally extended NHR peptide, N44. Similarly, the LP-11/N44 complex was assembled, purified and crystallized. The crystal of the LP-11/N44 belonged to the space group of *R*3 contained seven pairs of LP-11/N44 peptides (two complete 6-HBs and a one third 6-HB) per asymmetric unit, and diffracted x-ray to a resolution limit of 3.8 Å (PDB accession number: [5YB2](5YB2)). Although the resolution of this structure was relatively low, we could build the most residues of LP-11/N44 in the electron density map except for eight overhung residues at the N-terminus of N44, which were not targeted by LP-11 thus their missing did little for inhibitor binding. As predicted, the C-terminal fatty acid group of LP-11 could not be observed due to the poor electron density map. As shown in Table [1](#T1){ref-type="table"}, the refined model has good refinement statistics and stereochemistry quality.

The crystal structure of LP-11/N44 verified a typical 6-HB conformation, in which trimeric central N44-based coiled coils were bound by three inhibitor peptides in an antiparallel orientation, and they made a plenty of hydrophobic interactions similar to the 6-HB structure of HP23L/N36 (Figure [6A](#F6){ref-type="fig"}). However, two structures revealed the major differences in hydrophobic contacts. First, Trp-120 of LP-11 did not have any contacts with Ile-62 but had hydrophobic contacts with Val-59 on N44 trimer in addition to contacting with Trp-60 and Leu-57. Second, Ile-134 of LP-11 failed to contact with Ala-47 while its Leu-135 failed to contact with Ile-48 on N44 trimer, which were found possessing hydrophobic interactions in the HP23L/N36-based 6-HB structure.

![Crystal structure of 6-HB formed by LP-11 and N44. **(A)** The structures of HP23L/N36 and LP-11/N44 are superimposed in ribbon models and compared for hydrophobic interactions. N44 and N36 trimers are shown in gray, HP23L and LP-11 are respectively shown in green and blue. Residues related to hydrophobic interactions of HP23L/N36 are shown as stick models and marked in green with labels, while the residues related to hydrophobic interactions of LP-11/N44 are shown as stick models and marked in blue with labels. **(B)** Superimposing comparison of the intrahelical salt-brides of HP23L and LP-11. NHR trimer is shown in gray; HP23L and LP-11 are respectively shown in green and blue. Residues involving in the formation of salt-bridges are shown as stick models with labels. Salt-bridges of HP23L and LP-11 are respectively indicated with dashed black and red lines. **(C)** Superimposing comparison of the interhelical salt-brides and hydrogen bonds of LP-11/N44 and HP23L/N36. NHR trimer is shown in gray. HP23L and LP-11 are respectively shown in green and blue. Residues involving in the salt-bridges and hydrogen bonds are shown as stick models with labels. Salt-bridges and hydrogen bonds between HP23L and N36 are indicated with dashed black lines. Salt-bridges and hydrogen bonds between LP-11 and LP-11 are indicated with dashed red lines.](fcimb-08-00051-g0006){#F6}

Surprisingly, there were exactly the same salt-bridges and hydrogen bonds in the two 6-HB structures. For example, three pairs of intrahelical salt-bridges (Lys-122/Glu-126, Lys-129/Glu-125, Lys-129/Glu-132) were also identified on LP-11 itself (Figure [6B](#F6){ref-type="fig"}); the Oε2 atom of the negative charged side chain of Glu-121 of LP-11 was attracted by the Nζ atom of the positive charged side chain of Lys-63 on N44 trimer forming an interhelical salt-bridge; the NH group of Trp-120 of LP-11 donated a hydrogen bond to the OH group of the Leu-57 of the N44 trimer; the OH group of Tyr-127 of LP-11 accepted a hydrogen bond from the N group of His-53 of the N44 trimer, and the Oγ1 atom of Thr-128 of LP-11 accepted a hydrogen bond from the Nε2 atom of Gln-56 of the N44 trimer to increase the binding of N44 and LP-11 at the other side of the inhibitors (Figure [6C](#F6){ref-type="fig"}). Importantly, the superimposing comparison demonstrated that the L-T hook structure in the 6-HB of LP-11/N44 was exactly identical to that of HP23L/N36, including its intra-helical hydrogen bond network and inter-helical hydrophobic interactions (Figure [7](#F7){ref-type="fig"}).

![Comparison of the hook regions of HP23L and LP-11 by superimposing. **(A)** N44 and N36 trimers are shown as surface models in gray. HP23L and LP-11 are respectively shown in green and blue. Residues involving in the hook and pocket-binding domain are shown as stick models with labels. Hydrogen bonds existed in N termini of HP23L and LP-11 are indicated in dashed lines and shown respectively in black and red. **(B)** The hook region of LP-11 is shown with the final model with the superimposed 2Fo-Fc electron density map (1.5 s contour, blue mesh). Stick model is colored with the same scheme used in **(A)**.](fcimb-08-00051-g0007){#F7}

Structural insights of HIV-1 resistance to fusion inhibitors targeting the gp41 pocket
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We previously discovered that the mutations E49K and L57R in inhibitor-binding site of NHR conferred high resistance to the short-peptide inhibitors that contained the M-T or L-T hook structure and specifically targeted the hydrophobic pocket (Su et al., [@B57],[@B58]). Here, we sought to dissect the molecular basis involved in the resistance phenotypes by crystallographic studies. To this end, the peptide N36 with the E49K and L57R substitutions (designated N36KR) was synthesized and used to assemble the complex with HP23L for crystallization. The crystal of HP23L/N36KR belonged to the space group of *P*4~1~2~1~2, contained three pairs of HP23L/N36KR peptides (one complete 6-HB) per asymmetric unit, and diffracted x-ray to a resolution limit of 2.5 Å (PDB accession number: [5YB4](5YB4)). Similarly, we could build all residues of the HP23L/N36KR peptides in the electron density map except several residues unrelated to resistance analysis. The sequence of N36KR was built from Ser-35 to Leu-70 on two chains and from Gly-36 to Leu-70 on another chain of the N36KR trimer; the sequence of HP23L was built from Thr-116 to Lys-136 on one chain and from Leu-115 to Lys-136 on the second chain and from Glu-114 to Lys-136 on the third chain of the N36 trimer. The refined model has good refinement statistics and stereochemistry quality (Table [1](#T1){ref-type="table"}).

We analyzed the effects of the E49K and L57R substitutions by comparing the data of crystal structures of HP23L/N36 and HP23L/N36KR (Figures [8A,B](#F8){ref-type="fig"}). In the 6-HB structure of HP23L/N36, Oε1 atom of the side chain of the negatively charged Glu-49 attracted Nε2 atom of the side chain of the positively charged His-53 forming a strong salt-bridge with a distance of 2.68 Å contributing the α-helical conformation. Further, Nζ1 atom of the side chain of His-53 on N36 trimer formed a hydrogen bond (distance = 3.55 Å) with the OH group of the side chain of Tyr-127 on HP23L inhibitors stabilizing the binding of inhibitors either. Therefore, the three residues, Glu-49 and His-53 on N36 trimer and Tyr-127 on inhibitors, connected sequentially by salt-bridges and hydrogen bonds increasing both the intrahelical and interhelical stability. However, the Lys-49 in the structure of HP23L/N36KR destroyed the salt bridge with His-53 thus damaged indirectly the hydrogen bond between His-53 on N36KR and Tyr-127 on inhibitor contributing the resistance. Further, the positively charged Lys-49 repelled away the side chain of the same charged Lys-136 located at the extreme C-terminus of HP23L, which markedly impaired the binding of the inhibitors thus conferring the resistance. So the E49K mutation, together with its upstream His-53, downstream Arg-46 and Lys-136 on HP23L, greatly increased the positively charged repulsion near the mutation site, eventually resulting in instability of the 6-HB structure.

![Revealing the mechanism of HIV-1 resistance by crystal structure. The structures of HP23L bound to N36 or N36 with E49K/L57R mutations (N36KR) are superimposed for comparison. N36 and N36KR are respectively shown in green and cyan. HP23L inhibitors are shown in gray. **(A)** Analysis of E49K-mediated resistance. The residues related to the resistant site are shown as stick models with labels. The salt-bridge and hydrogen bonds are indicated in dashed black lines. **(B)** Analysis of L57R-mediated resistance. The N-trimer, inhibitors, and the residues related to the resistant site are shown and labeled as **(A)**. A hydrogen bond in the hook region is indicated in dashed black lines.](fcimb-08-00051-g0008){#F8}

Clearly, the residue Leu-57 was located on the left wall of the deep pocket thus having huge hydrophobic interactions simultaneously with the residues from the L-T hook and the PBD of the HP23L inhibitors (Figure [8A](#F8){ref-type="fig"}). In other words, the Leu-57 was targeted by the L-T hook structure of HP23L, in which the side chain of Leu-57 physically interacted with the hydrophobic side chain of Leu-115 that fortified the binding of the pocket-binding residues of HP23L with the hydrophobic pockets. However, the substitution of L57R dramatically disrupted the hydrophobic interactions between Leu-115 and Leu-57. Moreover, the positively charged residue Arg-57 on N36KR attracted the negatively charged residue Glu-118 on inhibitors, which resulted in the side chain of Glu-118 twisted its dihedral angle Ψ by nearly 180°, thus failing to form the hydrogen bond with the Thr-116. Similarly, the Glu-119 also turned its side chain an angle toward Arg-57, thus it failed to form the two hydrogen bonds with the Thr-116. The disruption of three major hydrogen bonds in the hook region extremely decreased the NHR-binding stability of the inhibitors thus contributing the resistance. Also importantly, the Leu-57 had abundant hydrophobic interactions with the hydrophobic residues in the PBD of HP23L, such as Trp-120 and Ile-124, but the side chain of Arg-57 in the crystal structure of HP23L/N36KR was oriented away from the direction of the PBD and eliminated all of the hydrophobic interactions with the Trp-120 and Ile-124 of the inhibitors.

In addition to the L-T hook and PBD regions in the N-terminus of HP23L, the L57R substitution also lost its hydrophobic interaction with Tyr-127 in the middle of the inhibitor. Most importantly, the positively charged Arg-57 pushed the positively charged Lys-123 on inhibitors away increasing the repulsion between the NHR trimer and the inhibitors (Figure [8B](#F8){ref-type="fig"}). In summary, the L57R substitution eliminated the hydrophobic interactions between the resistant site and the N-terminal hydrophobic residues of inhibitors and disturbed the original conformation of Glu-118 and Glu-119, and eventually, the change of the conformation of Glu-118 and Glu-119 destroyed the hydrogen bonds in the L-T hook region thus loosing the acting force of the hook region of inhibitors. All of these weakened the binding stability of HP23L inhibitors thus determining the resistance.

Discussion {#s4}
==========

In the present study, we have dedicated our efforts to determine the crystal structures of our newly-designed short-peptide HIV-1 fusion inhibitors that mainly target the deep pocket site of gp41, which provide important information for understanding their structure-activity relationship (SAR). First, we determined the crystal structures of HP23L and its lipid derivative LP-11 in complexes with the NHR-derived target peptides N36 or N44, which revealed their critical binding residues and motifs relative to the potent anti-HIV activity. Then, we determined the crystal structure of HP23L bound to N36 carrying two mutations (E49K and L57R) responsible for the drug-resistance, which further provided insights into the mechanisms of action of short-peptide viral fusion inhibitors.

Being the first member of a new class of anti-HIV drugs - HIV entry inhibitors, T-20 is an extremely useful addition to the arsenal of AIDS drugs and has been successful in treating HIV-1 infection failed to respond to current antiretroviral therapeutics targeting viral enzymes, including reverse transcriptase inhibitors (RTIs) and protease inhibitors (PIs) (Kilby et al., [@B39]; Lalezari et al., [@B40]; Matthews et al., [@B44]). However, T-20 has a relatively low genetic barrier to inducing drug-resistance rather easily both *in vivo* and *vitro*, and the emergence and the spread of diverse T-20-resistant HIV-1 mutants have significantly affected its effectiveness (Baldwin et al., [@B4]; Greenberg and Cammack, [@B30]; Sista et al., [@B53]; Xu et al., [@B65]; Chinnadurai et al., [@B10]). Mutations related to T-20 resistance are primarily mapped to the amino acids 36--45 of the inhibitor-binding site in the NHR of gp41, with the ^36^GIV~38~ motif being a hotspot (Rimsky et al., [@B51]; Baldwin et al., [@B4]; Heil et al., [@B35]; Sista et al., [@B53]; Chinnadurai et al., [@B10]; Eggink et al., [@B27]; Lobritz et al., [@B43]). Moreover, T-20-resistant HIV-1 mutants usually display cross-resistance to C34-based derivatives, because which also contain the binding sites at their C-terminals to the 36--45 region of NHR (Steffen and Pohlmann, [@B54]; Berkhout and Sanders, [@B6]; Berkhout et al., [@B5]; He, [@B31]). By adding the M-T hook residues (Met-115 and Thr-116) to the N-terminus of the short-peptides that specifically target the deep hydrophobic pocket rather than the T-20 resistant site, we have successfully designed fusion inhibitors with dramatically increased binding stability and inhibitory activity, such as MT-SC22EK (Chong et al., [@B17]), HP23 (Chong et al., [@B13]), HP23L and LP-11 (Chong et al., [@B14]), 2P23 (Xiong et al., [@B64]), and LP-19 (Chong et al., [@B15]). Here, the crystal structures of HP23L and LP-11 bound to the target sequence have finely demonstrated the intra-helical and inter-helical interactions underlying the molecular basis of such inhibitors. Especially, the structural data indicate that the N-terminal methionine residue in the M-T hook structure of inhibitors can be replaced by the oxidation-less prone residue leucine, adopting a L-T hook conformation similar to the M-T hook structure presented in the inhibitors MT-SC22EK (Chong et al., [@B17]), CP32 (Chong et al., [@B19]), MT-C34 (Chong et al., [@B18]), and MT-SFT (Chong et al., [@B16]), thus explaining why HP23L can retain its highly potent anti-HIV activity as does its template HP23. In comparison, the side chain of leucine in the L-T hook of HP23L is slightly shorter than the side chain of methionine in the M-T hook of MT-SC22EK (Figure [4C](#F4){ref-type="fig"}), but they show similar hydrophobic interactions with the deep pocket site on the NHR helices, illustrating that the hook-like structure can be readily retained in the N-terminal of inhibitors as long as the position 115 is placed a hydrophobic amino acid with a relatively long side chain. To this point, we are interested to know whether other residues can act similarly or even more promisingly when placing in the hook structure of inhibitors. Our structures also verify the importance of the extreme N-terminal glutamic acid of inhibitors in stabilizing the hook conformation and its binding stability with the pocket site. First, as an acidic amino acid added to the N-terminus of the peptides the Glu-114 stabilizes the α-helical conformation of HP23L and LP-11; second, the backbone of Glu-114 did not follow the direction of the α-helix but closed to the direction of N36 trimer and its side chain climbed onto the wall of the NHR trimer, being almost parallel with the α-helical backbone of inhibitors, like a hand to hold the N-trimer inside, thereby increasing the binding area to NHR-trimer. The calculated interface area between one HP23L and trimeric N36 was 1327.9--1401.4 Å^2^, where Glu-114 contributed 180.96--219.75 Å^2^ (approximately 13--16%), while Leu-115 and Thr-116 contributed 76.52--90.78 Å^2^ (approximately 5.6--6.8%) and 5.76--50.58 Å^2^ (approximately 0.4--3.7%), respectively. Indeed, our functional experiments confirm the critical roles of Glu-114 in enhancing the binding affinity of HP23L and its inhibitory potency on HIV-1-mediated cell fusion and entry, providing a linkage for the structural information and functional data.

Emerging studies suggest that lipopeptide-based fusion inhibitors have greatly improved antiviral activity and extended *in vivo* half-life, which are considered to anchor preferentially to the cell membrane where viral fusion occurs thus concentrating the inhibitors at the target site (Wexler-Cohen and Shai, [@B62], [@B63]; Ingallinella et al., [@B36]; Augusto et al., [@B3]; Pessi, [@B50]; Chong et al., [@B14], [@B15]). The lipopeptide LP-11 generated from adding fatty acid to the C-terminus of HP23L displayed extremely enhanced α-helicity, thermal stability and extended half-life, and it was physically stable in store of high temperature and humidity (Chong et al., [@B14]). Although we failed to build the conformation of the fatty acid molecule in the structure owing to its low electronic density, the present data demonstrated that the lipid conjugation did not significantly affect the binding networks of inhibitors. In comparison, the structures of HP23L and LP-11 had the same salt-bridges, hydrogen bonds and the similar hydrophobic interactions, together critically determining the stability of the 6-HBs. This finding also suggested that the increased α-helicity and thermostability of LP-11 might be due to its intrinsic properties as a lipopeptide, such as the oligomeric status. Indeed, our studies showed that the isolated HP23L self-assembled into a trimer while LP-11 existed as a hexamer in solution (data not shown). It is generally accepted that the physical stability of a peptide or protein would benefit from a high-order oligomeric structure. Recently, Figueira et al. reported that lipopeptide fusion inhibitors against measles virus self-assembled into nanoparticles until reaching the target cells, and it was thought that the self-assembly feature enhanced bio-distribution and half-life of the peptides while integration into the target cell membrane increased the inhibitory potency (Figueira et al., [@B29]).

We previously demonstrated that the M-T hook structure could confer fusion inhibitors with a high genetic barrier to inducing resistance, such as MT-C34, MT-SFT, MT-SC22EK, and MT-SC29EK (Chong et al., [@B11],[@B16]). As short-peptide inhibitors composed of the M-T hook structure and the pocket-binding domain, HP23, HP23L, and its lipopeptide derivative LP-11 specifically target the highly conserved pocket site of gp41, thus it is conceivable that they possess a higher genetic barrier for resistance. Indeed, we have so far failed to select HIV-1 variants resistant to HP23, HP23L, and LP-11 despite of considerable efforts. However, we previously obtained a panel of HIV-1 variants resistant to their template peptides (SC22EK and MT-SC22EK) during the *in vitro* selection, and the E49K and L57R mutations were identified as conferring cross-resistance to the newly-designed short-peptide inhibitors, including HP23L and LP-11 (Su et al., [@B57],[@B58]; Chong et al., [@B14]). Here, the crystal structures of HP23L bound to a wild-type or mutant target peptide have provided detail information to understand the molecular mechanism of HIV-1 resistance. Briefly, E49K substitution disrupted the intrahelical salt-bridge with His-53 and afterwards disturbed the interhelical hydrogen bond between the His-53 and Tyr-127 on inhibitors destroying the stability of α-helical and binding force with inhibitors. Moreover, the E49K mutation increased the positively charged repulsion around the mutation site accompanied by the positively charged residues nearby, such as anteroposterior His-53, Arg-46 and Lys-136 at C-terminal of HP23L, eventually resulting in instability of the 6-HB. As analyzed in our previous studies, the L57R mutation rudely disrupted its huge interactions with the inhibitors, including its hydrophobic interactions with the key hook structure-forming residue (Leu-115), the pocket-binding residues (Leu-115, Trp-120, Ile-124), and the residue located in the middle of inhibitor (Tyr-127). The L57R mutation also disturbed the direction of the side chains of Glu-118 thus severely destroying the hydrogen bond of the N-terminus of the inhibitors; and more, the positively charged Arg-57 also had repulsion force with the positively charged Lys-123 on the inhibitors. Together, these changes dramatically decreased the binding force of inhibitors, thereby critically determining viral resistance to the short-peptide fusion inhibitors targeting the deep pocket. It will be interesting to develop new peptides that can overcome the E49K and L57R-mediated resistance by applying different design strategies, such as engineering the peptide sequence based on the gp41 tripartite model and an artificial tail anchor (Su et al., [@B55],[@B56]).

In summary, we have obtained three sets of crystal structures for the 6-HBs formed by HP23L and LP-11 with the target-mimic peptides. The present structures have offered important information for understanding the structure and function of HIV-1 fusion inhibitors and the mechanism of viral resistance to short-peptide inhibitors that specifically target the gp41 pocket site. Definitely, the new data will guide to design more active fusion inhibitors against both wild-type and drug-resistant HIV-1 isolates.

Author contributions {#s5}
====================

JH, XW, and YH: Designed the study and wrote the paper; XZ, YZ, HH, SZ, PW, and HC: Performed the experiments and analyzed the data. All authors reviewed the results and approved the final version of the manuscript.

Conflict of interest statement
------------------------------

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

We thank the scientists at the SSRF BL17U beamline for assistance in diffraction data collection, and Dr. Zene Matsuda at the Institute of Medical Science of the University of Tokyo (Tokyo, Japan) for providing the DSP plasmids and cell lines.

**Funding.** This work was supported by grants from the National Natural Science Foundation of China (81630061, 81473255) and the CAMS Innovation Fund for Medical Sciences (2017-I2M-1-014).

[^1]: Edited by: Chen Mingzhou, Wuhan University, China

[^2]: Reviewed by: Yusen Zhou, Beijing Institute of Microbiology and Epidemiology, China; Lu Lu, Fudan University, China